PB2-Q591K Mutation Determines the Pathogenicity of Avian H9N2 Influenza Viruses for Mammalian Species

PubMed Central

Wang, Congrong; Lee, Horace Hok Yeung; Yang, Zi Feng; Mok, Chris Ka Pun; Zhang, Zhi

2016-01-01

Background Influenza A subtype H9N2 is widespread and prevalent in poultry. It has repeatedly transmitted zoonotically to cause mild influenza-like illness in humans and is regarded as a potential pandemic candidate. In additon, the six internal genes of H7N9 and H10N8 viruses which caused infection in human in China as well as some of the highly pathogenic H5N1 strains are origined from H9N2. Previous studies have shown that the mammalian adaptation PB2-Q591K contributes to the pathogenicity of H5N1 and H7N9 viruses. However, the role of the PB2-Q591K mutation in H9N2 subtype is still not well understood. Methods To define and compare the individual role of PB2-Q591K substitution in the PB2 gene segment of H9N2 in relation to polymerase activity, replication competence and the pathogenicity using in vitro and in vivo models. Results The PB2-Q591K mutation in H9N2 virus enhanced the polymerase activity and virus replication in human NHBE cells when compared to the wild type strain. Mice infected with the PB2 mutant showed significant weight loss, higher virus replication and immune responses in the lungs. Conclusions Our evidences suggest that the PB2-Q591K, in addition to the -E627K mutation in H9N2 enhanced the pathogenicity in mammalian host. PMID:27684944

Mutational analysis of varicella-zoster virus (VZV) immediate early protein (IE62) subdomains and their importance in viral replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khalil, Mohamed I., E-mail: mkhalil2@stanford.edu; Department of Molecular Biology, National Research Centre, El-Buhouth St., Cairo; Che, Xibing

VZV IE62 is an essential, immediate-early, tegument protein and consists of five domains. We generated recombinant viruses carrying mutations in the first three IE62 domains and tested their influence on VZV replication kinetics. The mutations in domain I did not affect replication kinetics while domain II mutations, disrupting the DNA binding and dimerization domain (DBD), were lethal for VZV replication. Mutations in domain III of the nuclear localization signal (NLS) and the two phosphorylation sites S686A/S722A resulted in slower growth in early and late infection respectively and were associated with IE62 accumulation in the cytoplasm and nucleus respectively. This studymore » mapped the functional domains of IE62 in context of viral infection, indicating that DNA binding and dimerization domain is essential for VZV replication. In addition, the correct localization of IE62, whether nuclear or cytoplasmic, at different points in the viral life cycle, is important for normal progression of VZV replication. - Highlights: • Mutation of IE62 domain I did not affect VZV replication in melanoma cells. • IE62 domain II and III are important for VZV replication in melanoma cells. • Mutations of IE62 domain II (DBD) were lethal for virus replication. • Mutations of IE62 NLS and phosphorylation sites inhibited VZV replication. • NLS and S686A/S722A mutations altered localization of IE62 during early and late infection.« less

Elucidation of the Molecular Mechanism Driving Duplication of the HIV-1 PTAP Late Domain.

PubMed

Martins, Angelica N; Waheed, Abdul A; Ablan, Sherimay D; Huang, Wei; Newton, Alicia; Petropoulos, Christos J; Brindeiro, Rodrigo D M; Freed, Eric O

2016-01-15

HIV-1 uses cellular machinery to bud from infected cells. This cellular machinery is comprised of several multiprotein complexes known as endosomal sorting complexes required for transport (ESCRTs). A conserved late domain motif, Pro-Thr-Ala-Pro (PTAP), located in the p6 region of Gag (p6(Gag)), plays a central role in ESCRT recruitment to the site of virus budding. Previous studies have demonstrated that PTAP duplications are selected in HIV-1-infected patients during antiretroviral therapy; however, the consequences of these duplications for HIV-1 biology and drug resistance are unclear. To address these questions, we constructed viruses carrying a patient-derived PTAP duplication with and without drug resistance mutations in the viral protease. We evaluated the effect of the PTAP duplication on viral release efficiency, viral infectivity, replication capacity, drug susceptibility, and Gag processing. In the presence of protease inhibitors, we observed that the PTAP duplication in p6(Gag) significantly increased the infectivity and replication capacity of the virus compared to those of viruses bearing only resistance mutations in protease. Our biochemical analysis showed that the PTAP duplication, in combination with mutations in protease, enhances processing between the nucleocapsid and p6 domains of Gag, resulting in more complete Gag cleavage in the presence of protease inhibitors. These results demonstrate that duplication of the PTAP motif in p6(Gag) confers a selective advantage in viral replication by increasing Gag processing efficiency in the context of protease inhibitor treatment, thereby enhancing the drug resistance of the virus. These findings highlight the interconnected role of PTAP duplications and protease mutations in the development of resistance to antiretroviral therapy. Resistance to current drug therapy limits treatment options in many HIV-1-infected patients. Duplications in a Pro-Thr-Ala-Pro (PTAP) motif in the p6 domain of Gag are frequently observed in viruses derived from patients on protease inhibitor (PI) therapy. However, the reason that these duplications arise and their consequences for virus replication remain to be established. In this study, we examined the effect of PTAP duplication on PI resistance in the context of wild-type protease or protease bearing PI resistance mutations. We observe that PTAP duplication markedly enhances resistance to a panel of PIs. Biochemical analysis reveals that the PTAP duplication reverses a Gag processing defect imposed by the PI resistance mutations in the context of PI treatment. The results provide a long-sought explanation for why PTAP duplications arise in PI-treated patients. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cell culture-adaptive mutations of NS5A affect replication of hepatitis C virus differentially depending on the viral genotypes.

PubMed

Chung, Aeri; Jin, Bora; Han, Kwang-Hyub; Ahn, Sang Hoon; Kim, Seungtaek

2017-01-01

Most of HCV RNAs require cell culture-adaptive mutations for efficient replication in cell culture and a number of such mutations have been described including a well-known S2204I substitution mutation in NS5A protein. In contrast, the replication of genotype 2a JFH1 RNA in cell culture does not require any cell culture-adaptive mutation. Rather, the presence of S2204I mutation impaired the JFH1 RNA replication. In this study, we examined the effect of reversions and substitutions of NS5A cell culture-adaptive mutations on virus replication in different genotypic backgrounds after either placing genotype 1a NS5A in the genotype 2a JFH1 or vice versa. The results from this investigation suggest that the S2204I mutation affects HCV RNA replication differentially depending on the viral genotypes but that the effect was not simply explained by the genotypic background. Perhaps, the effect of the S2204I mutation on HCV replication reflects both intra- and intergenic interactions of NS5A protein. J. Med. Virol. 89:146-152, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Emergence of Avian Influenza Viruses with Enhanced Transcription Activity by a Single Amino Acid Substitution in the Nucleoprotein during Replication in Chicken Brains ▿

PubMed Central

Tada, Tatsuya; Suzuki, Koutaro; Sakurai, Yu; Kubo, Masanori; Okada, Hironao; Itoh, Toshihiro; Tsukamoto, Kenji

2011-01-01

To explore the genetic basis of the pathogenesis and adaptation of avian influenza viruses (AIVs) to chickens, the A/duck/Yokohama/aq10/2003 (H5N1) (DkYK10) virus was passaged five times in the brains of chickens. The brain-passaged DkYK10-B5 caused quick death of chickens through rapid and efficient replication in tissues, accompanied by severe apoptosis. Genome sequence comparison of two viruses identified a single amino acid substitution at position 109 in NP from isoleucine to threonine (NP I109T). By analyzing viruses constructed by the reverse-genetic method, we established that the NP I109T substitution also contributed to increased viral replication and polymerase activity in chicken embryo fibroblasts, but not in duck embryo fibroblasts. Real-time RT-PCR analysis demonstrated that the NP I109T substitution enhances mRNA synthesis quickly and then cRNA and viral RNA (vRNA) synthesis slowly. Next, to determine the mechanism underlying the appearance of the NP I109T substitution during passages, four H5N1 highly pathogenic AIVs (HPAIVs) were passaged in the lungs and brains of chicken embryos. Single-nucleotide polymorphism analysis, together with a database search, suggests that the NP I109T mutation would be induced frequently during replication of HPAIVs in brains, but not in lungs. These results demonstrate that the amino acid at position 109 in NP enhances viral RNA synthesis and the pathogenicity of highly pathogenic avian influenza viruses in chickens and that the NP mutation emerges quickly during replication of the viruses in chicken brains. PMID:21795332

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ - dependent spontaneous mutagenesis.

PubMed

Stepchenkova, E I; Tarakhovskaya, E R; Siebler, H M; Pavlov, Y I

2017-01-01

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ. Copyright © 2016 Elsevier B.V. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G., E-mail: chinnag@slu.edu

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP–E1A interaction suppresses immortalization and Ras co-operativemore » transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP–E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. - Highlights: • Adenovirus E1A C-terminal region suppresses E1A/Ras co-transformation. • This E1A region binds with FOXK, DYRK1/HAN11 and CtBP cellular protein complexes. • We found that E1A–CtBP interaction suppresses immortalization and transformation. • The interaction enhances viral replication in human cells.« less

Double silencing of relevant genes suggests the existence of the direct link between DNA replication/repair and central carbon metabolism in human fibroblasts.

PubMed

Wieczorek, Aneta; Fornalewicz, Karolina; Mocarski, Łukasz; Łyżeń, Robert; Węgrzyn, Grzegorz

2018-04-15

Genetic evidence for a link between DNA replication and glycolysis has been demonstrated a decade ago in Bacillus subtilis, where temperature-sensitive mutations in genes coding for replication proteins could be suppressed by mutations in genes of glycolytic enzymes. Then, a strong influence of dysfunctions of particular enzymes from the central carbon metabolism (CCM) on DNA replication and repair in Escherichia coli was reported. Therefore, we asked if such a link occurs only in bacteria or it is a more general phenomenon. Here, we demonstrate that effects of silencing (provoked by siRNA) of expression of genes coding for proteins involved in DNA replication and repair (primase, DNA polymerase ι, ligase IV, and topoisomerase IIIβ) on these processes (less efficient entry into the S phase of the cell cycle and decreased level of DNA synthesis) could be suppressed by silencing of specific genes of enzymes from CMM. Silencing of other pairs of replication/repair and CMM genes resulted in enhancement of the negative effects of lower expression levels of replication/repair genes. We suggest that these results may be proposed as a genetic evidence for the link between DNA replication/repair and CMM in human cells, indicating that it is a common biological phenomenon, occurring from bacteria to humans. Copyright © 2018 Elsevier B.V. All rights reserved.

Interpreting the Dependence of Mutation Rates on Age and Time

PubMed Central

Gao, Ziyue; Wyman, Minyoung J.; Sella, Guy; Przeworski, Molly

2016-01-01

Mutations can originate from the chance misincorporation of nucleotides during DNA replication or from DNA lesions that arise between replication cycles and are not repaired correctly. We introduce a model that relates the source of mutations to their accumulation with cell divisions, providing a framework for understanding how mutation rates depend on sex, age, and cell division rate. We show that the accrual of mutations should track cell divisions not only when mutations are replicative in origin but also when they are non-replicative and repaired efficiently. One implication is that observations from diverse fields that to date have been interpreted as pointing to a replicative origin of most mutations could instead reflect the accumulation of mutations arising from endogenous reactions or exogenous mutagens. We further find that only mutations that arise from inefficiently repaired lesions will accrue according to absolute time; thus, unless life history traits co-vary, the phylogenetic “molecular clock” should not be expected to run steadily across species. PMID:26761240

Replication-associated mutational asymmetry in the human genome.

PubMed

Chen, Chun-Long; Duquenne, Lauranne; Audit, Benjamin; Guilbaud, Guillaume; Rappailles, Aurélien; Baker, Antoine; Huvet, Maxime; d'Aubenton-Carafa, Yves; Hyrien, Olivier; Arneodo, Alain; Thermes, Claude

2011-08-01

During evolution, mutations occur at rates that can differ between the two DNA strands. In the human genome, nucleotide substitutions occur at different rates on the transcribed and non-transcribed strands that may result from transcription-coupled repair. These mutational asymmetries generate transcription-associated compositional skews. To date, the existence of such asymmetries associated with replication has not yet been established. Here, we compute the nucleotide substitution matrices around replication initiation zones identified as sharp peaks in replication timing profiles and associated with abrupt jumps in the compositional skew profile. We show that the substitution matrices computed in these regions fully explain the jumps in the compositional skew profile when crossing initiation zones. In intergenic regions, we observe mutational asymmetries measured as differences between complementary substitution rates; their sign changes when crossing initiation zones. These mutational asymmetries are unlikely to result from cryptic transcription but can be explained by a model based on replication errors and strand-biased repair. In transcribed regions, mutational asymmetries associated with replication superimpose on the previously described mutational asymmetries associated with transcription. We separate the substitution asymmetries associated with both mechanisms, which allows us to determine for the first time in eukaryotes, the mutational asymmetries associated with replication and to reevaluate those associated with transcription. Replication-associated mutational asymmetry may result from unequal rates of complementary base misincorporation by the DNA polymerases coupled with DNA mismatch repair (MMR) acting with different efficiencies on the leading and lagging strands. Replication, acting in germ line cells during long evolutionary times, contributed equally with transcription to produce the present abrupt jumps in the compositional skew. These results demonstrate that DNA replication is one of the major processes that shape human genome composition.

Lysine residues K66, K109, and K110 in the bovine foamy virus transactivator protein are required for transactivation and viral replication.

PubMed

Zhang, Suzhen; Cui, Xiaoxu; Li, Jing; Liang, Zhibin; Qiao, Wentao; Tan, Juan

2016-04-01

Bovine foamy virus (BFV) is a complex retrovirus that infects cattle. Like all retroviruses, BFV encodes a transactivator Tas protein (BTas) that increases gene transcription from viral promoters. BFV encodes two promoters that can interact with BTas, a conserved promoter in the 5' long terminal repeat (LTR) and a unique internal promoter (IP). Our previous study showed that BTas is acetylated by p300 at residues K66, K109, and K110, which markedly enhanced the ability of BTas to bind to DNA. However, whether these residues are important for BFV replication was not determined. Therefore, in this study we provide direct evidence that BTas is required for BFV replication and demonstrate that residues K66, K109, and K110 are critical for BTas function and BFV replication. Full-length infectious clones were generated, which were BTas deficient or contained lysine to arginine (K→R) mutations at position 66, 109, and/or 110. In vivo data indicated that K→R mutations at positions 66, 109, and 110 in BTas impaired transactivation of both the LTR and IP promoters. In addition, the K→R mutations in full-length infectious clones reduced expression of viral proteins, and the triple mutant and BTas deletion completely abrogated viral replication. Taken together, these results indicate that lysine residues at positions 66, 109, and 110 in the BTas protein are crucial for BFV replication and suggest a potential role for BTas acetylation in regulating the viral life cycle.

Identifying Cancer Driver Genes Using Replication-Incompetent Retroviral Vectors

PubMed Central

Bii, Victor M.; Trobridge, Grant D.

2016-01-01

Identifying novel genes that drive tumor metastasis and drug resistance has significant potential to improve patient outcomes. High-throughput sequencing approaches have identified cancer genes, but distinguishing driver genes from passengers remains challenging. Insertional mutagenesis screens using replication-incompetent retroviral vectors have emerged as a powerful tool to identify cancer genes. Unlike replicating retroviruses and transposons, replication-incompetent retroviral vectors lack additional mutagenesis events that can complicate the identification of driver mutations from passenger mutations. They can also be used for almost any human cancer due to the broad tropism of the vectors. Replication-incompetent retroviral vectors have the ability to dysregulate nearby cancer genes via several mechanisms including enhancer-mediated activation of gene promoters. The integrated provirus acts as a unique molecular tag for nearby candidate driver genes which can be rapidly identified using well established methods that utilize next generation sequencing and bioinformatics programs. Recently, retroviral vector screens have been used to efficiently identify candidate driver genes in prostate, breast, liver and pancreatic cancers. Validated driver genes can be potential therapeutic targets and biomarkers. In this review, we describe the emergence of retroviral insertional mutagenesis screens using replication-incompetent retroviral vectors as a novel tool to identify cancer driver genes in different cancer types. PMID:27792127

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism

PubMed Central

Reynolds, John J; Bicknell, Louise S; Carroll, Paula; Higgs, Martin R; Shaheen, Ranad; Murray, Jennie E; Papadopoulos, Dimitrios K; Leitch, Andrea; Murina, Olga; Tarnauskaitė, Žygimantė; Wessel, Sarah R; Zlatanou, Anastasia; Vernet, Audrey; von Kriegsheim, Alex; Mottram, Rachel MA; Logan, Clare V; Bye, Hannah; Li, Yun; Brean, Alexander; Maddirevula, Sateesh; Challis, Rachel C; Skouloudaki, Kassiani; Almoisheer, Agaadir; Alsaif, Hessa S; Amar, Ariella; Prescott, Natalie J; Bober, Michael B; Duker, Angela; Faqeih, Eissa; Seidahmed, Mohammed Zain; Al Tala, Saeed; Alswaid, Abdulrahman; Ahmed, Saleem; Al-Aama, Jumana Yousuf; Altmüller, Janine; Al Balwi, Mohammed; Brady, Angela F; Chessa, Luciana; Cox, Helen; Fischetto, Rita; Heller, Raoul; Henderson, Bertram D; Hobson, Emma; Nürnberg, Peter; Percin, E Ferda; Peron, Angela; Spaccini, Luigina; Quigley, Alan J; Thakur, Seema; Wise, Carol A; Yoon, Grace; Alnemer, Maha; Tomancak, Pavel; Yigit, Gökhan; Taylor, A Malcolm R; Reijns, Martin AM; Simpson, Michael A; Cortez, David; Alkuraya, Fowzan S; Mathew, Christopher G; Jackson, Andrew P; Stewart, Grant S

2017-01-01

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication, and protect, repair and restart damaged forks. Here we identify DONSON as a novel fork protection factor, and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilises forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATR-dependent signalling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity, and potentiating chromosomal instability. Hypomorphic mutations substantially reduce DONSON protein levels and impair fork stability in patient cells, consistent with defective DNA replication underlying the disease phenotype. In summary, we identify mutations in DONSON as a common cause of microcephalic dwarfism, and establish DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability. PMID:28191891

The control of paramyxovirus genome hexamer length and mRNA editing.

PubMed

Matsumoto, Yusuke; Ohta, Keisuke; Kolakofsky, Daniel; Nishio, Machiko

2018-04-01

The unusual ability of a human parainfluenza virus type 2 (hPIV2) nucleoprotein point mutation (NP Q202A ) to strongly enhance minigenome replication was found to depend on the absence of a functional, internal element of the bipartite replication promoter (CRII). This point mutation allows relatively robust CRII-minus minigenome replication in a CRII-independent manner, under conditions in which NP wt is essentially inactive. The nature of the amino acid at position 202 apparently controls whether viral RNA-dependent RNA polymerase (vRdRp) can, or cannot, initiate RNA synthesis in a CRII-independent manner. By repressing genome synthesis when vRdRp cannot correctly interact with CRII, gln 202 of N, the only residue of the RNA-binding groove that contacts a nucleotide base in the N-RNA, acts as a gatekeeper for wild-type (CRII-dependent) RNA synthesis. This ensures that only hexamer-length genomes are replicated, and that the critical hexamer phase of the cis -acting mRNA editing sequence is maintained. © 2018 Matsumoto et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Autism-related neuroligin-3 mutation alters social behavior and spatial learning.

PubMed

Jaramillo, Thomas C; Liu, Shunan; Pettersen, Ami; Birnbaum, Shari G; Powell, Craig M

2014-04-01

Multiple candidate genes have been identified for autism spectrum disorders. While some of these genes reach genome-wide significance, others, such as the R451C point mutation in the synaptic cell adhesion molecule neuroligin-3, appear to be rare. Interestingly, two brothers with the same R451C point mutation in neuroligin-3 present clinically on seemingly disparate sides of the autism spectrum. These clinical findings suggest genetic background may play a role in modifying the penetrance of a particular autism-associated mutation. Animal models may contribute additional support for such mutations as functionally relevant and can provide mechanistic insights. Previously, in collaboration with the Südhof laboratory, we reported that mice with an R451C substitution in neuroligin-3 displayed social deficits and enhanced spatial learning. While some of these behavioral abnormalities have since been replicated independently in the Südhof laboratory, observations from the Crawley laboratory failed to replicate these findings in a similar neuroligin-3 mutant mouse model and suggested that genetic background may contribute to variation in observations across laboratories. Therefore, we sought to replicate our findings in the neuroligin-3 R451C point mutant knock-in mouse model (NL3R451C) in a different genetic background. We backcrossed our NL3R451C mouse line onto a 129S2/SvPasCrl genetic background and repeated a subset of our previous behavioral testing. NL3R451C mice on a 129S2/SvPasCrl displayed social deficits, enhanced spatial learning, and increased locomotor activity. These data extend our previous findings that NL3R451C mice exhibit autism-relevant behavioral abnormalities and further suggest that different genetic backgrounds can modify this behavioral phenotype through epistatic genetic interactions. © 2014 International Society for Autism Research, Wiley Periodicals, Inc.

Identification of novel subgroup A variants with enhanced receptor binding and replicative capacity in primary isolates of anaemogenic strains of feline leukaemia virus.

PubMed

Stewart, Hazel; Adema, Karen W; McMonagle, Elizabeth L; Hosie, Margaret J; Willett, Brian J

2012-05-31

The development of anaemia in feline leukaemia virus (FeLV)-infected cats is associated with the emergence of a novel viral subgroup, FeLV-C. FeLV-C arises from the subgroup that is transmitted, FeLV-A, through alterations in the amino acid sequence of the receptor binding domain (RBD) of the envelope glycoprotein that result in a shift in the receptor usage and the cell tropism of the virus. The factors that influence the transition from subgroup A to subgroup C remain unclear, one possibility is that a selective pressure in the host drives the acquisition of mutations in the RBD, creating A/C intermediates with enhanced abilities to interact with the FeLV-C receptor, FLVCR. In order to understand further the emergence of FeLV-C in the infected cat, we examined primary isolates of FeLV-C for evidence of FeLV-A variants that bore mutations consistent with a gradual evolution from FeLV-A to FeLV-C. Within each isolate of FeLV-C, we identified variants that were ostensibly subgroup A by nucleic acid sequence comparisons, but which bore mutations in the RBD. One such mutation, N91D, was present in multiple isolates and when engineered into a molecular clone of the prototypic FeLV-A (Glasgow-1), enhanced replication was noted in feline cells. Expression of the N91D Env on murine leukaemia virus (MLV) pseudotypes enhanced viral entry mediated by the FeLV-A receptor THTR1 while soluble FeLV-A Env bearing the N91D mutation bound more efficiently to mouse or guinea pig cells bearing the FeLV-A and -C receptors. Long-term in vitro culture of variants bearing the N91D substitution in the presence of anti-FeLV gp70 antibodies did not result in the emergence of FeLV-C variants, suggesting that additional selective pressures in the infected cat may drive the subsequent evolution from subgroup A to subgroup C. Our data support a model in which variants of FeLV-A, bearing subtle differences in the RBD of Env, may be predisposed towards enhanced replication in vivo and subsequent conversion to FeLV-C. The selection pressures in vivo that drive the emergence of FeLV-C in a proportion of infected cats remain to be established.

HBV subgenotypes F1b and F4 replication induces an incomplete autophagic process in hepatocytes: Role of BCP and preCore mutations.

PubMed

Elizalde, María Mercedes; Pérez, Paula Soledad; Sevic, Ina; Grasso, Daniel; Ropolo, Alejandro; Barbini, Luciana; Campos, Rodolfo Héctor; Vaccaro, María Inés; Flichman, Diego Martín

2018-01-01

Hepatitis B virus (HBV) genotypes and mutants have been associated with differences in clinical and virological characteristics. Autophagy is a cellular process that degrades long-lived proteins and damaged organelles. Viruses have evolved mechanisms to alter this process to survive in host cells. In this work, we studied the modulation of autophagy by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. HBV subgenotypes F1b and F4 replication induced accumulation of autophagosomes in hepatoma cells. However, no autophagic protein degradation was observed, indicating a blockage of autophagic flux at later stages. This inhibition of autophagy flux might be due to an impairment of lysosomal acidification in hepatoma cells. Moreover, HBV-mediated autophagy modulation was independent of the viral subgenotypes and enhanced in viruses with BCP and preCore naturally occurring mutations. These results contribute to understand the mechanisms by which different HBV variants contribute to the pathogenesis of HBV infections. In addition, this study is the first to describe the role that two highly prevalent naturally occurring mutations exert on the modulation of HBV-induced autophagy.

HBV subgenotypes F1b and F4 replication induces an incomplete autophagic process in hepatocytes: Role of BCP and preCore mutations

PubMed Central

Pérez, Paula Soledad; Sevic, Ina; Ropolo, Alejandro; Barbini, Luciana; Campos, Rodolfo Héctor; Vaccaro, María Inés; Flichman, Diego Martín

2018-01-01

Hepatitis B virus (HBV) genotypes and mutants have been associated with differences in clinical and virological characteristics. Autophagy is a cellular process that degrades long-lived proteins and damaged organelles. Viruses have evolved mechanisms to alter this process to survive in host cells. In this work, we studied the modulation of autophagy by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. HBV subgenotypes F1b and F4 replication induced accumulation of autophagosomes in hepatoma cells. However, no autophagic protein degradation was observed, indicating a blockage of autophagic flux at later stages. This inhibition of autophagy flux might be due to an impairment of lysosomal acidification in hepatoma cells. Moreover, HBV-mediated autophagy modulation was independent of the viral subgenotypes and enhanced in viruses with BCP and preCore naturally occurring mutations. These results contribute to understand the mechanisms by which different HBV variants contribute to the pathogenesis of HBV infections. In addition, this study is the first to describe the role that two highly prevalent naturally occurring mutations exert on the modulation of HBV-induced autophagy. PMID:29738548

Holes influence the mutation spectrum of human mitochondrial DNA

NASA Astrophysics Data System (ADS)

Villagran, Martha; Miller, John

Mutations drive evolution and disease, showing highly non-random patterns of variant frequency vs. nucleotide position. We use computational DNA hole spectroscopy [M.Y. Suarez-Villagran & J.H. Miller, Sci. Rep. 5, 13571 (2015)] to reveal sites of enhanced hole probability in selected regions of human mitochondrial DNA. A hole is a mobile site of positive charge created when an electron is removed, for example by radiation or contact with a mutagenic agent. The hole spectra are quantum mechanically computed using a two-stranded tight binding model of DNA. We observe significant correlation between spectra of hole probabilities and of genetic variation frequencies from the MITOMAP database. These results suggest that hole-enhanced mutation mechanisms exert a substantial, perhaps dominant, influence on mutation patterns in DNA. One example is where a trapped hole induces a hydrogen bond shift, known as tautomerization, which then triggers a base-pair mismatch during replication. Our results deepen overall understanding of sequence specific mutation rates, encompassing both hotspots and cold spots, which drive molecular evolution.

An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

PubMed

Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

2016-10-01

Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

Mutations in DONSON disrupt replication fork stability and cause microcephalic dwarfism.

PubMed

Reynolds, John J; Bicknell, Louise S; Carroll, Paula; Higgs, Martin R; Shaheen, Ranad; Murray, Jennie E; Papadopoulos, Dimitrios K; Leitch, Andrea; Murina, Olga; Tarnauskaitė, Žygimantė; Wessel, Sarah R; Zlatanou, Anastasia; Vernet, Audrey; von Kriegsheim, Alex; Mottram, Rachel M A; Logan, Clare V; Bye, Hannah; Li, Yun; Brean, Alexander; Maddirevula, Sateesh; Challis, Rachel C; Skouloudaki, Kassiani; Almoisheer, Agaadir; Alsaif, Hessa S; Amar, Ariella; Prescott, Natalie J; Bober, Michael B; Duker, Angela; Faqeih, Eissa; Seidahmed, Mohammed Zain; Al Tala, Saeed; Alswaid, Abdulrahman; Ahmed, Saleem; Al-Aama, Jumana Yousuf; Altmüller, Janine; Al Balwi, Mohammed; Brady, Angela F; Chessa, Luciana; Cox, Helen; Fischetto, Rita; Heller, Raoul; Henderson, Bertram D; Hobson, Emma; Nürnberg, Peter; Percin, E Ferda; Peron, Angela; Spaccini, Luigina; Quigley, Alan J; Thakur, Seema; Wise, Carol A; Yoon, Grace; Alnemer, Maha; Tomancak, Pavel; Yigit, Gökhan; Taylor, A Malcolm R; Reijns, Martin A M; Simpson, Michael A; Cortez, David; Alkuraya, Fowzan S; Mathew, Christopher G; Jackson, Andrew P; Stewart, Grant S

2017-04-01

To ensure efficient genome duplication, cells have evolved numerous factors that promote unperturbed DNA replication and protect, repair and restart damaged forks. Here we identify downstream neighbor of SON (DONSON) as a novel fork protection factor and report biallelic DONSON mutations in 29 individuals with microcephalic dwarfism. We demonstrate that DONSON is a replisome component that stabilizes forks during genome replication. Loss of DONSON leads to severe replication-associated DNA damage arising from nucleolytic cleavage of stalled replication forks. Furthermore, ATM- and Rad3-related (ATR)-dependent signaling in response to replication stress is impaired in DONSON-deficient cells, resulting in decreased checkpoint activity and the potentiation of chromosomal instability. Hypomorphic mutations in DONSON substantially reduce DONSON protein levels and impair fork stability in cells from patients, consistent with defective DNA replication underlying the disease phenotype. In summary, we have identified mutations in DONSON as a common cause of microcephalic dwarfism and established DONSON as a critical replication fork protein required for mammalian DNA replication and genome stability.

Genetic background effects in Neuroligin-3 mutant mice: Minimal behavioral abnormalities on C57 background.

PubMed

Jaramillo, Thomas C; Escamilla, Christine Ochoa; Liu, Shunan; Peca, Lauren; Birnbaum, Shari G; Powell, Craig M

2018-02-01

Neuroligin-3 (NLGN3) is a postsynaptic cell adhesion protein that interacts with presynaptic ligands including neurexin-1 (NRXN1) [Ichtchenko et al., Journal of Biological Chemistry, 271, 2676-2682, 1996]. Mice harboring a mutation in the NLGN3 gene (NL3R451C) mimicking a mutation found in two brothers with autism spectrum disorder (ASD) were previously generated and behaviorally phenotyped for autism-related behaviors. In these NL3R451C mice generated and tested on a hybrid C57BL6J/129S2/SvPasCrl background, we observed enhanced spatial memory and reduced social interaction [Tabuchi et al., Science, 318, 71-76, 2007]. Curiously, an independently generated second line of mice harboring the same mutation on a C57BL6J background exhibited minimal aberrant behavior, thereby providing apparently discrepant results. To investigate the origin of the discrepancy, we previously replicated the original findings of Tabuchi et al. by studying the same NL3R451C mutation on a pure 129S2/SvPasCrl genetic background. Here we complete the behavioral characterization of the NL3R451C mutation on a pure C57BL6J genetic background to determine if background genetics play a role in the discrepant behavioral outcomes involving NL3R451C mice. NL3R451C mutant mice on a pure C57BL6J background did not display spatial memory enhancements or social interaction deficits. We only observed a decreased startle response and mildly increased locomotor activity in these mice suggesting that background genetics influences behavioral outcomes involving the NL3R451C mutation. Autism Res 2018, 11: 234-244. © 2017 International Society for Autism Research, Wiley Periodicals, Inc. Behavioral symptoms of autism can be highly variable, even in cases that involve identical genetic mutations. Previous studies in mice with a mutation of the Neuroligin-3 gene showed enhanced learning and social deficits. We replicated these findings on the same and different genetic backgrounds. In this study, however, the same mutation in mice on a different genetic background did not reproduce our previous findings. Our results suggest that genetic background influences behavioral symptoms of this autism-associated mutation. © 2017 International Society for Autism Research, Wiley Periodicals, Inc.

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity.

PubMed

Xu, Guanlong; Zhang, Xuxiao; Gao, Weihua; Wang, Chenxi; Wang, Jinliang; Sun, Honglei; Sun, Yipeng; Guo, Lu; Zhang, Rui; Chang, Kin-Chow; Liu, Jinhua; Pu, Juan

2016-09-15

Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity

PubMed Central

Xu, Guanlong; Zhang, Xuxiao; Gao, Weihua; Wang, Chenxi; Wang, Jinliang; Sun, Honglei; Sun, Yipeng; Guo, Lu; Zhang, Rui; Chang, Kin-Chow; Liu, Jinhua

2016-01-01

ABSTRACT Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo. In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. IMPORTANCE Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo. Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity. PMID:27384648

A link among DNA replication, recombination, and gene expression revealed by genetic and genomic analysis of TEBICHI gene of Arabidopsis thaliana.

PubMed

Inagaki, Soichi; Nakamura, Kenzo; Morikami, Atsushi

2009-08-01

Spatio-temporal regulation of gene expression during development depends on many factors. Mutations in Arabidopsis thaliana TEBICHI (TEB) gene encoding putative helicase and DNA polymerase domains-containing protein result in defects in meristem maintenance and correct organ formation, as well as constitutive DNA damage response and a defect in cell cycle progression; but the molecular link between these phenotypes of teb mutants is unknown. Here, we show that mutations in the DNA replication checkpoint pathway gene, ATR, but not in ATM gene, enhance developmental phenotypes of teb mutants, although atr suppresses cell cycle defect of teb mutants. Developmental phenotypes of teb mutants are also enhanced by mutations in RAD51D and XRCC2 gene, which are involved in homologous recombination. teb and teb atr double mutants exhibit defects in adaxial-abaxial polarity of leaves, which is caused in part by the upregulation of ETTIN (ETT)/AUXIN RESPONSIVE FACTOR 3 (ARF3) and ARF4 genes. The Helitron transposon in the upstream of ETT/ARF3 gene is likely to be involved in the upregulation of ETT/ARF3 in teb. Microarray analysis indicated that teb and teb atr causes preferential upregulation of genes nearby the Helitron transposons. Furthermore, interestingly, duplicated genes, especially tandemly arrayed homologous genes, are highly upregulated in teb or teb atr. We conclude that TEB is required for normal progression of DNA replication and for correct expression of genes during development. Interplay between these two functions and possible mechanism leading to altered expression of specific genes will be discussed.

RNA Recombination Enhances Adaptability and Is Required for Virus Spread and Virulence.

PubMed

Xiao, Yinghong; Rouzine, Igor M; Bianco, Simone; Acevedo, Ashley; Goldstein, Elizabeth Faul; Farkov, Mikhail; Brodsky, Leonid; Andino, Raul

2016-04-13

Mutation and recombination are central processes driving microbial evolution. A high mutation rate fuels adaptation but also generates deleterious mutations. Recombination between two different genomes may resolve this paradox, alleviating effects of clonal interference and purging deleterious mutations. Here we demonstrate that recombination significantly accelerates adaptation and evolution during acute virus infection. We identified a poliovirus recombination determinant within the virus polymerase, mutation of which reduces recombination rates without altering replication fidelity. By generating a panel of variants with distinct mutation rates and recombination ability, we demonstrate that recombination is essential to enrich the population in beneficial mutations and purge it from deleterious mutations. The concerted activities of mutation and recombination are key to virus spread and virulence in infected animals. These findings inform a mathematical model to demonstrate that poliovirus adapts most rapidly at an optimal mutation rate determined by the trade-off between selection and accumulation of detrimental mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of HIV-1 determinants for replication in vivo.

PubMed

Su, L; Kaneshima, H; Bonyhadi, M L; Lee, R; Auten, J; Wolf, A; Du, B; Rabin, L; Hahn, B H; Terwilliger, E; Mccune, J M

1997-01-06

Pathogenic organisms are frequently attenuated after long-term culture in vitro. The mechanisms of the attenuation process are not clear, but probably involve mutations of functions required for replication and pathogenicity in vivo. To identify these functions, a direct comparison must be made between attenuated genomes and those that remain pathogenic in vivo. In this study, we used the heterochimeric SCID-hu Thy/Liv mouse as an in vivo model to define human immunodeficiency virus type 1 (HIV-1) determinants which are uniquely required for replication in vivo. The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T cell lines but failed to replicate in the SCID-hu Thy/Liv model. When a lab worker was accidentally infected by Lai/IIIB, however, HIV-1 was isolated only from infection of primary PBMC, and not from infection of T cell lines. We hypothesized that the lab worker was exposed to a heterogeneous viral stock which had been attenuated by passage in immortalized T cell lines. Either a rare family member from this stock was selected for in vivo replication or, alternatively, an attenuated genotype dominant in vitro may have reverted to become more infectious in vivo. To address this hypothesis, we have used the SCID-hu Thy/Liv model to study the replication of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolated from the infected lab worker. HXB2 showed no or very low levels of replication in the Thy/Liv organ. Replacement of its subgenomic fragment encoding the envelope gene with a corresponding fragment from the lab worker isolate generated a recombinant virus (HXB2/LW) which replicated actively in SCID-hu mice. The NEF mutation in the HXB2 genome is still present in HXB2/LW. Thus, the LW sequences encode HIV-1 determinants which enhance HIV replication in vivo in a NEF-independent mechanism. The specific determinants have been mapped to the V1-V3 regions of the HIV-1 genome. Six unique mutations in the V3 loop region of HXB2/LW have been identified which contribute to the increased replication in vivo.

Distinct contributions of replication and transcription to mutation rate variation of human genomes.

PubMed

Cui, Peng; Ding, Feng; Lin, Qiang; Zhang, Lingfang; Li, Ang; Zhang, Zhang; Hu, Songnian; Yu, Jun

2012-02-01

Here, we evaluate the contribution of two major biological processes--DNA replication and transcription--to mutation rate variation in human genomes. Based on analysis of the public human tissue transcriptomics data, high-resolution replicating map of Hela cells and dbSNP data, we present significant correlations between expression breadth, replication time in local regions and SNP density. SNP density of tissue-specific (TS) genes is significantly higher than that of housekeeping (HK) genes. TS genes tend to locate in late-replicating genomic regions and genes in such regions have a higher SNP density compared to those in early-replication regions. In addition, SNP density is found to be positively correlated with expression level among HK genes. We conclude that the process of DNA replication generates stronger mutational pressure than transcription-associated biological processes do, resulting in an increase of mutation rate in TS genes while having weaker effects on HK genes. In contrast, transcription-associated processes are mainly responsible for the accumulation of mutations in highly-expressed HK genes. Copyright © 2012 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

DNA-directed mutations. Leading and lagging strand specificity

NASA Technical Reports Server (NTRS)

Sinden, R. R.; Hashem, V. I.; Rosche, W. A.

1999-01-01

The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

Running on empty: does mitochondrial DNA mutation limit replicative lifespan in yeast?: Mutations that increase the division rate of cells lacking mitochondrial DNA also extend replicative lifespan in Saccharomyces cerevisiae.

PubMed

Dunn, Cory D

2011-10-01

Mitochondrial DNA (mtDNA) mutations escalate with increasing age in higher organisms. However, it has so far been difficult to experimentally determine whether mtDNA mutation merely correlates with age or directly limits lifespan. A recent study shows that budding yeast can also lose functional mtDNA late in life. Interestingly, independent studies of replicative lifespan (RLS) and of mtDNA-deficient cells show that the same mutations can increase both RLS and the division rate of yeast lacking the mitochondrial genome. These exciting, parallel findings imply a potential causal relationship between mtDNA mutation and replicative senescence. Furthermore, these results suggest more efficient methods for discovering genes that determine lifespan. Copyright © 2011 WILEY Periodicals, Inc.

Coordinate Deletion of N-Glycans from the Heptad Repeats of the Fusion F Protein of Newcastle Disease Virus Yields a Hyperfusogenic Virus with Increased Replication, Virulence, and Immunogenicity

PubMed Central

Samal, Sweety; Khattar, Sunil K.; Kumar, Sachin; Collins, Peter L.

2012-01-01

The role of N-linked glycosylation of the Newcastle disease virus (NDV) fusion (F) protein in viral replication and pathogenesis was examined by eliminating potential acceptor sites using a reverse genetics system for the moderately pathogenic strain Beaudette C (BC). The NDV-BC F protein contains six potential acceptor sites for N-linked glycosylation at residues 85, 191, 366, 447, 471, and 541 (sites Ng1 to Ng6, respectively). The sites at Ng2 and Ng5 are present in heptad repeat (HR) domains HR1 and HR2, respectively, and thus might affect fusion. Each N-glycosylation site was eliminated individually by replacing asparagine (N) with glutamine (Q), and a double mutant (Ng2 + 5) involving the two HR domains was also made. Each mutant was successfully recovered by reverse genetics except for the one involving Ng6, which is present in the cytoplasmic domain. All of the F proteins expressed by the recovered mutant viruses were efficiently cleaved and transported to the infected-cell surface. None of the individual mutations affected viral fusogenicity, but the double mutation at Ng2 and Ng5 in HR1 and HR2 increased fusogenicity >12-fold. The single mutations at sites Ng1, Ng2, and Ng5 resulted in modestly reduced multicycle growth in vitro. These three single mutations were also the most attenuating in eggs and 1-day-old chicks and were associated with decreased replication and spread in 2-week-old chickens. In contrast, the combination of the mutations at Ng2 and Ng5 yielded a virus that, compared to the BC parent, replicated >100-fold more efficiently in vitro, was more virulent in eggs and chicks, replicated more efficiently in chickens with enhanced tropism for the brain and gut, and elicited stronger humoral cell responses. These results illustrate the effects of N-glycosylation of the F protein on NDV pathobiology and suggest that the N-glycans in HR1 and HR2 coordinately downregulate viral fusion and virulence. PMID:22205748

Coordinate deletion of N-glycans from the heptad repeats of the fusion F protein of Newcastle disease virus yields a hyperfusogenic virus with increased replication, virulence, and immunogenicity.

PubMed

Samal, Sweety; Khattar, Sunil K; Kumar, Sachin; Collins, Peter L; Samal, Siba K

2012-03-01

The role of N-linked glycosylation of the Newcastle disease virus (NDV) fusion (F) protein in viral replication and pathogenesis was examined by eliminating potential acceptor sites using a reverse genetics system for the moderately pathogenic strain Beaudette C (BC). The NDV-BC F protein contains six potential acceptor sites for N-linked glycosylation at residues 85, 191, 366, 447, 471, and 541 (sites Ng1 to Ng6, respectively). The sites at Ng2 and Ng5 are present in heptad repeat (HR) domains HR1 and HR2, respectively, and thus might affect fusion. Each N-glycosylation site was eliminated individually by replacing asparagine (N) with glutamine (Q), and a double mutant (Ng2 + 5) involving the two HR domains was also made. Each mutant was successfully recovered by reverse genetics except for the one involving Ng6, which is present in the cytoplasmic domain. All of the F proteins expressed by the recovered mutant viruses were efficiently cleaved and transported to the infected-cell surface. None of the individual mutations affected viral fusogenicity, but the double mutation at Ng2 and Ng5 in HR1 and HR2 increased fusogenicity >12-fold. The single mutations at sites Ng1, Ng2, and Ng5 resulted in modestly reduced multicycle growth in vitro. These three single mutations were also the most attenuating in eggs and 1-day-old chicks and were associated with decreased replication and spread in 2-week-old chickens. In contrast, the combination of the mutations at Ng2 and Ng5 yielded a virus that, compared to the BC parent, replicated >100-fold more efficiently in vitro, was more virulent in eggs and chicks, replicated more efficiently in chickens with enhanced tropism for the brain and gut, and elicited stronger humoral cell responses. These results illustrate the effects of N-glycosylation of the F protein on NDV pathobiology and suggest that the N-glycans in HR1 and HR2 coordinately downregulate viral fusion and virulence.

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.

PubMed

Feitsma, Harma; de Bruijn, Ewart; van de Belt, Jose; Nijman, Isaac J; Cuppen, Edwin

2008-07-01

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

Identification of the ENT1 antagonists dipyridamole and dilazep as amplifiers of oncolytic herpes simplex virus-1 replication.

PubMed

Passer, Brent J; Cheema, Tooba; Zhou, Bingsen; Wakimoto, Hiroaki; Zaupa, Cecile; Razmjoo, Mani; Sarte, Jason; Wu, Shulin; Wu, Chin-lee; Noah, James W; Li, Qianjun; Buolamwini, John K; Yen, Yun; Rabkin, Samuel D; Martuza, Robert L

2010-05-15

Oncolytic herpes simplex virus-1 (oHSV) vectors selectively replicate in tumor cells, where they kill through oncolysis while sparing normal cells. One of the drawbacks of oHSV vectors is their limited replication and spread to neighboring cancer cells. Here, we report the outcome of a high-throughput chemical library screen to identify small-molecule compounds that augment the replication of oHSV G47Delta. Of the 2,640-screened bioactives, 6 compounds were identified and subsequently validated for enhanced G47Delta replication. Two of these compounds, dipyridamole and dilazep, interfered with nucleotide metabolism by potently and directly inhibiting the equilibrative nucleoside transporter-1 (ENT1). Replicative amplification promoted by dipyridamole and dilazep were dependent on HSV mutations in ICP6, the large subunit of ribonucleotide reductase. Our results indicate that ENT1 antagonists augment oHSV replication in tumor cells by increasing cellular ribonucleoside activity. (c)2010 AACR.

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication

PubMed Central

Shinbrot, Eve; Henninger, Erin E.; Weinhold, Nils; Covington, Kyle R.; Göksenin, A. Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M.; Gibbs, Richard A.; Sander, Chris; Pursell, Zachary F.

2014-01-01

Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication. PMID:25228659

Asynchronous accumulation of lettuce infectious yellows virus RNAs 1 and 2 and identification of an RNA 1 trans enhancer of RNA 2 accumulation.

PubMed

Yeh, H H; Tian, T; Rubio, L; Crawford, B; Falk, B W

2000-07-01

Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectious yellow virus (LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation.

Asynchronous Accumulation of Lettuce Infectious Yellows Virus RNAs 1 and 2 and Identification of an RNA 1 trans Enhancer of RNA 2 Accumulation

PubMed Central

Yeh, Hsin-Hung; Tian, Tongyan; Rubio, Luis; Crawford, Brett; Falk, Bryce W.

2000-01-01

Time course and mutational analyses were used to examine the accumulation in protoplasts of progeny RNAs of the bipartite Crinivirus, Lettuce infectious yellow virus (LIYV; family Closteroviridae). Hybridization analyses showed that simultaneous inoculation of LIYV RNAs 1 and 2 resulted in asynchronous accumulation of progeny LIYV RNAs. LIYV RNA 1 progeny genomic and subgenomic RNAs could be detected in protoplasts as early as 12 h postinoculation (p.i.) and accumulated to high levels by 24 h p.i. The LIYV RNA 1 open reading frame 2 (ORF 2) subgenomic RNA was the most abundant of all LIYV RNAs detected. In contrast, RNA 2 progeny were not readily detected until ca. 36 h p.i. Mutational analyses showed that in-frame stop codons introduced into five of seven RNA 2 ORFs did not affect accumulation of progeny LIYV RNA 1 or RNA 2, confirming that RNA 2 does not encode proteins necessary for LIYV RNA replication. Mutational analyses also supported that LIYV RNA 1 encodes proteins necessary for replication of LIYV RNAs 1 and 2. A mutation introduced into the LIYV RNA 1 region encoding the overlapping ORF 1B and ORF 2 was lethal. However, mutations introduced into only LIYV RNA 1 ORF 2 resulted in accumulation of progeny RNA 1 near or equal to wild-type RNA 1. In contrast, the RNA 1 ORF 2 mutants did not efficiently support the trans accumulation of LIYV RNA 2. Three distinct RNA 1 ORF 2 mutants were analyzed and all exhibited a similar phenotype for progeny LIYV RNA accumulation. These data suggest that the LIYV RNA 1 ORF 2 encodes a trans enhancer for RNA 2 accumulation. PMID:10846054

Mutagenic Effects of Ribavirin on Hepatitis E Virus-Viral Extinction versus Selection of Fitness-Enhancing Mutations.

PubMed

Todt, Daniel; Walter, Stephanie; Brown, Richard J P; Steinmann, Eike

2016-10-13

Hepatitis E virus (HEV), an important agent of viral hepatitis worldwide, can cause severe courses of infection in pregnant women and immunosuppressed patients. To date, HEV infections can only be treated with ribavirin (RBV). Major drawbacks of this therapy are that RBV is not approved for administration to pregnant women and that the virus can acquire mutations, which render the intra-host population less sensitive or even resistant to RBV. One of the proposed modes of action of RBV is a direct mutagenic effect on viral genomes, inducing mismatches and subsequent nucleotide substitutions. These transition events can drive the already error-prone viral replication beyond an error threshold, causing viral population extinction. In contrast, the expanded heterogeneous viral population can facilitate selection of mutant viruses with enhanced replication fitness. Emergence of these mutant viruses can lead to therapeutic failure. Consequently, the onset of RBV treatment in chronically HEV-infected individuals can result in two divergent outcomes: viral extinction versus selection of fitness-enhanced viruses. Following an overview of RNA viruses treated with RBV in clinics and a summary of the different antiviral modes of action of this drug, we focus on the mutagenic effect of RBV on HEV intrahost populations, and how HEV is able to overcome lethal mutagenesis.

Alkylating agent (MNU)-induced mutation in space environment

NASA Astrophysics Data System (ADS)

Ohnishi, T.; Takahashi, A.; Ohnishi, K.; Takahashi, S.; Masukawa, M.; Sekikawa, K.; Amano, T.; Nakano, T.; Nagaoka, S.

2001-01-01

In recent years, some contradictory data about the effects of microgravity on radiation-induced biological responses in space experiments have been reported. We prepared a damaged template DNA produced with an alkylating agent (N-methyl-N-nitroso urea; MNU) to measure incorrect base-incorporation during DNA replication in microgravity. We examined whether mutation frequency is affected by microgravity during DNA replication for a DNA template damaged by an alkylating agent. Using an in vitro enzymatic reaction system, DNA synthesis by Taq polymerase or polymerase III was done during a US space shuttle mission (Discovery, STS-91). After the flight, DNA replication and mutation frequencies were measured. We found that there was almost no effect of microgravity on DNA replication and mutation frequency. It is suggested that microgravity might not affect at the stage of substrate incorporation in induced-mutation frequency.

A Y527A mutation in the fusion protein of Newcastle disease virus strain LaSota leads to a hyperfusogenic virus with increased replication and immunogenicity.

PubMed

Manoharan, Vinoth K; Khattar, Sunil K; Paldurai, Anandan; Kim, Shin-Hee; Samal, Siba K

2016-02-01

Newcastle disease is a highly contagious and economically important disease of poultry. Low-virulence Newcastle disease virus (NDV) strains such as B1 and LaSota have been used as live vaccines, with a proven track record of safety and efficacy. However, these vaccines do not completely prevent infection or virus shedding. Therefore, there is a need to enhance the immunogenicity of these vaccine strains. In this study, the effect of mutations in the conserved tyrosine residues of the F protein of vaccine strain LaSota was investigated. Our results showed that substitution of tyrosine at position 527 by alanine resulted in a hyperfusogenic virus with increased replication and immunogenicity. Challenge study with highly virulent NDV strain Texas GB showed that immunization of chickens with Y527A mutant virus provided 100% protection and no shedding of the challenge virus. This study suggests that the strain LaSota harbouring the Y527A mutation may represent a more efficacious vaccine.

Evolution of 2009 H1N1 influenza viruses during the pandemic correlates with increased viral pathogenicity and transmissibility in the ferret model.

PubMed

Otte, Anna; Marriott, Anthony C; Dreier, Carola; Dove, Brian; Mooren, Kyra; Klingen, Thorsten R; Sauter, Martina; Thompson, Katy-Anne; Bennett, Allan; Klingel, Karin; van Riel, Debby; McHardy, Alice C; Carroll, Miles W; Gabriel, Gülsah

2016-06-24

There is increasing evidence that 2009 pandemic H1N1 influenza viruses have evolved after pandemic onset giving rise to severe epidemics in subsequent waves. However, it still remains unclear which viral determinants might have contributed to disease severity after pandemic initiation. Here, we show that distinct mutations in the 2009 pandemic H1N1 virus genome have occurred with increased frequency after pandemic declaration. Among those, a mutation in the viral hemagglutinin was identified that increases 2009 pandemic H1N1 virus binding to human-like α2,6-linked sialic acids. Moreover, these mutations conferred increased viral replication in the respiratory tract and elevated respiratory droplet transmission between ferrets. Thus, our data show that 2009 H1N1 influenza viruses have evolved after pandemic onset giving rise to novel virus variants that enhance viral replicative fitness and respiratory droplet transmission in a mammalian animal model. These findings might help to improve surveillance efforts to assess the pandemic risk by emerging influenza viruses.

Retroviral mutation rates and A-to-G hypermutations during different stages of retroviral replication.

PubMed Central

Kim, T; Mudry, R A; Rexrode, C A; Pathak, V K

1996-01-01

Retroviruses mutate at a high rate in vivo during viral replication. Mutations may occur during proviral transcription by RNA polymerase II, during minus-strand DNA synthesis (RNA template) by viral reverse transcriptase, or during plus-strand DNA synthesis (DNA template) by reverse transcriptase. To determine the contributions of different stages of replication to the retroviral mutation rates, we developed a spleen necrosis virus-based in vivo system to selectively identify mutations occurring during the early stage (RNA transcription plus minus-strand synthesis) and the late stage (plus-strand synthesis plus DNA repair). A lacZalpha reporter gene was inserted into the long terminal repeat (LTR) of a spleen necrosis virus shuttle vector, and proviruses were recovered from infected cells as plasmids containing either one or both LTRs. Plasmids containing both LTRs generated a mutant phenotype only if the lacZalpha genes in both LTRs were mutated, which is most likely to occur during the early stage. Mutant phenotypes were identified from plasmids containing one LTR regardless of the stage at which the mutations occurred. Thus, mutant frequencies obtained after recovery of plasmids containing both LTRs or one LTR provided early-stage and total mutation rates, respectively. Analysis of 56,409 proviruses suggested that the retroviral mutation rates during the early and late stages of replication were equal or within twofold of each other. In addition, two mutants with A-to-G hypermutations were discovered, suggesting a role for mammalian double-stranded RNA adenosine deaminase enzyme in retroviral mutations. These experiments provide a system to selectively identify mutations in the early stage of retroviral replication and to provide upper and lower limits to the in vivo mutation rates during minus-strand and plus-strand synthesis, respectively. PMID:8892879

RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells

PubMed Central

Date, Tomoko; Akazawa, Daisuke; Tian, Xiao; Suzuki, Tetsuro; Kato, Takanobu; Tanaka, Yasuhito; Mizokami, Masashi; Wakita, Takaji; Toyoda, Tetsuya

2010-01-01

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells. PMID:20442786

FISH-detected delay in replication timing of mutated FMR1 alleles on both active and inactive X-chromosomes.

PubMed

Yeshaya, J; Shalgi, R; Shohat, M; Avivi, L

1999-01-01

X-chromosome inactivation and the size of the CGG repeat number are assumed to play a role in the clinical, physical, and behavioral phenotype of female carriers of a mutated FMR1 allele. In view of the tight relationship between replication timing and the expression of a given DNA sequence, we have examined the replication timing of FMR1 alleles on active and inactive X-chromosomes in cell samples (lymphocytes or amniocytes) of 25 females: 17 heterozygous for a mutated FMR1 allele with a trinucleotide repeat number varying from 58 to a few hundred, and eight homozygous for a wild-type allele. We have applied two-color fluorescence in situ hybridization (FISH) with FMR1 and X-chromosome alpha-satellite probes to interphase cells of the various genotypes: the alpha-satellite probe was used to distinguish between early replicating (active) and late replicating (inactive) X-chromosomes, and the FMR1 probe revealed the replication pattern of this locus. All samples, except one with a large trinucleotide expansion, showed an early replicating FMR1 allele on the active X-chromosome and a late replicating allele on the inactive X-chromosome. In samples of mutation carriers, both the early and the late alleles showed delayed replication compared with normal alleles, regardless of repeat size. We conclude therefore that: (1) the FMR1 locus is subjected to X-inactivation; (2) mutated FMR1 alleles, regardless of repeat size, replicate later than wild-type alleles on both the active and inactive X-chromosomes; and (3) the delaying effect of the trinucleotide expansion, even with a low repeat size, is superimposed on the delay in replication associated with X-inactivation.

Differential regulation of hepatitis B virus core protein expression and genome replication by a small upstream open reading frame and naturally occurring mutations in the precore region.

PubMed

Zong, Li; Qin, Yanli; Jia, Haodi; Ye, Lei; Wang, Yongxiang; Zhang, Jiming; Wands, Jack R; Tong, Shuping; Li, Jisu

2017-05-01

Hepatitis B virus (HBV) transcribes two subsets of 3.5-kb RNAs: precore RNA for hepatitis B e antigen (HBeAg) expression, and pregenomic RNA for core and P protein translation as well as genome replication. HBeAg expression could be prevented by mutations in the precore region, while an upstream open reading frame (uORF) has been proposed as a negative regulator of core protein translation. We employed replication competent HBV DNA constructs and transient transfection experiments in Huh7 cells to verify the uORF effect and to explore the alternative function of precore RNA. Optimized Kozak sequence for the uORF or extra ATG codons as present in some HBV genotypes reduced core protein expression. G1896A nonsense mutation promoted more efficient core protein expression than mutated precore ATG, while a +1 frameshift mutation was ineffective. In conclusion, various HBeAg-negative precore mutations and mutations affecting uORF differentially regulate core protein expression and genome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

FANCJ localization by mismatch repair is vital to maintain genomic integrity after UV irradiation.

PubMed

Guillemette, Shawna; Branagan, Amy; Peng, Min; Dhruva, Aashana; Schärer, Orlando D; Cantor, Sharon B

2014-02-01

Nucleotide excision repair (NER) is critical for the repair of DNA lesions induced by UV radiation, but its contribution in replicating cells is less clear. Here, we show that dual incision by NER endonucleases, including XPF and XPG, promotes the S-phase accumulation of the BRCA1 and Fanconi anemia-associated DNA helicase FANCJ to sites of UV-induced damage. FANCJ promotes replication protein A phosphorylation and the arrest of DNA synthesis following UV irradiation. Interaction defective mutants of FANCJ reveal that BRCA1 binding is not required for FANCJ localization, whereas interaction with the mismatch repair (MMR) protein MLH1 is essential. Correspondingly, we find that FANCJ, its direct interaction with MLH1, and the MMR protein MSH2 function in a common pathway in response to UV irradiation. FANCJ-deficient cells are not sensitive to killing by UV irradiation, yet we find that DNA mutations are significantly enhanced. Thus, we considered that FANCJ deficiency could be associated with skin cancer. Along these lines, in melanoma we found several somatic mutations in FANCJ, some of which were previously identified in hereditary breast cancer and Fanconi anemia. Given that, mutations in XPF can also lead to Fanconi anemia, we propose collaborations between Fanconi anemia, NER, and MMR are necessary to initiate checkpoint activation in replicating human cells to limit genomic instability.

A role for the replication proteins PCNA, RF-C, polymerase epsilon and Cdc45 in transcriptional silencing in Saccharomyces cerevisiae.

PubMed Central

Ehrenhofer-Murray, A E; Kamakaka, R T; Rine, J

1999-01-01

Transcriptional silencing in the budding yeast Saccharomyces cerevisiae may be linked to DNA replication and cell cycle progression. In this study, we have surveyed the effect of 41 mutations in genes with a role in replication, the cell cycle, and DNA repair on silencing at HMR. Mutations in PCNA (POL30), RF-C (CDC44), polymerase epsilon (POL2, DPB2, DPB11), and CDC45 were found to restore silencing at a mutant HMR silencer allele that was still a chromosomal origin of replication. Replication timing experiments indicated that the mutant HMR locus was replicated late in S-phase, at the same time as wild-type HMR. Restoration of silencing by PCNA and CDC45 mutations required the origin recognition complex binding site of the HMR-E silencer. Several models for the precise role of these replication proteins in silencing are discussed. PMID:10545450

Addition of m6A to SV40 late mRNAs enhances viral structural gene expression and replication

PubMed Central

Courtney, David G.

2018-01-01

Polyomaviruses are a family of small DNA tumor viruses that includes several pathogenic human members, including Merkel cell polyomavirus, BK virus and JC virus. As is characteristic of DNA tumor viruses, gene expression in polyomaviruses is temporally regulated into an early phase, consisting of the viral regulatory proteins, and a late phase, consisting of the viral structural proteins. Previously, the late transcripts expressed by the prototypic polyomavirus simian virus 40 (SV40) were reported to contain several adenosines bearing methyl groups at the N6 position (m6A), although the precise location of these m6A residues, and their phenotypic effects, have not been investigated. Here, we first demonstrate that overexpression of the key m6A reader protein YTHDF2 induces more rapid viral replication, and larger viral plaques, in SV40 infected BSC40 cells, while mutational inactivation of the endogenous YTHDF2 gene, or the m6A methyltransferase METTL3, has the opposite effect, thus suggesting a positive role for m6A in the regulation of SV40 gene expression. To directly test this hypothesis, we mapped sites of m6A addition on SV40 transcripts and identified two m6A sites on the viral early transcripts and eleven m6A sites on the late mRNAs. Using synonymous mutations, we inactivated the majority of the m6A sites on the SV40 late mRNAs and observed that the resultant viral mutant replicated more slowly than wild type SV40. Alternative splicing of SV40 late mRNAs was unaffected by the reduction in m6A residues and our data instead suggest that m6A enhances the translation of viral late transcripts. Together, these data argue that the addition of m6A residues to the late transcripts encoded by SV40 plays an important role in enhancing viral gene expression and, hence, replication. PMID:29447282

Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells

PubMed Central

Nichol Edamura, Kerrie; Leonard, Michelle R.; Pearson, Christopher E.

2005-01-01

Instability of the fragile X CGG repeat involves both maternally derived expansions and deletions in the gametes of full-mutation males. It has also been suggested that the absence of aberrant CpG methylation may enhance repeat deletions through an unknown process. The effect of CGG tract length, DNA replication direction, location of replication initiation, and CpG methylation upon CGG stability were investigated using an SV40 primate replication system. Replication-dependant deletions with 53 CGG repeats were observed when replication was initiated proximal to the repeat, with CGG as the lagging-strand template. When we initiated replication further from the repeat, while maintaining CGG as the lagging-strand template or using CCG as the lagging-strand template, significant instability was not observed. CpG methylation of the unstable template stabilized the repeat, decreasing both the frequency and the magnitude of deletion events. Furthermore, CpG methylation slowed the efficiency of replication for all templates. Interestingly, replication forks displayed no evidence of a block at the CGG repeat tract, regardless of replication direction or CpG methylation status. Templates with 20 CGG repeats were stable under all circumstances. These results reveal that CGG deletions occur during replication and are sensitive to replication-fork dynamics, tract length, and CpG methylation. PMID:15625623

T antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus

PubMed Central

Shuda, Masahiro; Feng, Huichen; Kwun, Hyun Jin; Rosen, Steven T.; Gjoerup, Ole; Moore, Patrick S.; Chang, Yuan

2008-01-01

Merkel cell polyomavirus (MCV) is a virus discovered in our laboratory at the University of Pittsburgh that is monoclonally integrated into the genome of ≈80% of human Merkel cell carcinomas (MCCs). Transcript mapping was performed to show that MCV expresses transcripts in MCCs similar to large T (LT), small T (ST), and 17kT transcripts of SV40. Nine MCC tumor-derived LT genomic sequences have been examined, and all were found to harbor mutations prematurely truncating the MCV LT helicase. In contrast, four presumed episomal viruses from nontumor sources did not possess this T antigen signature mutation. Using coimmunoprecipitation and origin replication assays, we show that tumor-derived virus mutations do not affect retinoblastoma tumor suppressor protein (Rb) binding by LT but do eliminate viral DNA replication capacity. Identification of an MCC cell line (MKL-1) having monoclonal MCV integration and the signature LT mutation allowed us to functionally test both tumor-derived and wild type (WT) T antigens. Only WT LT expression activates replication of integrated MCV DNA in MKL-1 cells. Our findings suggest that MCV-positive MCC tumor cells undergo selection for LT mutations to prevent autoactivation of integrated virus replication that would be detrimental to cell survival. Because these mutations render the virus replication-incompetent, MCV is not a “passenger virus” that secondarily infects MCC tumors. PMID:18812503

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice

PubMed Central

Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai

2016-01-01

ABSTRACT H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. IMPORTANCE Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans. PMID:27558420

A Single Mutation at Position 190 in Hemagglutinin Enhances Binding Affinity for Human Type Sialic Acid Receptor and Replication of H9N2 Avian Influenza Virus in Mice.

PubMed

Teng, Qiaoyang; Xu, Dawei; Shen, Weixia; Liu, Qinfang; Rong, Guangyu; Li, Xuesong; Yan, Liping; Yang, Jianmei; Chen, Hongjun; Yu, Hai; Ma, Wenjun; Li, Zejun

2016-11-01

H9N2 avian influenza virus (AIV) has an extended host range, but the molecular basis underlying H9N2 AIV transmission to mammals remains unclear. We isolated more than 900 H9N2 AIVs in our 3-year surveillance in live bird markets in China from 2009 to 2012. Thirty-seven representative isolates were selected for further detailed characterization. These isolates were categorized into 8 genotypes (B64 to B71) and formed a distinct antigenic subgroup. Three isolates belonging to genotype B69, which is a predominant genotype circulating in China, replicated efficiently in mice, while the viruses tested in parallel in other genotypes replicated poorly, although they, like the three B69 isolates, have a leucine at position 226 in the hemagglutinin (HA) receptor binding site, which is critical for binding human type sialic acid receptors. Further molecular and single mutation analysis revealed that a valine (V) residue at position 190 in HA is responsible for efficient replication of these H9N2 viruses in mice. The 190V in HA does not affect virus receptor binding specificity but enhances binding affinity to human cells and lung tissues from mouse and humans. All these data indicate that the 190V in HA is one of the important determinants for H9N2 AIVs to cross the species barrier to infect mammals despite multiple genes conferring adaptation and replication of H9N2 viruses in mammals. Our findings provide novel insights on understanding host range expansion of H9N2 AIVs. Influenza virus hemagglutinin (HA) is responsible for binding to host cell receptors and therefore influences the viral host range and pathogenicity in different species. We showed that the H9N2 avian influenza viruses harboring 190V in the HA exhibit enhanced virus replication in mice. Further studies demonstrate that 190V in the HA does not change virus receptor binding specificity but enhances virus binding affinity of the H9N2 virus to human cells and attachment to lung tissues from humans and mouse. Our findings suggest that more attention should be given to the H9N2 AIVs with HA-190V during surveillance due to their potential threat to mammals, including humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

HLA-B27 Selects for Rare Escape Mutations that Significantly Impair Hepatitis C Virus Replication and Require Compensatory Mutations

PubMed Central

Neumann-Haefelin, Christoph; Oniangue-Ndza, Cesar; Kuntzen, Thomas; Schmidt, Julia; Nitschke, Katja; Sidney, John; Caillet-Saguy, Célia; Binder, Marco; Kersting, Nadine; Kemper, Michael W.; Power, Karen A.; Ingber, Susan; Reyor, Laura L.; Hills-Evans, Kelsey; Kim, Arthur Y.; Lauer, Georg M.; Lohmann, Volker; Sette, Alessandro; Henn, Matthew R.; Bressanelli, Stéphane; Thimme, Robert; Allen, Todd M.

2011-01-01

HLA-B27 is associated with spontaneous viral clearance in hepatitis C virus (HCV) infection. Viral escape within the immunodominant HLA-B27 restricted HCV-specific CD8+ T cell epitope NS5B2841-2849 (ARMILMTHF) has been shown to be limited by viral fitness costs as well as broad T cell cross-recognition, suggesting a potential mechanism of protection by HLA-B27. Here, we studied the subdominant HLA-B27 restricted epitope NS5B2936-2944 (GRAAICGKY) in order to further define the mechanisms of protection by HLA-B27. We identified a unique pattern of escape mutations within this epitope in a large cohort of HCV genotype 1a infected patients. The predominant escape mutations represented conservative substitutions at the main HLA-B27 anchor residue or a T cell receptor contact site, neither of which impaired viral replication capacity as assessed in a subgenomic HCV replicon system. In contrast, however, in a subset of HLA-B27+ subjects rare escape mutations arose at the HLA-B27 anchor residue R2937, which nearly abolished viral replication. Notably, these rare mutations only occurred in conjunction with the selection of two equally rare, and structurally proximal, upstream mutations. Co-expression of these upstream mutations with the rare escape mutations dramatically restored viral replication capacity from <5% to ≥70% of wild-type levels. Conclusion The selection of rare CTL escape mutations in this HLA-B27 restricted epitope dramatically impairs viral replicative fitness unless properly compensated. These data support a role for the targeting of highly-constrained regions by HLA-B27 in its ability to assert immune control of HCV and other highly variable pathogens. PMID:22006856

Influenza A Virus Acquires Enhanced Pathogenicity and Transmissibility after Serial Passages in Swine

PubMed Central

Wei, Kai; Sun, Honglei; Sun, Zhenhong; Sun, Yipeng; Kong, Weili; Pu, Juan; Ma, Guangpeng; Yin, Yanbo; Yang, Hanchun; Guo, Xin; Chang, Kin-Chow

2014-01-01

ABSTRACT Genetic and phylogenetic analyses suggest that the pandemic H1N1/2009 virus was derived from well-established swine influenza lineages; however, there is no convincing evidence that the pandemic virus was generated from a direct precursor in pigs. Furthermore, the evolutionary dynamics of influenza virus in pigs have not been well documented. Here, we subjected a recombinant virus (rH1N1) with the same constellation makeup as the pandemic H1N1/2009 virus to nine serial passages in pigs. The severity of infection sequentially increased with each passage. Deep sequencing of viral quasispecies from the ninth passage found five consensus amino acid mutations: PB1 A469T, PA 1129T, NA N329D, NS1 N205K, and NEP T48N. Mutations in the hemagglutinin (HA) protein, however, differed greatly between the upper and lower respiratory tracts. Three representative viral clones with the five consensus mutations were selected for functional evaluation. Relative to the parental virus, the three viral clones showed enhanced replication and polymerase activity in vitro and enhanced replication, pathogenicity, and transmissibility in pigs, guinea pigs, and ferrets in vivo. Specifically, two mutants of rH1N1 (PB1 A469T and a combination of NS1 N205K and NEP T48N) were identified as determinants of transmissibility in guinea pigs. Crucially, one mutant viral clone with the five consensus mutations, which also carried D187E, K211E, and S289N mutations in its HA, additionally was able to infect ferrets by airborne transmission as effectively as the pandemic virus. Our findings demonstrate that influenza virus can acquire viral characteristics that are similar to those of the pandemic virus after limited serial passages in pigs. IMPORTANCE We demonstrate here that an engineered reassortant swine influenza virus, with the same gene constellation pattern as the pandemic H1N1/2009 virus and subjected to only nine serial passages in pigs, acquired greatly enhanced virulence and transmissibility. In particular, one representative pathogenic passaged virus clone, which carried three mutations in the HA gene and five consensus mutations in PB1, PA, NA, NS1, and NEP genes, additionally was able to confer respiratory droplet transmission as effectively as the pandemic H1N1/2009 virus. Our findings suggest that pigs can readily induce adaptive mutational changes to a precursor pandemic-like virus to transform it into a highly virulent and infectious form akin to that of the pandemic H1N1/2009 virus, which underlines the potential direct role of pigs in promoting influenza A virus pathogenicity and transmissibility. PMID:25100840

Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers.

PubMed

Shlien, Adam; Campbell, Brittany B; de Borja, Richard; Alexandrov, Ludmil B; Merico, Daniele; Wedge, David; Van Loo, Peter; Tarpey, Patrick S; Coupland, Paul; Behjati, Sam; Pollett, Aaron; Lipman, Tatiana; Heidari, Abolfazl; Deshmukh, Shriya; Avitzur, Na'ama; Meier, Bettina; Gerstung, Moritz; Hong, Ye; Merino, Diana M; Ramakrishna, Manasa; Remke, Marc; Arnold, Roland; Panigrahi, Gagan B; Thakkar, Neha P; Hodel, Karl P; Henninger, Erin E; Göksenin, A Yasemin; Bakry, Doua; Charames, George S; Druker, Harriet; Lerner-Ellis, Jordan; Mistry, Matthew; Dvir, Rina; Grant, Ronald; Elhasid, Ronit; Farah, Roula; Taylor, Glenn P; Nathan, Paul C; Alexander, Sarah; Ben-Shachar, Shay; Ling, Simon C; Gallinger, Steven; Constantini, Shlomi; Dirks, Peter; Huang, Annie; Scherer, Stephen W; Grundy, Richard G; Durno, Carol; Aronson, Melyssa; Gartner, Anton; Meyn, M Stephen; Taylor, Michael D; Pursell, Zachary F; Pearson, Christopher E; Malkin, David; Futreal, P Andrew; Stratton, Michael R; Bouffet, Eric; Hawkins, Cynthia; Campbell, Peter J; Tabori, Uri

2015-03-01

DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.

Analysis of mutagenic DNA repair in a thermoconditional mutant of Saccharomyces cerevisiae. IV. Influence of DNA replication and excision repair on REV2 dependent UV-mutagenesis and repair.

PubMed

Siede, W; Eckardt, F

1986-01-01

A double mutant being thermoconditionally defective in mutation induction as well as in repair of pre-lethal UV-induced DNA damage (rev2ts) and deficient in excision repair (rad3-2) was studied in temperature-shift experiments. The influence of inhibitors of DNA replication (hydroxyurea, aphidicolin) was determined. Additionally, an analysis of the dose-response pattern of mutation induction ("mutation kinetics") at several ochre alleles was carried out. It was concluded that the UV-inducible REV2 dependent mutagenic repair process is not induced in excision-deficient cells. In excision-deficient cells, REV2 dependent mutation fixation is slow and mostly post-replicative though not dependent on DNA replication. The REV2 mediated mutagenic process could be separated from the repair function.

HIV-1 replication in cell lines harboring INI1/hSNF5 mutations.

PubMed

Sorin, Masha; Yung, Eric; Wu, Xuhong; Kalpana, Ganjam V

2006-08-31

INI1/hSNF5 is a cellular protein that directly interacts with HIV-1 integrase (IN). It is specifically incorporated into HIV-1 virions. A dominant negative mutant derived from INI1 inhibits HIV-1 replication. Recent studies indicate that INI1 is associated with pre-integration and reverse transcription complexes that are formed upon viral entry into the target cells. INI1 also is a tumor suppressor, biallelically deleted/mutated in malignant rhabdoid tumors. We have utilized cell lines derived from the rhabdoid tumors, MON and STA-WT1, that harbor either null or truncating mutations of INI1 respectively, to assess the effect of INI1 on HIV-1 replication. We found that while HIV-1 virions produced in 293T cells efficiently transduced MON and STA-WT1 cells, HIV-1 particle production was severely reduced in both of these cells. Reintroduction of INI1 into MON and STA-WT1 significantly enhanced the particle production in both cell lines. HIV-1 particles produced in MON cells were reduced for infectivity, while those produced in STA-WT1 were not. Further analysis indicated the presence of INI1 in those virions produced from STA-WT1 but not from those produced from MON cells. HIV-1 produced in MON cells were defective for synthesis of early and late reverse transcription products in the target cells. Furthermore, virions produced in MON cells were defective for exogenous reverse transcriptase activity carried out using exogenous template, primer and substrate. Our results suggest that INI1-deficient cells exhibit reduced particle production that can be partly enhanced by re-introduction of INI1. Infectivity of HIV-1 produced in some but not all INI1 defective cells, is affected and this defect may correlate to the lack of INI1 and/or some other proteins in these virions. The block in early events of virion produced from MON cells appears to be at the stage of reverse transcription. These studies suggest that presence of INI1 or some other host factor in virions and reverse transcription complexes may be important for early events of HIV-1 replication.

Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1.

PubMed

Murayama, Asako; Kato, Takanobu; Akazawa, Daisuke; Sugiyama, Nao; Date, Tomoko; Masaki, Takahiro; Nakamoto, Shingo; Tanaka, Yasuhito; Mizokami, Masashi; Yokosuka, Osamu; Nomoto, Akio; Wakita, Takaji

2012-02-01

To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.

A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding

PubMed Central

Yan, Yu; He, Ying; Boson, Bertrand; Wang, Xuesong; Cosset, François-Loïc

2017-01-01

ABSTRACT The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α0), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α0, which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix α0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly. PMID:28053108

SUMO Modification Stabilizes Enterovirus 71 Polymerase 3D To Facilitate Viral Replication

PubMed Central

Liu, Yan; Shu, Bo; Meng, Jin; Zhang, Yuan; Zheng, Caishang; Ke, Xianliang; Gong, Peng; Hu, Qinxue; Wang, Hanzhong

2016-01-01

ABSTRACT Accumulating evidence suggests that viruses hijack cellular proteins to circumvent the host immune system. Ubiquitination and SUMOylation are extensively studied posttranslational modifications (PTMs) that play critical roles in diverse biological processes. Cross talk between ubiquitination and SUMOylation of both host and viral proteins has been reported to result in distinct functional consequences. Enterovirus 71 (EV71), an RNA virus belonging to the family Picornaviridae, is a common cause of hand, foot, and mouth disease. Little is known concerning how host PTM systems interact with enteroviruses. Here, we demonstrate that the 3D protein, an RNA-dependent RNA polymerase (RdRp) of EV71, is modified by small ubiquitin-like modifier 1 (SUMO-1) both during infection and in vitro. Residues K159 and L150/D151/L152 were responsible for 3D SUMOylation as determined by bioinformatics prediction combined with site-directed mutagenesis. Also, primer-dependent polymerase assays indicated that mutation of SUMOylation sites impaired 3D polymerase activity and virus replication. Moreover, 3D is ubiquitinated in a SUMO-dependent manner, and SUMOylation is crucial for 3D stability, which may be due to the interplay between the two PTMs. Importantly, increasing the level of SUMO-1 in EV71-infected cells augmented the SUMOylation and ubiquitination levels of 3D, leading to enhanced replication of EV71. These results together suggested that SUMO and ubiquitin cooperatively regulated EV71 infection, either by SUMO-ubiquitin hybrid chains or by ubiquitin conjugating to the exposed lysine residue through SUMOylation. Our study provides new insight into how a virus utilizes cellular pathways to facilitate its replication. IMPORTANCE Infection with enterovirus 71 (EV71) often causes neurological diseases in children, and EV71 is responsible for the majority of fatalities. Based on a better understanding of interplay between virus and host cell, antiviral drugs against enteroviruses may be developed. As a dynamic cellular process of posttranslational modification, SUMOylation regulates global cellular protein localization, interaction, stability, and enzymatic activity. However, little is known concerning how SUMOylation directly influences virus replication by targeting viral polymerase. Here, we found that EV71 polymerase 3D was SUMOylated during EV71 infection and in vitro. Moreover, the SUMOylation sites were determined, and in vitro polymerase assays indicated that mutations at SUMOylation sites could impair polymerase synthesis. Importantly, 3D is ubiquitinated in a SUMOylation-dependent manner that enhances the stability of the viral polymerase. Our findings indicate that the two modifications likely cooperatively enhance virus replication. Our study may offer a new therapeutic strategy against virus replication. PMID:27630238

Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease.

PubMed

Louis, John M; Tözsér, József; Roche, Julien; Matúz, Krisztina; Aniana, Annie; Sayer, Jane M

2013-10-29

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis.

PubMed

Fenwick, Aimee L; Kliszczak, Maciej; Cooper, Fay; Murray, Jennie; Sanchez-Pulido, Luis; Twigg, Stephen R F; Goriely, Anne; McGowan, Simon J; Miller, Kerry A; Taylor, Indira B; Logan, Clare; Bozdogan, Sevcan; Danda, Sumita; Dixon, Joanne; Elsayed, Solaf M; Elsobky, Ezzat; Gardham, Alice; Hoffer, Mariette J V; Koopmans, Marije; McDonald-McGinn, Donna M; Santen, Gijs W E; Savarirayan, Ravi; de Silva, Deepthi; Vanakker, Olivier; Wall, Steven A; Wilson, Louise C; Yuregir, Ozge Ozalp; Zackai, Elaine H; Ponting, Chris P; Jackson, Andrew P; Wilkie, Andrew O M; Niedzwiedz, Wojciech; Bicknell, Louise S

2016-07-07

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast

PubMed Central

Sanchez, Joseph C.; Kwan, Elizabeth X.; Raghuraman, M. K.; Brewer, Bonita J.

2017-01-01

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways—DNA replication and ribosome biogenesis. PMID:29036220

Defective replication initiation results in locus specific chromosome breakage and a ribosomal RNA deficiency in yeast.

PubMed

Sanchez, Joseph C; Kwan, Elizabeth X; Pohl, Thomas J; Amemiya, Haley M; Raghuraman, M K; Brewer, Bonita J

2017-10-01

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.

In vitro resistance to the human immunodeficiency virus type 1 maturation inhibitor PA-457 (Bevirimat).

PubMed

Adamson, Catherine S; Ablan, Sherimay D; Boeras, Ioana; Goila-Gaur, Ritu; Soheilian, Ferri; Nagashima, Kunio; Li, Feng; Salzwedel, Karl; Sakalian, Michael; Wild, Carl T; Freed, Eric O

2006-11-01

3-O-(3',3'-dimethylsuccinyl)betulinic acid (PA-457 or bevirimat) potently inhibits human immunodeficiency virus type 1 (HIV-1) maturation by blocking a late step in the Gag processing pathway, specifically the cleavage of SP1 from the C terminus of capsid (CA). To gain insights into the mechanism(s) by which HIV-1 could evolve resistance to PA-457 and to evaluate the likelihood of such resistance arising in PA-457-treated patients, we sought to identify and characterize a broad spectrum of HIV-1 variants capable of conferring resistance to this compound. Numerous independent rounds of selection repeatedly identified six single-amino-acid substitutions that independently confer PA-457 resistance: three at or near the C terminus of CA (CA-H226Y, -L231F, and -L231M) and three at the first and third residues of SP1 (SP1-A1V, -A3T, and -A3V). We determined that mutations CA-H226Y, CA-L231F, CA-L231M, and SP1-A1V do not impose a significant replication defect on HIV-1 in culture. In contrast, mutations SP1-A3V and -A3T severely impaired virus replication and inhibited virion core condensation. The replication defect imposed by SP1-A3V was reversed by a second-site compensatory mutation in CA (CA-G225S). Intriguingly, high concentrations of PA-457 enhanced the maturation of SP1 residue 3 mutants. The different phenotypes associated with mutations that confer PA-457 resistance suggest the existence of multiple mechanisms by which HIV-1 can evolve resistance to this maturation inhibitor. These findings have implications for the ongoing development of PA-457 to treat HIV-1 infection in vivo.

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage

PubMed Central

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F. Peter; Zhang, Huidong

2017-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, E. coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. PMID:27234563

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

PubMed

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

2016-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Mutation Rates across Budding Yeast Chromosome VI Are Correlated with Replication Timing

PubMed Central

Lang, Gregory I.; Murray, Andrew W.

2011-01-01

Previous experimental studies suggest that the mutation rate is nonuniform across the yeast genome. To characterize this variation across the genome more precisely, we measured the mutation rate of the URA3 gene integrated at 43 different locations tiled across Chromosome VI. We show that mutation rate varies 6-fold across a single chromosome, that this variation is correlated with replication timing, and we propose a model to explain this variation that relies on the temporal separation of two processes for replicating past damaged DNA: error-free DNA damage tolerance and translesion synthesis. This model is supported by the observation that eliminating translesion synthesis decreases this variation. PMID:21666225

Combining magnetic sorting of mother cells and fluctuation tests to analyze genome instability during mitotic cell aging in Saccharomyces cerevisiae.

PubMed

Patterson, Melissa N; Maxwell, Patrick H

2014-10-16

Saccharomyces cerevisiae has been an excellent model system for examining mechanisms and consequences of genome instability. Information gained from this yeast model is relevant to many organisms, including humans, since DNA repair and DNA damage response factors are well conserved across diverse species. However, S. cerevisiae has not yet been used to fully address whether the rate of accumulating mutations changes with increasing replicative (mitotic) age due to technical constraints. For instance, measurements of yeast replicative lifespan through micromanipulation involve very small populations of cells, which prohibit detection of rare mutations. Genetic methods to enrich for mother cells in populations by inducing death of daughter cells have been developed, but population sizes are still limited by the frequency with which random mutations that compromise the selection systems occur. The current protocol takes advantage of magnetic sorting of surface-labeled yeast mother cells to obtain large enough populations of aging mother cells to quantify rare mutations through phenotypic selections. Mutation rates, measured through fluctuation tests, and mutation frequencies are first established for young cells and used to predict the frequency of mutations in mother cells of various replicative ages. Mutation frequencies are then determined for sorted mother cells, and the age of the mother cells is determined using flow cytometry by staining with a fluorescent reagent that detects bud scars formed on their cell surfaces during cell division. Comparison of predicted mutation frequencies based on the number of cell divisions to the frequencies experimentally observed for mother cells of a given replicative age can then identify whether there are age-related changes in the rate of accumulating mutations. Variations of this basic protocol provide the means to investigate the influence of alterations in specific gene functions or specific environmental conditions on mutation accumulation to address mechanisms underlying genome instability during replicative aging.

Accumulation of Human-Adapting Mutations during Circulation of A(H1N1)pdm09 Influenza Virus in Humans in the United Kingdom

PubMed Central

Elderfield, Ruth A.; Watson, Simon J.; Godlee, Alexandra; Adamson, Walt E.; Thompson, Catherine I.; Dunning, Jake; Fernandez-Alonso, Mirian; Blumenkrantz, Deena; Hussell, Tracy; Zambon, Maria; Openshaw, Peter; Kellam, Paul

2014-01-01

ABSTRACT The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves. IMPORTANCE Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection. PMID:25210166

Accumulation of human-adapting mutations during circulation of A(H1N1)pdm09 influenza virus in humans in the United Kingdom.

PubMed

Elderfield, Ruth A; Watson, Simon J; Godlee, Alexandra; Adamson, Walt E; Thompson, Catherine I; Dunning, Jake; Fernandez-Alonso, Mirian; Blumenkrantz, Deena; Hussell, Tracy; Zambon, Maria; Openshaw, Peter; Kellam, Paul; Barclay, Wendy S

2014-11-01

The influenza pandemic that emerged in 2009 provided an unprecedented opportunity to study adaptation of a virus recently acquired from an animal source during human transmission. In the United Kingdom, the novel virus spread in three temporally distinct waves between 2009 and 2011. Phylogenetic analysis of complete viral genomes showed that mutations accumulated over time. Second- and third-wave viruses replicated more rapidly in human airway epithelial (HAE) cells than did the first-wave virus. In infected mice, weight loss varied between viral isolates from the same wave but showed no distinct pattern with wave and did not correlate with viral load in the mouse lungs or severity of disease in the human donor. However, second- and third-wave viruses induced less alpha interferon in the infected mouse lungs. NS1 protein, an interferon antagonist, had accumulated several mutations in second- and third-wave viruses. Recombinant viruses with the third-wave NS gene induced less interferon in human cells, but this alone did not account for increased virus fitness in HAE cells. Mutations in HA and NA genes in third-wave viruses caused increased binding to α-2,6-sialic acid and enhanced infectivity in human mucus. A recombinant virus with these two segments replicated more efficiently in HAE cells. A mutation in PA (N321K) enhanced polymerase activity of third-wave viruses and also provided a replicative advantage in HAE cells. Therefore, multiple mutations allowed incremental changes in viral fitness, which together may have contributed to the apparent increase in severity of A(H1N1)pdm09 influenza virus during successive waves. Although most people infected with the 2009 pandemic influenza virus had mild or unapparent symptoms, some suffered severe and devastating disease. The reasons for this variability were unknown, but the numbers of severe cases increased during successive waves of human infection in the United Kingdom. To determine the causes of this variation, we studied genetic changes in virus isolates from individual hospitalized patients. There were no consistent differences between these viruses and those circulating in the community, but we found multiple evolutionary changes that in combination over time increased the virus's ability to infect human cells. These adaptations may explain the remarkable ability of A(H1N1)pdm09 virus to continue to circulate despite widespread immunity and the apparent increase in severity of influenza over successive waves of infection. Copyright © 2014 Elderfield et al.

Rates of spontaneous mutation.

PubMed Central

Drake, J W; Charlesworth, B; Charlesworth, D; Crow, J F

1998-01-01

Rates of spontaneous mutation per genome as measured in the laboratory are remarkably similar within broad groups of organisms but differ strikingly among groups. Mutation rates in RNA viruses, whose genomes contain ca. 10(4) bases, are roughly 1 per genome per replication for lytic viruses and roughly 0.1 per genome per replication for retroviruses and a retrotransposon. Mutation rates in microbes with DNA-based chromosomes are close to 1/300 per genome per replication; in this group, therefore, rates per base pair vary inversely and hugely as genome sizes vary from 6 x 10(3) to 4 x 10(7) bases or base pairs. Mutation rates in higher eukaryotes are roughly 0.1-100 per genome per sexual generation but are currently indistinguishable from 1/300 per cell division per effective genome (which excludes the fraction of the genome in which most mutations are neutral). It is now possible to specify some of the evolutionary forces that shape these diverse mutation rates. PMID:9560386

A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus.

PubMed

Qi, Xiaole; Gao, Xiang; Lu, Zhen; Zhang, Lizhou; Wang, Yongqiang; Gao, Li; Gao, Yulong; Li, Kai; Gao, Honglei; Liu, Changjun; Cui, Hongyu; Zhang, Yanping; Wang, Xiaomei

2016-07-01

To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (PBC) and S330R (PHI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.

Heterogeneity of spontaneous DNA replication errors in single isogenic Escherichia coli cells

PubMed Central

2018-01-01

Despite extensive knowledge of the molecular mechanisms that control mutagenesis, it is not known how spontaneous mutations are produced in cells with fully operative mutation-prevention systems. By using a mutation assay that allows visualization of DNA replication errors and stress response transcriptional reporters, we examined populations of isogenic Escherichia coli cells growing under optimal conditions without exogenous stress. We found that spontaneous DNA replication errors in proliferating cells arose more frequently in subpopulations experiencing endogenous stresses, such as problems with proteostasis, genome maintenance, and reactive oxidative species production. The presence of these subpopulations of phenotypic mutators is not expected to affect the average mutation frequency or to reduce the mean population fitness in a stable environment. However, these subpopulations can contribute to overall population adaptability in fluctuating environments by serving as a reservoir of increased genetic variability.

Molecular Determinants of Influenza Virus Pathogenesis in Mice

PubMed Central

Katz, Jaqueline M.; York, Ian A.

2015-01-01

Mice are widely used for studying influenza virus pathogenesis and immunology because of their low cost, the wide availability of mouse-specific reagents, and the large number of mouse strains available, including knockout and transgenic strains. However, mice do not fully recapitulate the signs of influenza infection of humans: transmission of influenza between mice is much less efficient than in humans, and influenza viruses often require adaptation before they are able to efficiently replicate in mice. In the process of mouse adaptation, influenza viruses acquire mutations that enhance their ability to attach to mouse cells, replicate within the cells, and suppress immunity, among other functions. Many such mouse-adaptive mutations have been identified, covering all 8 genomic segments of the virus. Identification and analysis of these mutations have provided insight into the molecular determinants of influenza virulence and pathogenesis, not only in mice but also in humans and other species. In particular, several mouse-adaptive mutations of avian influenza viruses have proved to be general mammalian-adaptive changes that are potential markers of pre-pandemic viruses. As well as evaluating influenza pathogenesis, mice have also been used as models for evaluation of novel vaccines and anti-viral therapies. Mice can be a useful animal model for studying influenza biology as long as differences between human and mice infections are taken into account. PMID:25038937

Significant contribution of the 3′→5′ exonuclease activity to the high fidelity of nucleotide incorporation catalyzed by human DNA polymerase ϵ

PubMed Central

Zahurancik, Walter J.; Klein, Seth J.; Suo, Zucai

2014-01-01

Most eukaryotic DNA replication is performed by A- and B-family DNA polymerases which possess a faithful polymerase activity that preferentially incorporates correct over incorrect nucleotides. Additionally, many replicative polymerases have an efficient 3′→5′ exonuclease activity that excises misincorporated nucleotides. Together, these activities contribute to overall low polymerase error frequency (one error per 106–108 incorporations) and support faithful eukaryotic genome replication. Eukaryotic DNA polymerase ϵ (Polϵ) is one of three main replicative DNA polymerases for nuclear genomic replication and is responsible for leading strand synthesis. Here, we employed pre-steady-state kinetic methods and determined the overall fidelity of human Polϵ (hPolϵ) by measuring the individual contributions of its polymerase and 3′→5′ exonuclease activities. The polymerase activity of hPolϵ has a high base substitution fidelity (10−4–10−7) resulting from large decreases in both nucleotide incorporation rate constants and ground-state binding affinities for incorrect relative to correct nucleotides. The 3′→5′ exonuclease activity of hPolϵ further enhances polymerization fidelity by an unprecedented 3.5 × 102 to 1.2 × 104-fold. The resulting overall fidelity of hPolϵ (10−6–10−11) justifies hPolϵ to be a primary enzyme to replicate human nuclear genome (0.1–1.0 error per round). Consistently, somatic mutations in hPolϵ, which decrease its exonuclease activity, are connected with mutator phenotypes and cancer formation. PMID:25414327

The R35 residue of the influenza A virus NS1 protein has minimal effects on nuclear localization but alters virus replication through disrupting protein dimerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lalime, Erin N.; Pekosz, Andrew, E-mail: apekosz@jhsph.edu

The influenza A virus NS1 protein has a nuclear localization sequence (NLS) in the amino terminal region. This NLS overlaps sequences that are important for RNA binding as well as protein dimerization. To assess the significance of the NS1 NLS on influenza virus replication, the NLS amino acids were individually mutated to alanines and recombinant viruses encoding these mutations were rescued. Viruses containing NS1 proteins with mutations at R37, R38 and K41 displayed minimal changes in replication or NS1 protein nuclear localization. Recombinant viruses encoding NS1 R35A were not recovered but viruses containing second site mutations at position D39 inmore » addition to the R35A mutation were isolated. The mutations at position 39 were shown to partially restore NS1 protein dimerization but had minimal effects on nuclear localization. These data indicate that the amino acids in the NS1 NLS region play a more important role in protein dimerization compared to nuclear localization. - Highlights: • Mutations were introduced into influenza NS1 NLS1. • NS1 R37A, R38A, K41A viruses had minimal changes in replication and NS1 localization. • Viruses from NS1 R35A rescue all contained additional mutations at D39. • NS1 R35A D39X mutations recover dimerization lost in NS1 R35A mutations. • These results reaffirm the importance of dimerization for NS1 protein function.« less

Resistance to cyclosporin A derives from mutations in hepatitis C virus nonstructural proteins.

PubMed

Arai, Masaaki; Tsukiyama-Kohara, Kyoko; Takagi, Asako; Tobita, Yoshimi; Inoue, Kazuaki; Kohara, Michinori

2014-05-23

Cyclosporine A (CsA) is an immunosuppressive drug that targets cyclophilins, cellular cofactors that regulate the immune system. Replication of hepatitis C virus (HCV) is suppressed by CsA, but the molecular basis of this suppression is still not fully understood. To investigate this suppression, we cultured HCV replicon cells (Con1, HCV genotype 1b, FLR-N cell) in the presence of CsA and obtained nine CsA-resistant FLR-N cell lines. We determined full-length HCV sequences for all nine clones, and chose two (clones #6 and #7) of the nine clones that have high replication activity in the presence of CsA for further analysis. Both clones showed two consensus mutations, one in NS3 (T1280V) and the other in NS5A (D2292E). Characterization of various mutants indicated that the D2292E mutation conferred resistance to high concentrations of CsA (up to 2 μM). In addition, the missense mutation T1280V contributed to the recovery of colony formation activity. The effects of these mutations are also evident in two established HCV replicon cell lines-HCV-RMT ([1], genotype 1a) and JFH1 (genotype 2a). Moreover, three other missense mutations in NS5A-D2303H, S2362G, and E2414K-enhanced the resistance to CsA conferred by D2292E; these double or all quadruple mutants could resist approximately 8- to 25-fold higher concentrations of CsA than could wild-type Con1. These four mutations, either as single or combinations, also made Con1 strain resistant to two other cyclophilin inhibitors, N-methyl-4-isoleucine-cyclosporin (NIM811) or Debio-025. Interestingly, the changes in IC50 values that resulted from each of these mutations were the lowest in the Debio-025-treated cells, indicating its highest resistant activity against the adaptive mutation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Molecular replication

NASA Technical Reports Server (NTRS)

Orgel, Leslie E.

1992-01-01

Recent experiments demonstrating nonenzymatic replication in molecular systems are reviewed. The difficulties facing nonenzymatic replication are discussed along with specificity, fidelity, and mutation in nonenzymatic replication. The prospects for research in this area are considered.

Neutrality of the canonical NF-kappaB-dependent pathway for human and murine cytomegalovirus transcription and replication in vitro.

PubMed

Benedict, Chris A; Angulo, Ana; Patterson, Ginelle; Ha, Sukwon; Huang, Huang; Messerle, Martin; Ware, Carl F; Ghazal, Peter

2004-01-01

Cytomegalovirus (CMV) is known to rapidly induce activation of nuclear factor kappaB (NF-kappaB) after infection of fibroblast and macrophage cells. NF-kappaB response elements are present in the enhancer region of the CMV major immediate-early promoter (MIEP), and activity of the MIEP is strongly upregulated by NF-kappaB in transient-transfection assays. Here we investigate whether the NF-kappaB-dependent pathway is required for initiating or potentiating human and murine CMV replication in vitro. We show that expression of a dominant negative mutant of the inhibitor of NF-kappaB-alpha (IkappaBalphaM) does not alter the replication kinetics of human or mouse CMV in cultured cells. In addition, mouse embryo fibroblasts genetically deficient for p65/RelA actually showed elevated levels of MCMV replication. Mutation of all NF-kappaB response elements within the enhancer of the MIEP in a recombinant mouse CMV containing the human MIEP (hMCMV-ES), which we have previously shown to replicate in murine fibroblasts with kinetics equivalent to that of wild-type mouse CMV, did not negatively affect replication in fibroblasts. Taken together, these data show that, for CMV replication in cultured fibroblasts activation of the canonical NF-kappaB pathway and binding of NF-kappaB to the MIEP are dispensable, and in the case of p65 may even interfere, thus uncovering a previously unrecognized level of complexity in the host regulatory network governing MIE gene expression in the context of a viral infection.

Impact of human immunodeficiency virus type 1 reverse transcriptase inhibitor drug resistance mutation interactions on phenotypic susceptibility.

PubMed

Trivedi, Vinod; Von Lindern, Jana; Montes-Walters, Miguel; Rojo, Daniel R; Shell, Elisabeth J; Parkin, Neil; O'Brien, William A; Ferguson, Monique R

2008-10-01

The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals.

Impact of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Inhibitor Drug Resistance Mutation Interactions on Phenotypic Susceptibility

PubMed Central

Trivedi, Vinod; Von Lindern, Jana; Montes-Walters, Miguel; Rojo, Daniel R.; Shell, Elisabeth J.; Parkin, Neil; O'Brien, William A.

2008-01-01

Abstract The role specific reverse transcriptase (RT) drug resistance mutations play in influencing phenotypic susceptibility to RT inhibitors in virus strains with complex resistance interaction patterns was assessed using recombinant viruses that consisted of RT-PCR-amplified pol fragments derived from plasma HIV-1 RNA from two treatment-experienced patients. Specific modifications of key RT amino acids were performed by site-directed mutagenesis. A panel of viruses with defined genotypic resistance mutations was assessed for phenotypic drug resistance. Introduction of M184V into several different clones expressing various RT resistance mutations uniformly decreased susceptibility to abacavir, lamivudine, and didanosine, and increased susceptibility to zidovudine, stavudine, and tenofovir; replication capacity was decreased. The L74V mutation had similar but slightly different effects, contributing to decreased susceptibility to abacavir, lamivudine, and didanosine and increased susceptibility to zidovudine and tenofovir, but in contrast to M184V, L74V contributed to decreased susceptibility to stavudine. In virus strains with the nonnucleoside reverse transcriptase inhibitor (NNRTI) mutations K101E and G190S, the L74V mutation increased replication capacity, consistent with published observations, but replication capacity was decreased in strains without NNRTI resistance mutations. K101E and G190S together tend to decrease susceptibility to all nucleoside RT inhibitors, but the K103N mutation had little effect on nucleoside RT inhibitor susceptibility. Mutational interactions can have a substantial impact on drug resistance phenotype and replication capacity, and this has been exploited in clinical practice with the development of fixed-dose combination pills. However, we are the first to report these mutational interactions using molecularly cloned recombinant strains derived from viruses that occur naturally in HIV-infected individuals. PMID:18844463

The DnaK Chaperone Uses Different Mechanisms To Promote and Inhibit Replication of Vibrio cholerae Chromosome 2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jha, Jyoti K.; Li, Mi; Ghirlando, Rodolfo

Replication of Vibrio cholerae chromosome 2 (Chr2) depends on molecular chaperone DnaK to facilitate binding of the initiator (RctB) to the replication origin. The binding occurs at two kinds of site, 12-mers and 39-mers, which promote and inhibit replication, respectively. Here we show that DnaK employs different mechanisms to enhance the two kinds of binding. We found that mutations inrctBthat reduce DnaK binding also reduce 12-mer binding and initiation. The initiation defect is suppressed by second-site mutations that increase 12-mer binding only marginally. Instead, they reduce replication inhibitory mechanisms: RctB dimerization and 39-mer binding. One suppressing change was in amore » dimerization domain which is folded similarly to the initiator of an iteron plasmid—the presumed progenitor of Chr2. In plasmids, DnaK promotes initiation by reducing dimerization. A different mutation was in the 39-mer binding domain of RctB and inactivated it, indicating an alternative suppression mechanism. Paradoxically, although DnaK increases 39-mer binding, the increase was also achieved by inactivating the DnaK binding site of RctB. This result suggests that the site inhibits the 39-mer binding domain (via autoinhibition) when prevented from binding DnaK. Taken together, our results reveal an important feature of the transition from plasmid to chromosome: the Chr2 initiator retains the plasmid-like dimerization domain and its control by chaperones but uses the chaperones in an unprecedented way to control the inhibitory 39-mer binding. IMPORTANCE The capacity of proteins to undergo remodeling provides opportunities to control their function. However, remodeling remains a poorly understood aspect of the structure-function paradigm due to its dynamic nature. Here we have studied remodeling of the initiator of replication ofVibrio choleraeChr2 by the molecular chaperone, DnaK. We show that DnaK binds to a site on the Chr2 initiator (RctB) that promotes initiation by reducing the initiator’s propensity to dimerize. Dimerization of the initiator of the putative plasmid progenitor of Chr2 is also reduced by DnaK, which promotes initiation. Paradoxically, the DnaK binding also promotes replication inhibition by reducing an autoinhibitory activity of RctB. In the plasmid-to-chromosome transition, it appears that the initiator has acquired an autoinhibitory activity and along with it a new chaperone activity that apparently helps to control replication inhibition independently of replication promotion.« less

Genome complexity, robustness and genetic interactions in digital organisms

NASA Astrophysics Data System (ADS)

Lenski, Richard E.; Ofria, Charles; Collier, Travis C.; Adami, Christoph

1999-08-01

Digital organisms are computer programs that self-replicate, mutate and adapt by natural selection. They offer an opportunity to test generalizations about living systems that may extend beyond the organic life that biologists usually study. Here we have generated two classes of digital organism: simple programs selected solely for rapid replication, and complex programs selected to perform mathematical operations that accelerate replication through a set of defined `metabolic' rewards. To examine the differences in their genetic architecture, we introduced millions of single and multiple mutations into each organism and measured the effects on the organism's fitness. The complex organisms are more robust than the simple ones with respect to the average effects of single mutations. Interactions among mutations are common and usually yield higher fitness than predicted from the component mutations assuming multiplicative effects; such interactions are especially important in the complex organisms. Frequent interactions among mutations have also been seen in bacteria, fungi and fruitflies. Our findings support the view that interactions are a general feature of genetic systems.

Genome complexity, robustness and genetic interactions in digital organisms.

PubMed

Lenski, R E; Ofria, C; Collier, T C; Adami, C

1999-08-12

Digital organisms are computer programs that self-replicate, mutate and adapt by natural selection. They offer an opportunity to test generalizations about living systems that may extend beyond the organic life that biologists usually study. Here we have generated two classes of digital organism: simple programs selected solely for rapid replication, and complex programs selected to perform mathematical operations that accelerate replication through a set of defined 'metabolic' rewards. To examine the differences in their genetic architecture, we introduced millions of single and multiple mutations into each organism and measured the effects on the organism's fitness. The complex organisms are more robust than the simple ones with respect to the average effects of single mutations. Interactions among mutations are common and usually yield higher fitness than predicted from the component mutations assuming multiplicative effects; such interactions are especially important in the complex organisms. Frequent interactions among mutations have also been seen in bacteria, fungi and fruitflies. Our findings support the view that interactions are a general feature of genetic systems.

A mutation in the hepatitis E virus RNA polymerase promotes its replication and associates with ribavirin treatment failure in organ transplant recipients.

PubMed

Debing, Yannick; Gisa, Anett; Dallmeier, Kai; Pischke, Sven; Bremer, Birgit; Manns, Michael; Wedemeyer, Heiner; Suneetha, Pothakamuri Venkata; Neyts, Johan

2014-11-01

We analyzed blood samples collected from 15 patients with chronic hepatitis E who were recipients of solid-organ transplants. All patients cleared the hepatitis E virus (HEV) except for 2 (nonresponders); 1 patient died. A G1634R mutation in viral polymerase was detected in the HEV RNA of the nonresponders; this mutation did not provide the virus with resistance to ribavirin in vitro. However, the mutant form of a subgenomic replicon of genotype 3 HEV replicated more efficiently in vitro than HEV without this mutation, and the same was true for infectious virus, including in competition assays. Similar results were obtained for genotype 1 HEV. The G1634R mutation therefore appears to increase the replicative capacity of HEV in the human liver and hence reduce the efficacy of ribavirin. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

Replication-Competent Simian Immunodeficiency Virus (SIV) Gag Escape Mutations Archived in Latent Reservoirs during Antiretroviral Treatment of SIV-Infected Macaques▿

PubMed Central

Queen, Suzanne E.; Mears, Brian M.; Kelly, Kathleen M.; Dorsey, Jamie L.; Liao, Zhaohao; Dinoso, Jason B.; Gama, Lucio; Adams, Robert J.; Zink, M. Christine; Clements, Janice E.; Kent, Stephen J.; Mankowski, Joseph L.

2011-01-01

In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8+ T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8+ T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4+ T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8+ T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted. PMID:21715484

Impacts of different expressions of PA-X protein on 2009 pandemic H1N1 virus replication, pathogenicity and host immune responses

PubMed Central

Lee, Jinhwa; Yu, Hai; Li, Yonghai; Ma, Jingjiao; Lang, Yuekun; Duff, Michael; Henningson, Jamie; Liu, Qinfang; Li, Yuhao; Nagy, Abdou; Bawa, Bhupinder; Li, Zejun; Tong, Guangzhi; Richt, Juergen A.; Ma, Wenjun

2017-01-01

Although several studies have investigated the functions of influenza PA-X, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on virus replication, pathogenicity and host response remains unclear. Herein, we generated two mutated viruses expressing a full-length or deficient PA-X protein based on the A/California/04/2009 (H1N1) virus that expresses a truncated PA-X to understand three different expressions of PA-X protein on virus replication, pathogenicity and host immune responses. The results showed that expression of either full-length or truncated PA-X protein enhanced viral replication and pathogenicity as well as reduced host innate immune response in mice by host shutoff activity when compared to the virus expressing the deficient PA-X form. Furthermore, the full-length PA-X expression exhibited a greater effect on virus pathogenicity than the truncated PA-X form. Our results provide novel insights of PA-X on viral replication, pathogenicity and host immune responses. PMID:28142079

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation

PubMed Central

Nikolaitchik, Olga A.; Burdick, Ryan C.; Gorelick, Robert J.; Keele, Brandon F.; Hu, Wei-Shau; Pathak, Vinay K.

2016-01-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10−5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10−21 and1 × 10−11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication. PMID:27186986

Minimal Contribution of APOBEC3-Induced G-to-A Hypermutation to HIV-1 Recombination and Genetic Variation.

PubMed

Delviks-Frankenberry, Krista A; Nikolaitchik, Olga A; Burdick, Ryan C; Gorelick, Robert J; Keele, Brandon F; Hu, Wei-Shau; Pathak, Vinay K

2016-05-01

Although the predominant effect of host restriction APOBEC3 proteins on HIV-1 infection is to block viral replication, they might inadvertently increase retroviral genetic variation by inducing G-to-A hypermutation. Numerous studies have disagreed on the contribution of hypermutation to viral genetic diversity and evolution. Confounding factors contributing to the debate include the extent of lethal (stop codon) and sublethal hypermutation induced by different APOBEC3 proteins, the inability to distinguish between G-to-A mutations induced by APOBEC3 proteins and error-prone viral replication, the potential impact of hypermutation on the frequency of retroviral recombination, and the extent to which viral recombination occurs in vivo, which can reassort mutations in hypermutated genomes. Here, we determined the effects of hypermutation on the HIV-1 recombination rate and its contribution to genetic variation through recombination to generate progeny genomes containing portions of hypermutated genomes without lethal mutations. We found that hypermutation did not significantly affect the rate of recombination, and recombination between hypermutated and wild-type genomes only increased the viral mutation rate by 3.9 × 10-5 mutations/bp/replication cycle in heterozygous virions, which is similar to the HIV-1 mutation rate. Since copackaging of hypermutated and wild-type genomes occurs very rarely in vivo, recombination between hypermutated and wild-type genomes does not significantly contribute to the genetic variation of replicating HIV-1. We also analyzed previously reported hypermutated sequences from infected patients and determined that the frequency of sublethal mutagenesis for A3G and A3F is negligible (4 × 10-21 and1 × 10-11, respectively) and its contribution to viral mutations is far below mutations generated during error-prone reverse transcription. Taken together, we conclude that the contribution of APOBEC3-induced hypermutation to HIV-1 genetic variation is substantially lower than that from mutations during error-prone replication.

Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera[OPEN

PubMed Central

Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S.; Johnson, Marc T.J.; Wright, Stephen I.; Pellizzer, Tommaso; Sobanski, Johanna; Greiner, Stephan

2016-01-01

Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. PMID:27053421

Processing closely spaced lesions during Nucleotide Excision Repair triggers mutagenesis in E. coli

PubMed Central

Isogawa, Asako; Fujii, Shingo

2017-01-01

It is generally assumed that most point mutations are fixed when damage containing template DNA undergoes replication, either right at the fork or behind the fork during gap filling. Here we provide genetic evidence for a pathway, dependent on Nucleotide Excision Repair, that induces mutations when processing closely spaced lesions. This pathway, referred to as Nucleotide Excision Repair-induced Mutagenesis (NERiM), exhibits several characteristics distinct from mutations that occur within the course of replication: i) following UV irradiation, NER-induced mutations are fixed much more rapidly (t ½ ≈ 30 min) than replication dependent mutations (t ½ ≈ 80–100 min) ii) NERiM specifically requires DNA Pol IV in addition to Pol V iii) NERiM exhibits a two-hit dose-response curve that suggests processing of closely spaced lesions. A mathematical model let us define the geometry (infer the structure) of the toxic intermediate as being formed when NER incises a lesion that resides in close proximity of another lesion in the complementary strand. This critical NER intermediate requires Pol IV / Pol II for repair, it is either lethal if left unrepaired or mutation-prone when repaired. Finally, NERiM is found to operate in stationary phase cells providing an intriguing possibility for ongoing evolution in the absence of replication. PMID:28686598

A Point Mutation in the N-Terminal Amphipathic Helix α0 in NS3 Promotes Hepatitis C Virus Assembly by Altering Core Localization to the Endoplasmic Reticulum and Facilitating Virus Budding.

PubMed

Yan, Yu; He, Ying; Boson, Bertrand; Wang, Xuesong; Cosset, François-Loïc; Zhong, Jin

2017-03-15

The assembly of hepatitis C virus (HCV), a complicated process in which many viral and cellular factors are involved, has not been thoroughly deciphered. NS3 is a multifunctional protein that contains an N-terminal amphipathic α helix (designated helix α 0 ), which is crucial for the membrane association and stability of NS3 protein, followed by a serine protease domain and a C-terminal helicase/NTPase domain. NS3 participates in HCV assembly likely through its C-terminal helicase domain, in which all reported adaptive mutations enhancing virion assembly reside. In this study, we determined that the N-terminal helix α 0 of NS3 may contribute to HCV assembly. We identified a single mutation from methionine to threonine at amino acid position 21 (M21T) in helix α 0 , which significantly promoted viral production while having no apparent effect on the membrane association and protease activity of NS3. Subsequent analyses demonstrated that the M21T mutation did not affect HCV genome replication but rather promoted virion assembly. Further study revealed a shift in the subcellular localization of core protein from lipid droplets (LD) to the endoplasmic reticulum (ER). Finally, we showed that the M21T mutation increased the colocalization of core proteins and viral envelope proteins, leading to a more efficient envelopment of viral nucleocapsids. Collectively, the results of our study revealed a new function of NS3 helix α 0 and aid understanding of the role of NS3 in HCV virion morphogenesis. IMPORTANCE HCV NS3 protein possesses the protease activity in its N-terminal domain and the helicase activity in its C-terminal domain. The role of NS3 in virus assembly has been mainly attributed to its helicase domain, because all adaptive mutations enhancing progeny virus production are found to be within this domain. Our study identified, for the first time to our knowledge, an adaptive mutation within the N-terminal helix α 0 domain of NS3 that significantly enhanced virus assembly while having no effect on viral genome replication. The mechanistic studies suggested that this mutation promoted the relocation of core proteins from LD to the ER, leading to a more efficient envelopment of viral nucleocapsids. Our results revealed a possible new function of helix α 0 in the HCV life cycle and provided new clues to understanding the molecular mechanisms for the action of NS3 in HCV assembly. Copyright © 2017 American Society for Microbiology.

Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice.

PubMed

Yang, W; Punyadarsaniya, D; Lambertz, R L O; Lee, D C C; Liang, C H; Höper, D; Leist, S R; Hernández-Cáceres, A; Stech, J; Beer, M; Wu, C Y; Wong, C H; Schughart, K; Meng, F; Herrler, G

2017-04-15

The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized α2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with α2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs. IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by passaging the virus three times in differentiated porcine respiratory epithelial cells. Among the four mutations detected, the two HA mutations were analyzed by generating recombinant viruses. Depending on the infection system used, the mutations differed in their phenotypic expression, e.g., sialic acid binding activity, replication kinetics, plaque size, and pathogenicity in inbred mice. However, none of the mutations affected the ciliary activity which serves as a virulence marker. Thus, early adaptive mutation enhances the replication kinetics, but more mutations are required for IAV of the H9N2 subtype to become virulent. Copyright © 2017 American Society for Microbiology.

Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

PubMed Central

Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek

2012-01-01

Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates. PMID:22811532

Epistatic Interactions within the Influenza A Virus Polymerase Complex Mediate Mutagen Resistance and Replication Fidelity

PubMed Central

Pauly, Matthew D.; Lyons, Daniel M.; Fitzsimmons, William J.

2017-01-01

ABSTRACT Lethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza A virus using three nucleoside analogs as well as the virus’s high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil (2,4-dihydroxy-5-fluoropyrimidine) treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C-to-U and G-to-A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB1 T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza virus polymerase, our data suggest that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin {1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide} resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza A virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates. IMPORTANCE RNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able to tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis. PMID:28815216

Mutation rate evolution in replicator dynamics.

PubMed

Allen, Benjamin; Rosenbloom, Daniel I Scholes

2012-11-01

The mutation rate of an organism is itself evolvable. In stable environments, if faithful replication is costless, theory predicts that mutation rates will evolve to zero. However, positive mutation rates can evolve in novel or fluctuating environments, as analytical and empirical studies have shown. Previous work on this question has focused on environments that fluctuate independently of the evolving population. Here we consider fluctuations that arise from frequency-dependent selection in the evolving population itself. We investigate how the dynamics of competing traits can induce selective pressure on the rates of mutation between these traits. To address this question, we introduce a theoretical framework combining replicator dynamics and adaptive dynamics. We suppose that changes in mutation rates are rare, compared to changes in the traits under direct selection, so that the expected evolutionary trajectories of mutation rates can be obtained from analysis of pairwise competition between strains of different rates. Depending on the nature of frequency-dependent trait dynamics, we demonstrate three possible outcomes of this competition. First, if trait frequencies are at a mutation-selection equilibrium, lower mutation rates can displace higher ones. Second, if trait dynamics converge to a heteroclinic cycle-arising, for example, from "rock-paper-scissors" interactions-mutator strains succeed against non-mutators. Third, in cases where selection alone maintains all traits at positive frequencies, zero and nonzero mutation rates can coexist indefinitely. Our second result suggests that relatively high mutation rates may be observed for traits subject to cyclical frequency-dependent dynamics.

The Spectrum of Replication Errors in the Absence of Error Correction Assayed Across the Whole Genome of Escherichia coli.

PubMed

Niccum, Brittany A; Lee, Heewook; MohammedIsmail, Wazim; Tang, Haixu; Foster, Patricia L

2018-06-15

When the DNA polymerase that replicates the Escherichia coli chromosome, DNA Pol III, makes an error, there are two primary defenses against mutation: proofreading by the epsilon subunit of the holoenzyme and mismatch repair. In proofreading deficient strains, mismatch repair is partially saturated and the cell's response to DNA damage, the SOS response, may be partially induced. To investigate the nature of replication errors, we used mutation accumulation experiments and whole genome sequencing to determine mutation rates and mutational spectra across the entire chromosome of strains deficient in proofreading, mismatch repair, and the SOS response. We report that a proofreading-deficient strain has a mutation rate 4,000-fold greater than wild-type strains. While the SOS response may be induced in these cells, it does not contribute to the mutational load. Inactivating mismatch repair in a proofreading-deficient strain increases the mutation rate another 1.5-fold. DNA polymerase has a bias for converting G:C to A:T base pairs, but proofreading reduces the impact of these mutations, helping to maintain the genomic G:C content. These findings give an unprecedented view of how polymerase and error-correction pathways work together to maintain E. coli' s low mutation rate of 1 per thousand generations. Copyright © 2018, Genetics.

Cellular replication limits in the Luria-Delbrück mutation model

NASA Astrophysics Data System (ADS)

Rodriguez-Brenes, Ignacio A.; Wodarz, Dominik; Komarova, Natalia L.

2016-08-01

Originally developed to elucidate the mechanisms of natural selection in bacteria, the Luria-Delbrück model assumed that cells are intrinsically capable of dividing an unlimited number of times. This assumption however, is not true for human somatic cells which undergo replicative senescence. Replicative senescence is thought to act as a mechanism to protect against cancer and the escape from it is a rate-limiting step in cancer progression. Here we introduce a Luria-Delbrück model that explicitly takes into account cellular replication limits in the wild type cell population and models the emergence of mutants that escape replicative senescence. We present results on the mean, variance, distribution, and asymptotic behavior of the mutant population in terms of three classical formulations of the problem. More broadly the paper introduces the concept of incorporating replicative limits as part of the Luria-Delbrück mutational framework. Guidelines to extend the theory to include other types of mutations and possible applications to the modeling of telomere crisis and fluctuation analysis are also discussed.

ORF4-protein deficient PCV2 mutants enhance virus-induced apoptosis and show differential expression of mRNAs in vitro.

PubMed

Gao, Zhangzhao; Dong, Qinfang; Jiang, Yonghou; Opriessnig, Tanja; Wang, Jingxiu; Quan, Yanping; Yang, Zongqi

2014-04-01

Porcine circovirus type 2 (PCV2) is the essential infectious agent of PCV associated disease (PCVAD). During previous in vitro studies, 11 RNAs and four viral proteins have been detected in PCV2-infected cells. Open reading frame (ORF) 4 is 180bp in length and has been identified at the transcription and the translation level. It overlaps completely with ORF3, which has a role in virus-induced apoptosis. In this study, start codon mutations (M1-PCV2) or in-frame termination mutations (M2-PCV2) were utilized to construct two ORF4-protein deficient viruses aiming to investigate its role in viral infection. The abilities of M1-PCV2 and M2-PCV2 to replicate, transcribe, express viral proteins, and to cause cellular apoptosis were evaluated. Viral DNA replication curves supported that the ORF4 protein is not essential for viral replication, but inhibits viral replication in the early stage of infection. Comparison of the expression level of ORF3 mRNA among wild-type and ORF4-deficient viruses in infected PK-15 cell demonstrated enhanced ORF3 transcription of both ORF4 mutants suggesting that the ORF4 protein may play an important role by restricting ORF3 transcription thereby preventing virus-induced apoptosis. This is further confirmed by the significantly higher caspase 3 and 8 activities in M1-PCV2 and M2-PCV2 compared to wild-type PCV2. Furthermore, the role of ORF4 in cell apoptosis and a possible interaction with the ORF1 associated Rep protein could perhaps explain the rapid viral growth in the early stage of infection and the higher expression level of ORF1 mRNA in ORF4 protein deficient PCV2 mutants. Copyright © 2014 Elsevier B.V. All rights reserved.

Structural basis for DNA binding by replication initiator Mcm10

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warren, Eric M.; Vaithiyalingam, Sivaraja; Haworth, Justin

2009-06-30

Mcm10 is an essential eukaryotic DNA replication protein required for assembly and progression of the replication fork. The highly conserved internal domain (Mcm10-ID) has been shown to physically interact with single-stranded (ss) DNA, DNA polymerase alpha, and proliferating cell nuclear antigen (PCNA). The crystal structure of Xenopus laevis Mcm10-ID presented here reveals a DNA binding architecture composed of an oligonucleotide/oligosaccharide-fold followed in tandem by a variant and highly basic zinc finger. NMR chemical shift perturbation and mutational studies of DNA binding activity in vitro reveal how Mcm10 uses this unique surface to engage ssDNA. Corresponding mutations in Saccharomyces cerevisiae resultmore » in increased sensitivity to replication stress, demonstrating the functional importance of DNA binding by this region of Mcm10 to replication. In addition, mapping Mcm10 mutations known to disrupt PCNA, polymerase alpha, and DNA interactions onto the crystal structure provides insight into how Mcm10 might coordinate protein and DNA binding within the replisome.« less

Replication fork collapse is a major cause of the high mutation frequency at three-base lesion clusters

PubMed Central

Sedletska, Yuliya; Radicella, J. Pablo; Sage, Evelyne

2013-01-01

Unresolved repair of clustered DNA lesions can lead to the formation of deleterious double strand breaks (DSB) or to mutation induction. Here, we investigated the outcome of clusters composed of base lesions for which base excision repair enzymes have different kinetics of excision/incision. We designed multiply damaged sites (MDS) composed of a rapidly excised uracil (U) and two oxidized bases, 5-hydroxyuracil (hU) and 8-oxoguanine (oG), excised more slowly. Plasmids harboring these U-oG/hU MDS-carrying duplexes were introduced into Escherichia coli cells either wild type or deficient for DNA n-glycosylases. Induction of DSB was estimated from plasmid survival and mutagenesis determined by sequencing of surviving clones. We show that a large majority of MDS is converted to DSB, whereas almost all surviving clones are mutated at hU. We demonstrate that mutagenesis at hU is correlated with excision of the U placed on the opposite strand. We propose that excision of U by Ung initiates the loss of U-oG-carrying strand, resulting in enhanced mutagenesis at the lesion present on the opposite strand. Our results highlight the importance of the kinetics of excision by base excision repair DNA n-glycosylases in the processing and fate of MDS and provide evidence for the role of strand loss/replication fork collapse during the processing of MDS on their mutational consequences. PMID:23945941

Mutational signatures reveal the role of RAD52 in p53-independent p21-driven genomic instability.

PubMed

Galanos, Panagiotis; Pappas, George; Polyzos, Alexander; Kotsinas, Athanassios; Svolaki, Ioanna; Giakoumakis, Nickolaos N; Glytsou, Christina; Pateras, Ioannis S; Swain, Umakanta; Souliotis, Vassilis L; Georgakilas, Alexandros G; Geacintov, Nicholas; Scorrano, Luca; Lukas, Claudia; Lukas, Jiri; Livneh, Zvi; Lygerou, Zoi; Chowdhury, Dipanjan; Sørensen, Claus Storgaard; Bartek, Jiri; Gorgoulis, Vassilis G

2018-03-16

Genomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential for the design of appropriate therapeutic strategies. In a previous study, we reported an unexpected oncogenic property of p21 WAF1/Cip1 , showing that its chronic expression in a p53-deficient environment causes genomic instability by deregulation of the replication licensing machinery. We now demonstrate that p21 WAF1/Cip1 can further fuel genomic instability by suppressing the repair capacity of low- and high-fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we find that the DSBs are repaired by Rad52-dependent break-induced replication (BIR) and single-strand annealing (SSA) repair pathways. Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route is deficient. Surprisingly, Rad52 is activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation. Our results signify the importance of mutational signatures as guides to disclose the repair history leading to genomic instability. We unveil how chronic p21 WAF1/Cip1 expression rewires the repair process and identifies Rad52 as a source of genomic instability and a candidate therapeutic target.

Function of the Plant DNA Polymerase Epsilon in Replicative Stress Sensing, a Genetic Analysis.

PubMed

Pedroza-García, José-Antonio; Mazubert, Christelle; Del Olmo, Ivan; Bourge, Mickael; Domenichini, Séverine; Bounon, Rémi; Tariq, Zakia; Delannoy, Etienne; Piñeiro, Manuel; Jarillo, José A; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

2017-03-01

Faithful transmission of the genetic information is essential in all living organisms. DNA replication is therefore a critical step of cell proliferation, because of the potential occurrence of replication errors or DNA damage when progression of a replication fork is hampered causing replicative stress. Like other types of DNA damage, replicative stress activates the DNA damage response, a signaling cascade allowing cell cycle arrest and repair of lesions. The replicative DNA polymerase ε (Pol ε) was shown to activate the S-phase checkpoint in yeast in response to replicative stress, but whether this mechanism functions in multicellular eukaryotes remains unclear. Here, we explored the genetic interaction between Pol ε and the main elements of the DNA damage response in Arabidopsis ( Arabidopsis thaliana ). We found that mutations affecting the polymerase domain of Pol ε trigger ATR-dependent signaling leading to SOG1 activation, WEE1-dependent cell cycle inhibition, and tolerance to replicative stress induced by hydroxyurea, but result in enhanced sensitivity to a wide range of DNA damaging agents. Using knock-down lines, we also provide evidence for the direct role of Pol ε in replicative stress sensing. Together, our results demonstrate that the role of Pol ε in replicative stress sensing is conserved in plants, and provide, to our knowledge, the first genetic dissection of the downstream signaling events in a multicellular eukaryote. © 2017 American Society of Plant Biologists. All Rights Reserved.

The topography of mutational processes in breast cancer genomes.

PubMed

Morganella, Sandro; Alexandrov, Ludmil B; Glodzik, Dominik; Zou, Xueqing; Davies, Helen; Staaf, Johan; Sieuwerts, Anieta M; Brinkman, Arie B; Martin, Sancha; Ramakrishna, Manasa; Butler, Adam; Kim, Hyung-Yong; Borg, Åke; Sotiriou, Christos; Futreal, P Andrew; Campbell, Peter J; Span, Paul N; Van Laere, Steven; Lakhani, Sunil R; Eyfjord, Jorunn E; Thompson, Alastair M; Stunnenberg, Hendrik G; van de Vijver, Marc J; Martens, John W M; Børresen-Dale, Anne-Lise; Richardson, Andrea L; Kong, Gu; Thomas, Gilles; Sale, Julian; Rada, Cristina; Stratton, Michael R; Birney, Ewan; Nik-Zainal, Serena

2016-05-02

Somatic mutations in human cancers show unevenness in genomic distribution that correlate with aspects of genome structure and function. These mutations are, however, generated by multiple mutational processes operating through the cellular lineage between the fertilized egg and the cancer cell, each composed of specific DNA damage and repair components and leaving its own characteristic mutational signature on the genome. Using somatic mutation catalogues from 560 breast cancer whole-genome sequences, here we show that each of 12 base substitution, 2 insertion/deletion (indel) and 6 rearrangement mutational signatures present in breast tissue, exhibit distinct relationships with genomic features relating to transcription, DNA replication and chromatin organization. This signature-based approach permits visualization of the genomic distribution of mutational processes associated with APOBEC enzymes, mismatch repair deficiency and homologous recombinational repair deficiency, as well as mutational processes of unknown aetiology. Furthermore, it highlights mechanistic insights including a putative replication-dependent mechanism of APOBEC-related mutagenesis.

Mechanisms of viral mutation.

PubMed

Sanjuán, Rafael; Domingo-Calap, Pilar

2016-12-01

The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be introduced by some virus-encoded diversity-generating elements, as well as by host-encoded cytidine/adenine deaminases. Our current knowledge of viral mutation rates indicates that viral genetic diversity is determined by multiple virus- and host-dependent processes, and that viral mutation rates can evolve in response to specific selective pressures.

PB2 mutations D701N and S714R promote adaptation of an influenza H5N1 virus to a mammalian host.

PubMed

Czudai-Matwich, Volker; Otte, Anna; Matrosovich, Mikhail; Gabriel, Gülsah; Klenk, Hans-Dieter

2014-08-01

Mutation D701N in the PB2 protein is known to play a prominent role in the adaptation of avian influenza A viruses to mammalian hosts. In contrast, little is known about the nearby mutations S714I and S714R, which have been observed in some avian influenza viruses highly pathogenic for mammals. We have generated recombinant H5N1 viruses with PB2 displaying the avian signature 701D or the mammalian signature 701N and serine, isoleucine, and arginine at position 714 and compared them for polymerase activity and virus growth in avian and mammalian cells, as well as for pathogenicity in mice. Mutation D701N led to an increase in polymerase activity and replication efficiency in mammalian cells and in mouse pathogenicity, and this increase was significantly enhanced when mutation D701N was combined with mutation S714R. Stimulation by mutation S714I was less distinct. These observations indicate that PB2 mutation S714R, in combination with the mammalian signature at position 701, has the potential to promote the adaptation of an H5N1 virus to a mammalian host. Influenza A/H5N1 viruses are avian pathogens that have pandemic potential, since they are spread over large parts of Asia, Africa, and Europe and are occasionally transmitted to humans. It is therefore of high scientific interest to understand the mechanisms that determine the host specificity and pathogenicity of these viruses. It is well known that the PB2 subunit of the viral polymerase is an important host range determinant and that PB2 mutation D701N plays an important role in virus adaptation to mammalian cells. In the present study, we show that mutation S714R is also involved in adaptation and that it cooperates with D701N in exposing a nuclear localization signal that mediates importin-α binding and entry of PB2 into the nucleus, where virus replication and transcription take place. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies

PubMed Central

Bratic, Ana; Kauppila, Timo E. S.; Macao, Bertil; Grönke, Sebastian; Siibak, Triinu; Stewart, James B.; Baggio, Francesca; Dols, Jacqueline; Partridge, Linda; Falkenberg, Maria; Wredenberg, Anna; Larsson, Nils-Göran

2015-01-01

Replication errors are the main cause of mitochondrial DNA (mtDNA) mutations and a compelling approach to decrease mutation levels would therefore be to increase the fidelity of the catalytic subunit (POLγA) of the mtDNA polymerase. Here we genomically engineer the tamas locus, encoding fly POLγA, and introduce alleles expressing exonuclease- (exo−) and polymerase-deficient (pol−) POLγA versions. The exo− mutant leads to accumulation of point mutations and linear deletions of mtDNA, whereas pol− mutants cause mtDNA depletion. The mutant tamas alleles are developmentally lethal but can complement each other in trans resulting in viable flies with clonally expanded mtDNA mutations. Reconstitution of human mtDNA replication in vitro confirms that replication is a highly dynamic process where POLγA goes on and off the template to allow complementation during proofreading and elongation. The created fly models are valuable tools to study germ line transmission of mtDNA and the pathophysiology of POLγA mutation disease. PMID:26554610

Spontaneous Chloroplast Mutants Mostly Occur by Replication Slippage and Show a Biased Pattern in the Plastome of Oenothera.

PubMed

Massouh, Amid; Schubert, Julia; Yaneva-Roder, Liliya; Ulbricht-Jones, Elena S; Zupok, Arkadiusz; Johnson, Marc T J; Wright, Stephen I; Pellizzer, Tommaso; Sobanski, Johanna; Bock, Ralph; Greiner, Stephan

2016-04-01

Spontaneous plastome mutants have been used as a research tool since the beginning of genetics. However, technical restrictions have severely limited their contributions to research in physiology and molecular biology. Here, we used full plastome sequencing to systematically characterize a collection of 51 spontaneous chloroplast mutants in Oenothera (evening primrose). Most mutants carry only a single mutation. Unexpectedly, the vast majority of mutations do not represent single nucleotide polymorphisms but are insertions/deletions originating from DNA replication slippage events. Only very few mutations appear to be caused by imprecise double-strand break repair, nucleotide misincorporation during replication, or incorrect nucleotide excision repair following oxidative damage. U-turn inversions were not detected. Replication slippage is induced at repetitive sequences that can be very small and tend to have high A/T content. Interestingly, the mutations are not distributed randomly in the genome. The underrepresentation of mutations caused by faulty double-strand break repair might explain the high structural conservation of seed plant plastomes throughout evolution. In addition to providing a fully characterized mutant collection for future research on plastid genetics, gene expression, and photosynthesis, our work identified the spectrum of spontaneous mutations in plastids and reveals that this spectrum is very different from that in the nucleus. © 2016 American Society of Plant Biologists. All rights reserved.

The PA and HA Gene-Mediated High Viral Load and Intense Innate Immune Response in the Brain Contribute to the High Pathogenicity of H5N1 Avian Influenza Virus in Mallard Ducks

PubMed Central

Hu, Jiao; Hu, Zenglei; Mo, Yiqun; Wu, Qiwen; Cui, Zhu; Duan, Zhiqiang; Huang, Junqing; Chen, Hongzhi; Chen, Yuxin; Gu, Min; Wang, Xiaoquan; Hu, Shunlin; Liu, Huimou; Liu, Wenbo; Liu, Xiaowen

2013-01-01

Most highly pathogenic avian influenza A viruses cause only mild clinical signs in ducks, serving as an important natural reservoir of influenza A viruses. However, we isolated two H5N1 viruses that are genetically similar but differ greatly in virulence in ducks. A/Chicken/Jiangsu/k0402/2010 (CK10) is highly pathogenic, whereas A/Goose/Jiangsu/k0403/2010 (GS10) is low pathogenic. To determine the genetic basis for the high virulence of CK10 in ducks, we generated a series of single-gene reassortants between CK10 and GS10 and tested their virulence in ducks. Expression of the CK10 PA or hemagglutinin (HA) gene in the GS10 context resulted in increased virulence and virus replication. Conversely, inclusion of the GS10 PA or HA gene in the CK10 background attenuated the virulence and virus replication. Moreover, the PA gene had a greater contribution. We further determined that residues 101G and 237E in the PA gene contribute to the high virulence of CK10. Mutations at these two positions produced changes in virulence, virus replication, and polymerase activity of CK10 or GS10. Position 237 plays a greater role in determining these phenotypes. Moreover, the K237E mutation in the GS10 PA gene increased PA nuclear accumulation. Mutant GS10 viruses carrying the CK10 HA gene or the PA101G or PA237E mutation induced an enhanced innate immune response. A sustained innate response was detected in the brain rather than in the lung and spleen. Our results suggest that the PA and HA gene-mediated high virus replication and the intense innate immune response in the brain contribute to the high virulence of H5N1 virus in ducks. PMID:23926340

Substitution of D701N in the PB2 protein could enhance the viral replication and pathogenicity of Eurasian avian-like H1N1 swine influenza viruses.

PubMed

Liu, Suli; Zhu, Wenfei; Feng, Zhaomin; Gao, Rongbao; Guo, Junfeng; Li, Xiyan; Liu, Jia; Wang, Dayan; Shu, Yuelong

2018-05-02

Eurasian avian-like H1N1 (EA H1N1) swine influenza viruses (SIVs) have become predominant in pig populations in China and have recently been reported to have the most potential to raise the next pandemic in humans. The mutation D701N in the PB2 protein, which accounts for 31% of H1N1 SIVs, has previously been shown to contribute to the adaptation of the highly pathogenic H5N1 or H7N7 avian influenza viruses in mammals. However, little is known of the effects of this substitution on the EA H1N1 viruses. Herein, we investigated the contributions of 701N in the PB2 protein to an EA H1N1 SIV (A/Hunan/42443/2015(H1N1), HuN EA-H1N1), which had 701D in the PB2 protein. Our results found that viral polymerase activity, viral replication, and pathogenicity in mice were indeed enhanced due to the introduction of 701N into the PB2 protein, and the increased viral growth was partly mediated by the host factor importin-α7. Thus, substantial attention should be paid to the D701N mutation in pig populations.

Links between DNA Replication, Stem Cells and Cancer

PubMed Central

Vassilev, Alex; DePamphilis, Melvin L.

2017-01-01

Cancers can be categorized into two groups: those whose frequency increases with age, and those resulting from errors during mammalian development. The first group is linked to DNA replication through the accumulation of genetic mutations that occur during proliferation of developmentally acquired stem cells that give rise to and maintain tissues and organs. These mutations, which result from DNA replication errors as well as environmental insults, fall into two categories; cancer driver mutations that initiate carcinogenesis and genome destabilizing mutations that promote aneuploidy through excess genome duplication and chromatid missegregation. Increased genome instability results in accelerated clonal evolution leading to the appearance of more aggressive clones with increased drug resistance. The second group of cancers, termed germ cell neoplasia, results from the mislocation of pluripotent stem cells during early development. During normal development, pluripotent stem cells that originate in early embryos give rise to all of the cell lineages in the embryo and adult, but when they mislocate to ectopic sites, they produce tumors. Remarkably, pluripotent stem cells, like many cancer cells, depend on the Geminin protein to prevent excess DNA replication from triggering DNA damage-dependent apoptosis. This link between the control of DNA replication during early development and germ cell neoplasia reveals Geminin as a potential chemotherapeutic target in the eradication of cancer progenitor cells. PMID:28125050

Mutations that alter a repeated ACCA element located at the 5' end of the Potato virus X genome affect RNA accumulation.

PubMed

Park, Mi-Ri; Kwon, Sun-Jung; Choi, Hong-Soo; Hemenway, Cynthia L; Kim, Kook-Hyung

2008-08-15

The repeated ACCA or AC-rich sequence and structural (SL1) elements in the 5' non-translated region (NTR) of the Potato virus X (PVX) RNA play vital roles in the PVX life cycle by controlling translation, RNA replication, movement, and assembly. It has already been shown that the repeated ACCA or AC-rich sequence affect both gRNA and sgRNA accumulation, while not affecting minus-strand RNA accumulation, and are also required for host protein binding. The functional significance of the repeated ACCA sequence elements in the 5' NTR region was investigated by analyzing the effects of deletion and site-directed mutations on PVX replication in Nicotiana benthamiana plants and NT1 protoplasts. Substitution (ACCA into AAAA or UUUU) mutations introduced in the first (nt 10-13) element in the 5' NTR of the PVX RNA significantly affected viral replication, while mutations introduced in the second (nt 17-20) and third (nt 20-23) elements did not. The fourth (nt 29-32) ACCA element weakly affected virus replication, whereas mutations in the fifth (nt 38-41) significantly reduced virus replication due to the structure disruption of SL1 by AAAA and/or UUUU substitutions. Further characterization of the first ACCA element indicated that duplication of ACCA at nt 10-13 (nt 10-17, ACCAACCA) caused severe symptom development as compared to that of wild type, while deletion of the single element (nt 10-13), DeltaACCA) or tripling of this element caused reduced symptom development. Single- and double-nucleotide substitutions introduced into the first ACCA element revealed the importance of CC located at nt positions 11 and 12. Altogether, these results indicate that the first ACCA element is important for PVX replication.

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

PubMed Central

Ayyagari, R; Impellizzeri, K J; Yoder, B L; Gary, S L; Burgers, P M

1995-01-01

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication. PMID:7623835

Rates of spontaneous mutation among RNA viruses.

PubMed Central

Drake, J W

1993-01-01

Simple methods are presented to estimate rates of spontaneous mutation from mutant frequencies and population parameters in RNA viruses. Published mutant frequencies yield a wide range of mutation rates per genome per replication, mainly because mutational targets have usually been small and, thus, poor samples of the mutability of the average base. Nevertheless, there is a clear central tendency for lytic RNA viruses (bacteriophage Q beta, poliomyelitis, vesicular stomatitis, and influenza A) to display rates of spontaneous mutation of approximately 1 per genome per replication. This rate is some 300-fold higher than previously reported for DNA-based microbes. Lytic RNA viruses thus mutate at a rate close to the maximum value compatible with viability. Retroviruses (spleen necrosis, murine leukemia, Rous sarcoma), however, mutate at an average rate about an order of magnitude lower than lytic RNA viruses. PMID:8387212

CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells

PubMed Central

Russo, Giorgio; Corradi, Francesca; Siteni, Silvia; Musella, Martina; Vitale, Sara; De Angelis, Maria Laura; Pallocca, Matteo; Amoreo, Carla Azzurra; Sperati, Francesca; Di Franco, Simone; Barresi, Sabina; Policicchio, Eleonora; De Luca, Gabriele; De Nicola, Francesca; Mottolese, Marcella; Zeuner, Ann; Fanciulli, Maurizio; Stassi, Giorgio; Maugeri-Saccà, Marcello; Baiocchi, Marta; Tartaglia, Marco

2018-01-01

Objective Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies. Design To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents. Results The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368. Conclusions LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC. PMID:28389531

Effects of Mutations on Replicative Fitness and Major Histocompatibility Complex Class I Binding Affinity Are Among the Determinants Underlying Cytotoxic-T-Lymphocyte Escape of HIV-1 Gag Epitopes.

PubMed

Du, Yushen; Zhang, Tian-Hao; Dai, Lei; Zheng, Xiaojuan; Gorin, Aleksandr M; Oishi, John; Wu, Ting-Ting; Yoshizawa, Janice M; Li, Xinmin; Yang, Otto O; Martinez-Maza, Otoniel; Detels, Roger; Sun, Ren

2017-11-28

Certain "protective" major histocompatibility complex class I (MHC-I) alleles, such as B*57 and B*27, are associated with long-term control of HIV-1 in vivo mediated by the CD8 + cytotoxic-T-lymphocyte (CTL) response. However, the mechanism of such superior protection is not fully understood. Here we combined high-throughput fitness profiling of mutations in HIV-1 Gag, in silico prediction of MHC-peptide binding affinity, and analysis of intraperson virus evolution to systematically compare differences with respect to CTL escape mutations between epitopes targeted by protective MHC-I alleles and those targeted by nonprotective MHC-I alleles. We observed that the effects of mutations on both viral replication and MHC-I binding affinity are among the determinants of CTL escape. Mutations in Gag epitopes presented by protective MHC-I alleles are associated with significantly higher fitness cost and lower reductions in binding affinity with respect to MHC-I. A linear regression model accounting for the effect of mutations on both viral replicative capacity and MHC-I binding can explain the protective efficacy of MHC-I alleles. Finally, we found a consistent pattern in the evolution of Gag epitopes in long-term nonprogressors versus progressors. Overall, our results suggest that certain protective MHC-I alleles allow superior control of HIV-1 by targeting epitopes where mutations typically incur high fitness costs and small reductions in MHC-I binding affinity. IMPORTANCE Understanding the mechanism of viral control achieved in long-term nonprogressors with protective HLA alleles provides insights for developing functional cure of HIV infection. Through the characterization of CTL escape mutations in infected persons, previous researchers hypothesized that protective alleles target epitopes where escape mutations significantly reduce viral replicative capacity. However, these studies were usually limited to a few mutations observed in vivo Here we utilized our recently developed high-throughput fitness profiling method to quantitatively measure the fitness of mutations across the entirety of HIV-1 Gag. The data enabled us to integrate the results with in silico prediction of MHC-peptide binding affinity and analysis of intraperson virus evolution to systematically determine the differences in CTL escape mutations between epitopes targeted by protective HLA alleles and those targeted by nonprotective HLA alleles. We observed that the effects of Gag epitope mutations on HIV replicative fitness and MHC-I binding affinity are among the major determinants of CTL escape. Copyright © 2017 Du et al.

Electron holes appear to trigger cancer-implicated mutations

NASA Astrophysics Data System (ADS)

Miller, John; Villagran, Martha

Malignant tumors are caused by mutations, which also affect their subsequent growth and evolution. We use a novel approach, computational DNA hole spectroscopy [M.Y. Suarez-Villagran & J.H. Miller, Sci. Rep. 5, 13571 (2015)], to compute spectra of enhanced hole probability based on actual sequence data. A hole is a mobile site of positive charge created when an electron is removed, for example by radiation or contact with a mutagenic agent. Peaks in the hole spectrum depict sites where holes tend to localize and potentially trigger a base pair mismatch during replication. Our studies of reveal a correlation between hole spectrum peaks and spikes in human mutation frequencies. Importantly, we also find that hole peak positions that do not coincide with large variant frequencies often coincide with cancer-implicated mutations and/or (for coding DNA) encoded conserved amino acids. This enables combining hole spectra with variant data to identify critical base pairs and potential cancer `driver' mutations. Such integration of DNA hole and variance spectra could also prove invaluable for pinpointing critical regions, and sites of driver mutations, in the vast non-protein-coding genome. Supported by the State of Texas through the Texas Ctr. for Superconductivity.

De Novo GMNN Mutations Cause Autosomal-Dominant Primordial Dwarfism Associated with Meier-Gorlin Syndrome.

PubMed

Burrage, Lindsay C; Charng, Wu-Lin; Eldomery, Mohammad K; Willer, Jason R; Davis, Erica E; Lugtenberg, Dorien; Zhu, Wenmiao; Leduc, Magalie S; Akdemir, Zeynep C; Azamian, Mahshid; Zapata, Gladys; Hernandez, Patricia P; Schoots, Jeroen; de Munnik, Sonja A; Roepman, Ronald; Pearring, Jillian N; Jhangiani, Shalini; Katsanis, Nicholas; Vissers, Lisenka E L M; Brunner, Han G; Beaudet, Arthur L; Rosenfeld, Jill A; Muzny, Donna M; Gibbs, Richard A; Eng, Christine M; Xia, Fan; Lalani, Seema R; Lupski, James R; Bongers, Ernie M H F; Yang, Yaping

2015-12-03

Meier-Gorlin syndrome (MGS) is a genetically heterogeneous primordial dwarfism syndrome known to be caused by biallelic loss-of-function mutations in one of five genes encoding pre-replication complex proteins: ORC1, ORC4, ORC6, CDT1, and CDC6. Mutations in these genes cause disruption of the origin of DNA replication initiation. To date, only an autosomal-recessive inheritance pattern has been described in individuals with this disorder, with a molecular etiology established in about three-fourths of cases. Here, we report three subjects with MGS and de novo heterozygous mutations in the 5' end of GMNN, encoding the DNA replication inhibitor geminin. We identified two truncating mutations in exon 2 (the 1(st) coding exon), c.16A>T (p.Lys6(∗)) and c.35_38delTCAA (p.Ile12Lysfs(∗)4), and one missense mutation, c.50A>G (p.Lys17Arg), affecting the second-to-last nucleotide of exon 2 and possibly RNA splicing. Geminin is present during the S, G2, and M phases of the cell cycle and is degraded during the metaphase-anaphase transition by the anaphase-promoting complex (APC), which recognizes the destruction box sequence near the 5' end of the geminin protein. All three GMNN mutations identified alter sites 5' to residue Met28 of the protein, which is located within the destruction box. We present data supporting a gain-of-function mechanism, in which the GMNN mutations result in proteins lacking the destruction box and hence increased protein stability and prolonged inhibition of replication leading to autosomal-dominant MGS. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

De Novo GMNN Mutations Cause Autosomal-Dominant Primordial Dwarfism Associated with Meier-Gorlin Syndrome

PubMed Central

Burrage, Lindsay C.; Charng, Wu-Lin; Eldomery, Mohammad K.; Willer, Jason R.; Davis, Erica E.; Lugtenberg, Dorien; Zhu, Wenmiao; Leduc, Magalie S.; Akdemir, Zeynep C.; Azamian, Mahshid; Zapata, Gladys; Hernandez, Patricia P.; Schoots, Jeroen; de Munnik, Sonja A.; Roepman, Ronald; Pearring, Jillian N.; Jhangiani, Shalini; Katsanis, Nicholas; Vissers, Lisenka E.L.M.; Brunner, Han G.; Beaudet, Arthur L.; Rosenfeld, Jill A.; Muzny, Donna M.; Gibbs, Richard A.; Eng, Christine M.; Xia, Fan; Lalani, Seema R.; Lupski, James R.; Bongers, Ernie M.H.F.; Yang, Yaping

2015-01-01

Meier-Gorlin syndrome (MGS) is a genetically heterogeneous primordial dwarfism syndrome known to be caused by biallelic loss-of-function mutations in one of five genes encoding pre-replication complex proteins: ORC1, ORC4, ORC6, CDT1, and CDC6. Mutations in these genes cause disruption of the origin of DNA replication initiation. To date, only an autosomal-recessive inheritance pattern has been described in individuals with this disorder, with a molecular etiology established in about three-fourths of cases. Here, we report three subjects with MGS and de novo heterozygous mutations in the 5′ end of GMNN, encoding the DNA replication inhibitor geminin. We identified two truncating mutations in exon 2 (the 1st coding exon), c.16A>T (p.Lys6∗) and c.35_38delTCAA (p.Ile12Lysfs∗4), and one missense mutation, c.50A>G (p.Lys17Arg), affecting the second-to-last nucleotide of exon 2 and possibly RNA splicing. Geminin is present during the S, G2, and M phases of the cell cycle and is degraded during the metaphase-anaphase transition by the anaphase-promoting complex (APC), which recognizes the destruction box sequence near the 5′ end of the geminin protein. All three GMNN mutations identified alter sites 5′ to residue Met28 of the protein, which is located within the destruction box. We present data supporting a gain-of-function mechanism, in which the GMNN mutations result in proteins lacking the destruction box and hence increased protein stability and prolonged inhibition of replication leading to autosomal-dominant MGS. PMID:26637980

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

PubMed Central

Tuplin, A.; Evans, D. J.; Buckley, A.; Jones, I. M.; Gould, E. A.; Gritsun, T. S.

2011-01-01

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents. PMID:21622960

Male Mutation Bias Is the Main Force Shaping Chromosomal Substitution Rates in Monotreme Mammals

PubMed Central

Link, Vivian; Aguilar-Gómez, Diana; Ramírez-Suástegui, Ciro; Hurst, Laurence D.

2017-01-01

Abstract In many species, spermatogenesis involves more cell divisions than oogenesis, and the male germline, therefore, accumulates more DNA replication errors, a phenomenon known as male mutation bias. The extent of male mutation bias (α) is estimated by comparing substitution rates of the X, Y, and autosomal chromosomes, as these chromosomes spend different proportions of their time in the germlines of the two sexes. Male mutation bias has been characterized in placental and marsupial mammals as well as birds, but analyses in monotremes failed to detect any such bias. Monotremes are an ancient lineage of egg-laying mammals with distinct biological properties, which include unique germline features. Here, we sought to assess the presence and potential characteristics of male mutation bias in platypus and the short-beaked echidna based on substitution rate analyses of X, Y, and autosomes. We established the presence of moderate male mutation bias in monotremes, corresponding to an α value of 2.12–3.69. Given that it has been unclear what proportion of the variation in substitution rates on the different chromosomal classes is really due to differential number of replications, we analyzed the influence of other confounding forces (selection, replication-timing, etc.) and found that male mutation bias is the main force explaining the between-chromosome classes differences in substitution rates. Finally, we estimated the proportion of variation at the gene level in substitution rates that is owing to replication effects and found that this phenomenon can explain >68% of these variations in monotremes, and in control species, rodents, and primates. PMID:28922870

DNA replication stress and cancer: cause or cure?

PubMed

Taylor, Elaine M; Lindsay, Howard D

2016-01-01

There is an extensive and growing body of evidence that DNA replication stress is a major driver in the development and progression of many cancers, and that these cancers rely heavily on replication stress response pathways for their continued proliferation. This raises the possibility that the pathways that ordinarily protect cells from the accumulation of cancer-causing mutations may actually prove to be effective therapeutic targets for a wide range of malignancies. In this review, we explore the mechanisms by which sustained proliferation can lead to replication stress and genome instability, and discuss how the pattern of mutations observed in human cancers is supportive of this oncogene-induced replication stress model. Finally, we go on to consider the implications of replication stress both as a prognostic indicator and, more encouragingly, as a potential target in cancer treatment.

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism.

PubMed

Harley, Margaret E; Murina, Olga; Leitch, Andrea; Higgs, Martin R; Bicknell, Louise S; Yigit, Gökhan; Blackford, Andrew N; Zlatanou, Anastasia; Mackenzie, Karen J; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A M; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B; Nürnberg, Peter; Jackson, Stephen P; Hurles, Matthew E; Wollnik, Bernd; Stewart, Grant S; Jackson, Andrew P

2016-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

A Single Amino Acid Change in the Marburg Virus Matrix Protein VP40 Provides a Replicative Advantage in a Species-Specific Manner

PubMed Central

Koehler, Alexander; Kolesnikova, Larissa; Welzel, Ulla; Schudt, Gordian; Herwig, Astrid

2015-01-01

ABSTRACT Marburg virus (MARV) induces severe hemorrhagic fever in humans and nonhuman primates but only transient nonlethal disease in rodents. However, sequential passages of MARV in rodents boosts infection leading to lethal disease. Guinea pig-adapted MARV contains one mutation in the viral matrix protein VP40 at position 184 (VP40D184N). The contribution of the D184N mutation to the efficacy of replication in a new host is unknown. In the present study, we demonstrated that recombinant MARV containing the D184N mutation in VP40 [rMARVVP40(D184N)] grew to higher titers than wild-type recombinant MARV (rMARVWT) in guinea pig cells. Moreover, rMARVVP40(D184N) displayed higher infectivity in guinea pig cells. Comparative analysis of VP40 functions indicated that neither the interferon (IFN)-antagonistic function nor the membrane binding capabilities of VP40 were affected by the D184N mutation. However, the production of VP40-induced virus-like particles (VLPs) and the recruitment of other viral proteins to the budding site was improved by the D184N mutation in guinea pig cells, which resulted in the higher infectivity of VP40D184N-induced infectious VLPs (iVLPs) compared to that of VP40-induced iVLPs. In addition, the function of VP40 in suppressing viral RNA synthesis was influenced by the D184N mutation specifically in guinea pig cells, thus allowing greater rates of transcription and replication. Our results showed that the improved viral fitness of rMARVVP40(D184N) in guinea pig cells was due to the better viral assembly function of VP40D184N and its lower inhibitory effect on viral transcription and replication rather than modulation of the VP40-mediated suppression of IFN signaling. IMPORTANCE The increased virulence achieved by virus passaging in a new host was accompanied by mutations in the viral genome. Analyzing how these mutations affect the functions of viral proteins and the ability of the virus to grow within new host cells helps in the understanding of the molecular mechanisms increasing virulence. Using a reverse genetics approach, we demonstrated that a single mutation in MARV VP40 detected in a guinea pig-adapted MARV provided a replicative advantage of rMARVVP40(D184N) in guinea pig cells. Our studies show that this replicative advantage of rMARV VP40D184N was based on the improved functions of VP40 in iVLP assembly and in the regulation of transcription and replication rather than on the ability of VP40 to combat the host innate immunity. PMID:26581998

Influenza A Virus Polymerase Is a Site for Adaptive Changes during Experimental Evolution in Bat Cells

PubMed Central

Poole, Daniel S.; Yú, Shuǐqìng; Caì, Yíngyún; Dinis, Jorge M.; Müller, Marcel A.; Jordan, Ingo; Friedrich, Thomas C.; Kuhn, Jens H.

2014-01-01

ABSTRACT The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus with the PA M285K mutation completely phenocopied the adapted virus. Adaptation of an avian virus-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the gene for viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel mutation and a well-known adaptive mutation in the viral polymerase to support replication. IMPORTANCE Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigated the replication of human influenza A virus in bat cell lines and the barriers that the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and evolutionarily diverse New and Old World bats. Viruses mutated during infections in bat cells, resulting in increased replication and cytopathic effects. These mutations were mapped to the viral polymerase and shown to be solely responsible for adaptation to bat cells. Our data suggest that replication of human influenza A viruses in a nonnative host drives the evolution of new variants and may be an important source of genetic diversity. PMID:25142579

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader.

PubMed

Rock, Jeremy M; Lang, Ulla F; Chase, Michael R; Ford, Christopher B; Gerrick, Elias R; Gawande, Richa; Coscolla, Mireia; Gagneux, Sebastien; Fortune, Sarah M; Lamers, Meindert H

2015-06-01

The DNA replication machinery is an important target for antibiotic development in increasingly drug-resistant bacteria, including Mycobacterium tuberculosis. Although blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In Escherichia coli, the proofreading subunit of the replisome, the ɛ exonuclease, is essential for high-fidelity DNA replication; however, we find that the corresponding subunit is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase DnaE1 itself encodes an editing function that proofreads DNA replication, mediated by an intrinsic 3'-5' exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by more than 3,000-fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP domain-mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader.

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader

PubMed Central

Rock, Jeremy M.; Lang, Ulla F.; Chase, Michael R.; Ford, Christopher B.; Gerrick, Elias R.; Gawande, Richa; Coscolla, Mireia; Gagneux, Sebastien; Fortune, Sarah M.; Lamers, Meindert H.

2015-01-01

The DNA replication machinery is an important target for antibiotic development for increasingly drug resistant bacteria including Mycobacterium tuberculosis1. While blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In E. coli, the proofreading subunit of the replisome, the ε-exonuclease, is essential for high fidelity DNA replication2; however, we find that it is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase, DnaE1, encodes a novel editing function that proofreads DNA replication, mediated by an intrinsic 3′-5′ exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by greater than 3,000 fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP-domain mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader. PMID:25894501

The topography of mutational processes in breast cancer genomes

DOE PAGES

Morganella, Sandro; Alexandrov, Ludmil B.; Glodzik, Dominik; ...

2016-01-01

Somatic mutations in human cancers show unevenness in genomic distribution that correlate with aspects of genome structure and function. These mutations are, however, generated by multiple mutational processes operating through the cellular lineage between the fertilized egg and the cancer cell, each composed of specific DNA damage and repair components and leaving its own characteristic mutational signature on the genome. Using somatic mutation catalogues from 560 breast cancer whole-genome sequences, here we show that each of 12 base substitution, 2 insertion/deletion (indel) and 6 rearrangement mutational signatures present in breast tissue, exhibit distinct relationships with genomic features relating to transcription,more » DNA replication and chromatin organization. This signature-based approach permits visualization of the genomic distribution of mutational processes associated with APOBEC enzymes, mismatch repair deficiency and homologous recombinational repair deficiency, as well as mutational processes of unknown aetiology. Lastly, it highlights mechanistic insights including a putative replication-dependent mechanism of APOBEC-related mutagenesis.« less

Resistance and cross-resistance profile of the diaryltriazine NNRTI and candidate microbicide UAMC01398.

PubMed

Ariën, Kevin K; Venkatraj, Muthusamy; Michiels, Johan; Joossens, Jurgen; Vereecken, Katleen; Van der Veken, Pieter; Heeres, Jan; De Winter, Hans; Heyndrickx, Leo; Augustyns, Koen; Vanham, Guido

2016-05-01

The resistance development, cross-resistance to other NNRTIs and the impact of resistance on viral replicative fitness were studied for the new and potent NNRTI UAMC01398. Resistance was selected by dose escalation and by single high-dose selection against a comprehensive panel of NNRTIs used as therapeutics and NNRTIs under investigation for pre-exposure prophylaxis of sexual HIV transmission. A panel of 27 site-directed mutants with single mutations or combinations of mutations involved in reverse transcriptase (RT) inhibitor-mediated resistance was developed and used to confirm resistance to UAMC01398. Cross-resistance to other NNRTIs was assessed, as well as susceptibility of UAMC01398-resistant HIV to diarylpyrimidine-resistant viruses. Finally, the impact of UAMC01398 resistance on HIV replicative fitness was studied. We showed that UAMC01398 has potent activity against dapivirine-resistant HIV, that at least four mutations in the RT are required in concert for resistance and that the resistance profile is similar to rilpivirine, both genotypically and phenotypically. Resistance development to UAMC01398 is associated with a severe fitness cost. These data, together with the enhanced safety profile and good solubility in aqueous gels, make UAMC01398 an excellent candidate for HIV topical prevention. © The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

CEP152 is a genome maintenance protein disrupted in Seckel syndrome

PubMed Central

Kalay, Ersan; Yigit, Gökhan; Aslan, Yakup; Brown, Karen E; Pohl, Esther; Bicknell, Louise S; Kayserili, Hülya; Li, Yun; Tüysüz, Beyhan; Nürnberg, Gudrun; Kiess, Wieland; Koegl, Manfred; Baessmann, Ingelore; Buruk, Kurtulus; Toraman, Bayram; Kayipmaz, Saadettin; Kul, Sibel; Ikbal, Mevlit; Turner, Daniel J; Taylor, Martin S; Aerts, Jan; Scott, Carol; Milstein, Karen; Dollfus, Helene; Wieczorek, Dagmar; Brunner, Han G; Hurles, Matthew; Jackson, Andrew P; Rauch, Anita; Nürnberg, Peter; Karagüzel, Ahmet; Wollnik, Bernd

2012-01-01

Functional impairment of DNA damage response pathways leads to increased genomic instability. Here we describe the centrosomal protein CEP152 as a new regulator of genomic integrity and cellular response to DNA damage. Using homozygosity mapping and exome sequencing, we identified CEP152 mutations in Seckel syndrome and showed that impaired CEP152 function leads to accumulation of genomic defects resulting from replicative stress through enhanced activation of ATM signaling and increased H2AX phosphorylation. PMID:21131973

Drosophila model of Meier-Gorlin syndrome based on the mutation in a conserved C-Terminal domain of Orc6.

PubMed

Balasov, Maxim; Akhmetova, Katarina; Chesnokov, Igor

2015-11-01

Meier-Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, primordial dwarfism, small ears, and skeletal abnormalities. Patients with MGS often carry mutations in the genes encoding the components of the pre-replicative complex such as Origin Recognition Complex (ORC) subunits Orc1, Orc4, Orc6, and helicase loaders Cdt1 and Cdc6. Orc6 is an important component of ORC and has functions in both DNA replication and cytokinesis. Mutation in conserved C-terminal motif of Orc6 associated with MGS impedes the interaction of Orc6 with core ORC. In order to study the effects of MGS mutation in an animal model system we introduced MGS mutation in Orc6 and established Drosophila model of MGS. Mutant flies die at third instar larval stage with abnormal chromosomes and DNA replication defects. The lethality can be rescued by elevated expression of mutant Orc6 protein. Rescued MGS flies are unable to fly and display multiple planar cell polarity defects. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seetharam, S.; Protic-Sabljic, M.; Seidman, M.M.

1987-12-01

A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher frequency of plasmids with mutations, fewer plasmids with two or more mutations in the marker gene, and a new mutagenic hotspot. The major type of base substitution mutation was the G:C tomore » A:T transition with both cell lines. These results, together with similar findings published earlier with cells from a xeroderma pigmentosum patient in complementation group A, suggest that isolated G:C to A:T somatic mutations may be particularly important in generation of human skin cancer by UV radiation.« less

Impact of human immunodeficiency virus type 1 resistance to protease inhibitors on evolution of resistance to the maturation inhibitor bevirimat (PA-457).

PubMed

Adamson, Catherine S; Waki, Kayoko; Ablan, Sherimay D; Salzwedel, Karl; Freed, Eric O

2009-05-01

The maturation inhibitor bevirimat [3-O-(3',3'dimethysuccinyl)betulinic acid; BVM; also known as PA-457 or DSB] potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking protease (PR)-mediated cleavage at the junction between capsid (CA) and spacer peptide 1 (SP1) in Gag. We previously isolated a panel of single-amino-acid substitutions that confer resistance to BVM in vitro (C. S. Adamson, S. D. Ablan, I. Boeras, R. Goila-Gaur, F. Soheilian, K. Nagashima, F. Li, K. Salzwedel, M. Sakalian, C. T. Wild, and E. O. Freed, J. Virol. 80:10957-10971, 2006). The BVM resistance mutations cluster at or near the CA-SP1 cleavage site. Because BVM likely will be used clinically in patients harboring viruses resistant to PR inhibitors (PIs), in this study we evaluated the interplay between a PI-resistant (PIR) PR and the BVM resistance mutations in Gag. As expected, the PIR mutations had no effect on inhibition by BVM; however, we observed general processing defects and a slight delay in viral replication in Jurkat T cells associated with the PIR mutations, even in the absence of compound. When combined, most BVM resistance and PIR mutations acted additively to impair viral replication, particularly in the presence of BVM. The BVM-resistant mutant SP1-A1V was an exception, as it supported robust replication in the context of either wild-type (WT) or PIR PR, even at high BVM concentrations. Significantly, the emergence of BVM resistance was delayed in the context of the PIR PR, and the SP1-A1V mutation was acquired most frequently with either WT or PIR PR. These results suggest that resistance to BVM is less likely to emerge in patients who have failed PIs than in patients who are PI naive. We predict that the SP1-A1V substitution is the most likely to emerge in vivo, as this mutant replicates robustly independently of PR mutations or BVM. These findings offer insights into the effect of PIR mutations on the evolution of BVM resistance in PI-experienced patients.

Prereplicative repair of oxidized bases in the human genome is mediated by NEIL1 DNA glycosylase together with replication proteins

PubMed Central

Hegde, Muralidhar L.; Hegde, Pavana M.; Bellot, Larry J.; Mandal, Santi M.; Hazra, Tapas K.; Li, Guo-Min; Boldogh, Istvan; Tomkinson, Alan E.; Mitra, Sankar

2013-01-01

Base oxidation by endogenous and environmentally induced reactive oxygen species preferentially occurs in replicating single-stranded templates in mammalian genomes, warranting prereplicative repair of the mutagenic base lesions. It is not clear how such lesions (which, unlike bulky adducts, do not block replication) are recognized for repair. Furthermore, strand breaks caused by base excision from ssDNA by DNA glycosylases, including Nei-like (NEIL) 1, would generate double-strand breaks during replication, which are not experimentally observed. NEIL1, whose deficiency causes a mutator phenotype and is activated during the S phase, is present in the DNA replication complex isolated from human cells, with enhanced association with DNA in S-phase cells and colocalization with replication foci containing DNA replication proteins. Furthermore, NEIL1 binds to 5-hydroxyuracil, the oxidative deamination product of C, in replication protein A-coated ssDNA template and inhibits DNA synthesis by DNA polymerase δ. We postulate that, upon encountering an oxidized base during replication, NEIL1 initiates prereplicative repair by acting as a “cowcatcher” and preventing nascent chain growth. Regression of the stalled replication fork, possibly mediated by annealing helicases, then allows lesion repair in the reannealed duplex. This model is supported by our observations that NEIL1, whose deficiency slows nascent chain growth in oxidatively stressed cells, is stimulated by replication proteins in vitro. Furthermore, deficiency of the closely related NEIL2 alone does not affect chain elongation, but combined NEIL1/2 deficiency further inhibits DNA replication. These results support a mechanism of NEIL1-mediated prereplicative repair of oxidized bases in the replicating strand, with NEIL2 providing a backup function. PMID:23898192

Darwinian Evolution of Mutualistic RNA Replicators with Different Genes

NASA Astrophysics Data System (ADS)

Mizuuchi, R.; Ichihashi, N.

2017-07-01

We report a sustainable long-term replication and evolution of two distinct cooperative RNA replicators encoding different genes. One of the RNAs evolved to maintain or increase the cooperativity, despite selective advantage of selfish mutations.

dNTP pool levels modulate mutator phenotypes of error-prone DNA polymerase ε variants.

PubMed

Williams, Lindsey N; Marjavaara, Lisette; Knowels, Gary M; Schultz, Eric M; Fox, Edward J; Chabes, Andrei; Herr, Alan J

2015-05-12

Mutator phenotypes create genetic diversity that fuels tumor evolution. DNA polymerase (Pol) ε mediates leading strand DNA replication. Proofreading defects in this enzyme drive a number of human malignancies. Here, using budding yeast, we show that mutator variants of Pol ε depend on damage uninducible (Dun)1, an S-phase checkpoint kinase that maintains dNTP levels during a normal cell cycle and up-regulates dNTP synthesis upon checkpoint activation. Deletion of DUN1 (dun1Δ) suppresses the mutator phenotype of pol2-4 (encoding Pol ε proofreading deficiency) and is synthetically lethal with pol2-M644G (encoding altered Pol ε base selectivity). Although pol2-4 cells cycle normally, pol2-M644G cells progress slowly through S-phase. The pol2-M644G cells tolerate deletions of mediator of the replication checkpoint (MRC) 1 (mrc1Δ) and radiation sensitive (Rad) 9 (rad9Δ), which encode mediators of checkpoint responses to replication stress and DNA damage, respectively. The pol2-M644G mutator phenotype is partially suppressed by mrc1Δ but not rad9Δ; neither deletion suppresses the pol2-4 mutator phenotype. Thus, checkpoint activation augments the Dun1 effect on replication fidelity but is not required for it. Deletions of genes encoding key Dun1 targets that negatively regulate dNTP synthesis, suppress the dun1Δ pol2-M644G synthetic lethality and restore the mutator phenotype of pol2-4 in dun1Δ cells. DUN1 pol2-M644G cells have constitutively high dNTP levels, consistent with checkpoint activation. In contrast, pol2-4 and POL2 cells have similar dNTP levels, which decline in the absence of Dun1 and rise in the absence of the negative regulators of dNTP synthesis. Thus, dNTP pool levels correlate with Pol ε mutator severity, suggesting that treatments targeting dNTP pools could modulate mutator phenotypes for therapy.

Deletion Mutagenesis Downstream of the 5′ Long Terminal Repeat of Human Immunodeficiency Virus Type 1 Is Compensated for by Point Mutations in both the U5 Region and gag Gene

PubMed Central

Liang, Chen; Rong, Liwei; Russell, Rodney S.; Wainberg, Mark A.

2000-01-01

We have studied the role of an RNA region at nucleotides (nt) +200 to +233, just downstream of the 5′ long terminal repeat, in encapsidation of human immunodeficiency virus type 1 genomic RNA. Three deletion mutations, namely, BH-D0, BH-D1, and BH-D2, were generated to eliminate sequences at positions nt +200 to +219, +200 to +226, and +200 to +233. The result in each case was decreased levels of packaging of viral RNA into the mutated viruses, with the BH-D2 virus being the most severely affected. Consistently, all three deletions resulted in impaired viral infectiousness and the BH-D2 mutation showed the most dramatic impact in this regard. Further analysis revealed additional defects in Gag precursor processing and in the extension efficiency of the tRNA3Lys primer in reverse transcription reactions performed with these mutated viruses. To shed further light on the function of these deleted sequences in viral replication, the mutated viruses were cultured in MT-2 cells over prolonged periods to enable them to reacquire wild-type replication kinetics. Sequencing of the reverted viruses revealed point mutations in both the noncoding region and the gag gene. In the case of the BH-D0 revertant, two mutations were observed at positions G112A in the U5 region, termed M1, and T24I in the nucleocapsid protein, termed MNC, respectively. Either of these two mutations was able to confer wild-type replication capacity on BH-D0. In the case of BH-D1, each of the M1 mutations, a mutation termed M2, i.e., C227T, just downstream of the primer binding site, a mutation termed MP2 (T12I) in the p2 protein, and the MNC mutation were observed. A combination of either M1 and M2 or MP2 and MNC was able to rescue BH-D1. In the case of the BH-D2 deletion-containing viruses, three point mutations, i.e., M1, MP2, and MNC, were observed and the presence of all three was required to restore viral replication to wild-type levels. PMID:10864634

An application of the Caputo-Fabrizio operator to replicator-mutator dynamics: Bifurcation, chaotic limit cycles and control

NASA Astrophysics Data System (ADS)

Doungmo Goufo, Emile Franc

2018-02-01

The physical behaviors of replicator-mutator processes found in theoretical biophysics, physical chemistry, biochemistry and population biology remain complex with unlimited expressibility. People languages, for instance, have impressively and unpredictably changed over the time in human history. This is mainly due to the collection of small changes and collaboration with other languages. In this paper, the Caputo-Fabrizio operator is applied to a replicator-mutator dynamic taking place in midsts with movement. The model is fully analyzed and solved numerically via the Crank-Nicolson scheme. Stability and convergence results are provided. A concrete application to replicator-mutator dynamics for a population with three strategies is performed with numerical simulations provided for some fixed values of the physical position of the population symbolized by r and the grid points. Physically, it happens that limit cycles appear, not only in function of the mutation parameter μ but also in function of the values given to r . The amplitudes of limit cycles also appear to be proportional to r but the stability of the system remains unaffected. However, those limit cycles instead of disappearing as expected, are immediately followed by chaotic and unpredictable behaviors certainly due to the non-singular kernel used in the model and suitable to non-linear dynamics. Hence, the appearance and disappearance of limit cycles might be controlled by the position variable r which can also apprehend chaos.

TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism

PubMed Central

Leitch, Andrea; Higgs, Martin R.; Bicknell, Louise S.; Yigit, Gökhan; Blackford, Andrew N.; Zlatanou, Anastasia; Mackenzie, Karen J.; Reddy, Kaalak; Halachev, Mihail; McGlasson, Sarah; Reijns, Martin A. M.; Fluteau, Adeline; Martin, Carol-Anne; Sabbioneda, Simone; Elcioglu, Nursel H.; Altmüller, Janine; Thiele, Holger; Greenhalgh, Lynn; Chessa, Luciana; Maghnie, Mohamad; Salim, Mahmoud; Bober, Michael B.; Nürnberg, Peter; Jackson, Stephen P.; Hurles, Matthew E.; Wollnik, Bernd; Stewart, Grant S.; Jackson, Andrew P.

2015-01-01

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism/Seckel syndrome. We establish that TRAIP relocalizes to sites of DNA damage where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to UV irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a novel component of the DNA damage response to replication-blocking DNA lesions. PMID:26595769

Male Mutation Bias Is the Main Force Shaping Chromosomal Substitution Rates in Monotreme Mammals.

PubMed

Link, Vivian; Aguilar-Gómez, Diana; Ramírez-Suástegui, Ciro; Hurst, Laurence D; Cortez, Diego

2017-09-01

In many species, spermatogenesis involves more cell divisions than oogenesis, and the male germline, therefore, accumulates more DNA replication errors, a phenomenon known as male mutation bias. The extent of male mutation bias (α) is estimated by comparing substitution rates of the X, Y, and autosomal chromosomes, as these chromosomes spend different proportions of their time in the germlines of the two sexes. Male mutation bias has been characterized in placental and marsupial mammals as well as birds, but analyses in monotremes failed to detect any such bias. Monotremes are an ancient lineage of egg-laying mammals with distinct biological properties, which include unique germline features. Here, we sought to assess the presence and potential characteristics of male mutation bias in platypus and the short-beaked echidna based on substitution rate analyses of X, Y, and autosomes. We established the presence of moderate male mutation bias in monotremes, corresponding to an α value of 2.12-3.69. Given that it has been unclear what proportion of the variation in substitution rates on the different chromosomal classes is really due to differential number of replications, we analyzed the influence of other confounding forces (selection, replication-timing, etc.) and found that male mutation bias is the main force explaining the between-chromosome classes differences in substitution rates. Finally, we estimated the proportion of variation at the gene level in substitution rates that is owing to replication effects and found that this phenomenon can explain >68% of these variations in monotremes, and in control species, rodents, and primates. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer.

PubMed

Ruark, Elise; Snape, Katie; Humburg, Peter; Loveday, Chey; Bajrami, Ilirjana; Brough, Rachel; Rodrigues, Daniel Nava; Renwick, Anthony; Seal, Sheila; Ramsay, Emma; Duarte, Silvana Del Vecchio; Rivas, Manuel A; Warren-Perry, Margaret; Zachariou, Anna; Campion-Flora, Adriana; Hanks, Sandra; Murray, Anne; Ansari Pour, Naser; Douglas, Jenny; Gregory, Lorna; Rimmer, Andrew; Walker, Neil M; Yang, Tsun-Po; Adlard, Julian W; Barwell, Julian; Berg, Jonathan; Brady, Angela F; Brewer, Carole; Brice, Glen; Chapman, Cyril; Cook, Jackie; Davidson, Rosemarie; Donaldson, Alan; Douglas, Fiona; Eccles, Diana; Evans, D Gareth; Greenhalgh, Lynn; Henderson, Alex; Izatt, Louise; Kumar, Ajith; Lalloo, Fiona; Miedzybrodzka, Zosia; Morrison, Patrick J; Paterson, Joan; Porteous, Mary; Rogers, Mark T; Shanley, Susan; Walker, Lisa; Gore, Martin; Houlston, Richard; Brown, Matthew A; Caufield, Mark J; Deloukas, Panagiotis; McCarthy, Mark I; Todd, John A; Turnbull, Clare; Reis-Filho, Jorge S; Ashworth, Alan; Antoniou, Antonis C; Lord, Christopher J; Donnelly, Peter; Rahman, Nazneen

2013-01-17

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer

PubMed Central

Ruark, Elise; Snape, Katie; Humburg, Peter; Loveday, Chey; Bajrami, Ilirjana; Brough, Rachel; Rodrigues, Daniel Nava; Renwick, Anthony; Seal, Sheila; Ramsay, Emma; Duarte, Silvana Del Vecchio; Rivas, Manuel A.; Warren-Perry, Margaret; Zachariou, Anna; Campion-Flora, Adriana; Hanks, Sandra; Murray, Anne; Pour, Naser Ansari; Douglas, Jenny; Gregory, Lorna; Rimmer, Andrew; Walker, Neil M.; Yang, Tsun-Po; Adlard, Julian W.; Barwell, Julian; Berg, Jonathan; Brady, Angela F.; Brewer, Carole; Brice, Glen; Chapman, Cyril; Cook, Jackie; Davidson, Rosemarie; Donaldson, Alan; Douglas, Fiona; Eccles, Diana; Evans, D. Gareth; Greenhalgh, Lynn; Henderson, Alex; Izatt, Louise; Kumar, Ajith; Lalloo, Fiona; Miedzybrodzka, Zosia; Morrison, Patrick J.; Paterson, Joan; Porteous, Mary; Rogers, Mark T.; Shanley, Susan; Walker, Lisa; Gore, Martin; Houlston, Richard; Brown, Matthew A.; Caufield, Mark J.; Deloukas, Panagiotis; McCarthy, Mark I.; Todd, John A.; Turnbull, Clare; Reis-Filho, Jorge S.; Ashworth, Alan; Antoniou, Antonis C.; Lord, Christopher J.; Donnelly, Peter; Rahman, Nazneen

2013-01-01

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication1. Here, using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focussed on protein truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, we show that rare PTVs in the p53 inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and to ovarian cancer. PPM1D PTV mutations were present in 25/7781 cases vs 1/5861 controls; P=1.12×10−5, which included 18 mutations in 6,912 individuals with breast cancer; P = 2.42×10−4 and 12 mutations in 1,121 individuals with ovarian cancer; P = 3.10×10−9. Notably, all the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370 bp region in the final exon of the gene, C-terminal to the phosphatase catalytic domain. Functional studies demonstrated that the mutations result in enhanced suppression of p53 in response to ionising radiation exposure, suggesting the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function typically associated with this class of variant, but instead likely have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the utility of sequencing in their identification. PMID:23242139

Amino Acid Substitutions in Polymerase Basic Protein 2 Gene Contribute to the Pathogenicity of the Novel A/H7N9 Influenza Virus in Mammalian Hosts

PubMed Central

Mok, Chris Ka Pun; Lee, Horace Hok Yeung; Lestra, Maxime; Nicholls, John Malcolm; Chan, Michael Chi Wai; Sia, Sin Fun; Zhu, Huachen; Poon, Leo Lit Man; Guan, Yi

2014-01-01

ABSTRACT A novel avian-origin influenza A/H7N9 virus emerged in 2013 to cause more than 130 cases of zoonotic human disease, with an overall case fatality rate of around 30% in cases detected. It has been shown that an E-to-K amino acid change at residue 627 of polymerase basic protein 2 (PB2) occurred frequently in the H7N9 isolates obtained from humans but not in viruses isolated from poultry. Although this mutation has been reported to confer increased mammalian pathogenicity in other avian influenza subtypes, it has not been experimentally investigated in the H7N9 virus. In this study, we determined the contribution of PB2-E627K in H7N9 virus to its pathogenicity in mammalian hosts. In addition, the compensatory role of the PB2 mutations T271A, Q591K, and D701N in H7N9 virus was investigated. We characterized the activity of polymerase complexes with these PB2 mutations and found that they enhance the polymerase activity in human 293T cells. The rescued mutants enhanced growth in mammalian cells in vitro. Mice infected with the H7N9 mutant containing the avian signature protein PB2-627E showed a marked decrease in disease severity (weight loss) and pathology compared to mice infected with the wild-type strain (PB2-627K) or other PB2 mutants. Also, mutants with PB2-627E showed lower virus replication and proinflammatory cytokine responses in the lungs of the virus-infected mice, which may contribute to pathogenicity. Our results suggest that these amino acid substitutions contribute to mouse pathogenicity and mammalian adaptation. IMPORTANCE A novel avian H7N9 influenza A virus emerged in east China in 2013 to cause zoonotic human disease associated with significant mortality. It is important to understand the viral genetic markers of mammalian adaptation and disease severity in this H7N9 virus. Since many human (but not avian) H7N9 virus isolates have an amino acid substitution at position E627K in the polymerase basic protein 2 (PB2) gene, we investigated the role of this and other functionally related mutations for polymerase activity in vitro, virus replication competence, and pathogenicity in the mouse model. We found that E627K and functionally related mutations are associated with increased polymerase activity, increased viral replication competence, and increased disease severity in mice. PMID:24403592

The Cytoplasmic C-Tail of the Mouse Cytomegalovirus 7 Transmembrane Receptor Homologue, M78, Regulates Endocytosis of the Receptor and Modulates Virus Replication in Different Cell Types

PubMed Central

2016-01-01

Virus homologues of seven-transmembrane receptors (7TMR) are encoded by all beta- and gammaherpesviruses, suggesting important functional roles. M78 of mouse cytomegalovirus (MCMV) is representative of a family of 7TMR conserved in all betaherpesviruses. M78 family members have been found to exhibit cell-type specific effects upon virus replication in tissue culture and to affect virus pathogenesis in vivo. We reported previously that M78, for which no ligands are known, undergoes rapid, constitutive endocytosis. In this study, we have investigated the role of the M78 cytoplasmic C-tail in mediating endocytosis and consequences of C-tail deletion upon replication and pathogenesis. Mutations of M78 (C-tail truncations or point mutations) and CCR5-M78 chimeras identified two distinct regions affecting endocytosis. The first was a classical acidic di-leucine motif (DDxxxLL), located close to the C-terminus. The second region, the activity of which was suppressed by downstream sequences, included the putative 8th helix, located close to the 7th transmembrane domain. A recombinant MCMV expressing an endocytosis-deficient M78, lacking most of the C-tail (M78_CΔ155), had a cell-type specific replication phenotype. M78_CΔ155 had restricted replication in bone marrow macrophages, indistinguishable from an M78-null recombinant. In contrast, M78_CΔ155 replicated normally or with enhanced titres to wild type virus in other tested cell-types, whereas M78-null was attenuated. Distinct phenotypes for M78_CΔ155 and M78-null suggest that the C-tail deletion resulted in M78 dysfunction, rather than complete loss of function; furthermore, they highlight a cell-type specific role of M78 during replication. Infection of mice (intranasal) demonstrated that M78_CΔ155, similar to M78-null, was cleared more rapidly from the lungs than wild type virus and was severely attenuated for replication in salivary glands. It may be speculated that attenuation of both M78_CΔ155 and M78-null for replication in macrophages may have contributed to their similar pathogenic phenotypes. PMID:27760189

Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication.

PubMed

Brustel, Julien; Kirstein, Nina; Izard, Fanny; Grimaud, Charlotte; Prorok, Paulina; Cayrou, Christelle; Schotta, Gunnar; Abdelsamie, Alhassan F; Déjardin, Jérôme; Méchali, Marcel; Baldacci, Giuseppe; Sardet, Claude; Cadoret, Jean-Charles; Schepers, Aloys; Julien, Eric

2017-09-15

Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila , partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein-Barr virus-derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se , but rather serves as an enhancer for MCM2-7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes. © 2017 The Authors.

West Nile virus (WNV) genome RNAs with up to three adjacent mutations that disrupt long distance 5'-3' cyclization sequence basepairs are viable

DOE Office of Scientific and Technical Information (OSTI.GOV)

Basu, Mausumi; Brinton, Margo A., E-mail: mbrinton@gsu.ed

2011-03-30

Mosquito-borne flavivirus genomes contain conserved 5' and 3' cyclization sequences (CYC) that facilitate long distance RNA-RNA interactions. In previous studies, flavivirus replicon RNA replication was completely inhibited by single or multiple mismatching CYC nt substitutions. In the present study, full-length WNV genomes with one, two or three mismatching CYC substitutions showed reduced replication efficiencies but were viable and generated revertants with increased replication efficiency. Several different three adjacent mismatching CYC substitution mutant RNAs were rescued by a second site mutation that created an additional basepair (nts 147-10913) on the internal genomic side of the 5'-3' CYC. The finding that full-lengthmore » genomes with up to three mismatching CYC mutations are viable and can be rescued by a single nt spontaneous mutation indicates that more than three adjacent CYC basepair substitutions would be required to increase the safety of vaccine genomes by creating mismatches in inter-genomic recombinants.« less

Replicative DNA polymerase mutations in cancer☆

PubMed Central

Heitzer, Ellen; Tomlinson, Ian

2014-01-01

Three DNA polymerases — Pol α, Pol δ and Pol ɛ — are essential for DNA replication. After initiation of DNA synthesis by Pol α, Pol δ or Pol ɛ take over on the lagging and leading strand respectively. Pol δ and Pol ɛ perform the bulk of replication with very high fidelity, which is ensured by Watson–Crick base pairing and 3′exonuclease (proofreading) activity. Yeast models have shown that mutations in the exonuclease domain of Pol δ and Pol ɛ homologues can cause a mutator phenotype. Recently, we identified germline exonuclease domain mutations (EDMs) in human POLD1 and POLE that predispose to ‘polymerase proofreading associated polyposis’ (PPAP), a disease characterised by multiple colorectal adenomas and carcinoma, with high penetrance and dominant inheritance. Moreover, somatic EDMs in POLE have also been found in sporadic colorectal and endometrial cancers. Tumors with EDMs are microsatellite stable and show an ‘ultramutator’ phenotype, with a dramatic increase in base substitutions. PMID:24583393

Natural and Unanticipated Modifiers of RNAi Activity in Caenorhabditis elegans

PubMed Central

Asad, Nadeem; Aw, Wen Yih; Timmons, Lisa

2012-01-01

Organisms used as model genomics systems are maintained as isogenic strains, yet evidence of sequence differences between independently maintained wild-type stocks has been substantiated by whole-genome resequencing data and strain-specific phenotypes. Sequence differences may arise from replication errors, transposon mobilization, meiotic gene conversion, or environmental or chemical assault on the genome. Low frequency alleles or mutations with modest effects on phenotypes can contribute to natural variation, and it has proven possible for such sequences to become fixed by adapted evolutionary enrichment and identified by resequencing. Our objective was to identify and analyze single locus genetic defects leading to RNAi resistance in isogenic strains of Caenorhabditis elegans. In so doing, we uncovered a mutation that arose de novo in an existing strain, which initially frustrated our phenotypic analysis. We also report experimental, environmental, and genetic conditions that can complicate phenotypic analysis of RNAi pathway defects. These observations highlight the potential for unanticipated mutations, coupled with genetic and environmental phenomena, to enhance or suppress the effects of known mutations and cause variation between wild-type strains. PMID:23209671

BRCA2 and RAD51 promote double-strand break formation and cell death in response to gemcitabine.

PubMed

Jones, Rebecca M; Kotsantis, Panagiotis; Stewart, Grant S; Groth, Petra; Petermann, Eva

2014-10-01

Replication inhibitors cause replication fork stalling and double-strand breaks (DSB) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitize cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here, we investigate why, paradoxically, mutations in HR genes protect cells from killing by gemcitabine. Using DNA replication and DNA damage assays in mammalian cells, we show that even short gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression, and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at gemcitabine-stalled forks are converted into DSBs and thus contribute to gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumors. ©2014 American Association for Cancer Research.

BRCA2 and RAD51 promote double-strand break formation and cell death in response to Gemcitabine

PubMed Central

Jones, Rebecca M.; Kotsantis, Panagiotis; Stewart, Grant S.; Groth, Petra; Petermann, Eva

2014-01-01

Replication inhibitors cause replication fork stalling and double-strand breaks (DSBs) that result from processing of stalled forks. During recovery from replication blocks, the homologous recombination (HR) factor RAD51 mediates fork restart and DSB repair. HR defects therefore sensitise cells to replication inhibitors, with clear implications for cancer therapy. Gemcitabine is a potent replication inhibitor used to treat cancers with mutations in HR genes such as BRCA2. Here we investigate why, paradoxically, mutations in HR genes protect cells from killing by Gemcitabine. Using DNA replication and -damage assays in mammalian cells, we show that even short Gemcitabine treatments cause persistent replication inhibition. BRCA2 and RAD51 are recruited to chromatin early after removal of the drug, actively inhibit replication fork progression and promote the formation of MUS81- and XPF-dependent DSBs that remain unrepaired. Our data suggest that HR intermediates formed at Gemcitabine-stalled forks are converted into DSBs and thus contribute to Gemcitabine-induced cell death, which could have implications for the treatment response of HR-deficient tumours. PMID:25053826

On the mutagenicity of homologous recombination and double-strand break repair in bacteriophage.

PubMed

Shcherbakov, Victor P; Plugina, Lidiya; Shcherbakova, Tamara; Sizova, Svetlana; Kudryashova, Elena

2011-01-02

The double-strand break (DSB) repair via homologous recombination is generally construed as a high-fidelity process. However, some molecular genetic observations show that the recombination and the recombinational DSB repair may be mutagenic and even highly mutagenic. Here we developed an effective and precise method for studying the fidelity of DSB repair in vivo by combining DSBs produced site-specifically by the SegC endonuclease with the famous advantages of the recombination analysis of bacteriophage T4 rII mutants. The method is based on the comparison of the rate of reversion of rII mutation in the presence and in the absence of a DSB repair event initiated in the proximity of the mutation. We observed that DSB repair may moderately (up to 6-fold) increase the apparent reversion frequency, the effect of being dependent on the mutation structure. We also studied the effect of the T4 recombinase deficiency (amber mutation in the uvsX gene) on the fidelity of DSB repair. We observed that DSBs are still repaired via homologous recombination in the uvsX mutants, and the apparent fidelity of this repair is higher than that seen in the wild-type background. The mutator effect of the DSB repair may look unexpected given that most of the normal DNA synthesis in bacteriophage T4 is performed via a recombination-dependent replication (RDR) pathway, which is thought to be indistinguishable from DSB repair. There are three possible explanations for the observed mutagenicity of DSB repair: (1) the origin-dependent (early) DNA replication may be more accurate than the RDR; (2) the step of replication initiation may be more mutagenic than the process of elongation; and (3) the apparent mutagenicity may just reflect some non-randomness in the pool of replicating DNA, i.e., preferential replication of the sequences already involved in replication. We discuss the DSB repair pathway in the absence of UvsX recombinase. Copyright © 2010 Elsevier B.V. All rights reserved.

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

PubMed Central

Paulovich, A G; Armour, C D; Hartwell, L H

1998-01-01

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication. PMID:9725831

The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage.

PubMed

Paulovich, A G; Armour, C D; Hartwell, L H

1998-09-01

In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rudneva, Irina A.; Timofeeva, Tatiana A.; Ignatieva, Anna V.

In the present study we assessed pleiotropic characteristics of the antibody-selected mutations. We examined pH optimum of fusion, temperatures of HA heat inactivation, and in vitro and in vivo replication kinetics of the previously obtained influenza H5 escape mutants. Our results showed that HA1 N142K mutation significantly lowered the pH of fusion optimum. Mutations of the escape mutants located in the HA lateral loop significantly affected H5 HA thermostability (P<0.05). HA changes at positions 131, 144, 145, and 156 and substitutions at positions 131, 142, 145, and 156 affected the replicative ability of H5 escape mutants in vitro and inmore » vivo, respectively. Overall, a co-variation between antigenic specificity and different HA phenotypic properties has been demonstrated. We believe that the monitoring of pleiotropic effects of the HA mutations found in H5 escape mutants is essential for accurate prediction of mutants with pandemic potential. - Highlights: • HA1 N142K mutation significantly lowered the pH of fusion optimum. • Mutations located in the HA lateral loop significantly affected H5 HA thermostability. • HA changes at positions 131, 142, 144, 145, and 156 affected the replicative ability of H5 mutants. • Acquisition of glycosylation site could lead to the emergence of multiple pleiotropic effects.« less

Regulation of Viral RNA Synthesis by the V Protein of Parainfluenza Virus 5

PubMed Central

Yang, Yang; Zengel, James; Sun, Minghao; Sleeman, Katrina; Timani, Khalid Amine; Aligo, Jason; Rota, Paul

2015-01-01

ABSTRACT Paramyxoviruses include many important animal and human pathogens. The genome of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, encodes a V protein that inhibits viral RNA synthesis. In this work, the mechanism of inhibition was investigated. Using mutational analysis and a minigenome system, we identified regions in the N and C termini of the V protein that inhibit viral RNA synthesis: one at the very N terminus of V and the second at the C terminus of V. Furthermore, we determined that residues L16 and I17 are critical for the inhibitory function of the N-terminal region of the V protein. Both regions interact with the nucleocapsid protein (NP), an essential component of the viral RNA genome complex (RNP). Mutations at L16 and I17 abolished the interaction between NP and the N-terminal domain of V. This suggests that the interaction between NP and the N-terminal domain plays a critical role in V inhibition of viral RNA synthesis by the N-terminal domain. Both the N- and C-terminal regions inhibited viral RNA replication. The C terminus inhibited viral RNA transcription, while the N-terminal domain enhanced viral RNA transcription, suggesting that the two domains affect viral RNA through different mechanisms. Interestingly, V also inhibited the synthesis of the RNA of other paramyxoviruses, such as Nipah virus (NiV), human parainfluenza virus 3 (HPIV3), measles virus (MeV), mumps virus (MuV), and respiratory syncytial virus (RSV). This suggests that a common host factor may be involved in the replication of these paramyxoviruses. IMPORTANCE We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the rational design of vaccines and potential antiviral drugs. PMID:26378167

Single-Molecule Sequencing Reveals Complex Genome Variation of Hepatitis B Virus during 15 Years of Chronic Infection following Liver Transplantation

PubMed Central

Betz-Stablein, B. D.; Töpfer, A.; Littlejohn, M.; Yuen, L.; Colledge, D.; Sozzi, V.; Angus, P.; Thompson, A.; Revill, P.; Beerenwinkel, N.; Warner, N.

2016-01-01

ABSTRACT Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome. IMPORTANCE Single-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available. PMID:27252524

POB3 is required for both transcription and replication in the yeast Saccharomyces cerevisiae.

PubMed Central

Schlesinger, M B; Formosa, T

2000-01-01

Spt16 and Pob3 form stable heterodimers in Saccharomyces cerevisiae, and homologous proteins have also been purified as complexes from diverse eukaryotes. This conserved factor has been implicated in both transcription and replication and may affect both by altering the characteristics of chromatin. Here we describe the isolation and properties of a set of pob3 mutants and confirm that they have defects in both replication and transcription. Mutation of POB3 caused the Spt(-) phenotype, spt16 and pob3 alleles displayed severe synthetic defects, and elevated levels of Pob3 suppressed some spt16 phenotypes. These results are consistent with previous reports that Spt16 and Pob3 act in a complex that modulates transcription. Additional genetic interactions were observed between pob3 mutations and the genes encoding several DNA replication factors, including POL1, CTF4, DNA2, and CHL12. pob3 alleles caused sensitivity to the ribonucleotide reductase inhibitor hydroxyurea, indicating a defect in a process requiring rapid dNTP synthesis. Mutation of the S phase checkpoint gene MEC1 caused pob3 mutants to lose viability rapidly under restrictive conditions, revealing defects in a process monitored by Mec1. Direct examination of DNA contents by flow cytometry showed that S phase onset and progression were delayed when POB3 was mutated. We conclude that Pob3 is required for normal replication as well as for transcription. PMID:10924459

Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

PubMed

Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

2015-05-08

Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Foamy virus reverse transcriptase is expressed independently from the Gag protein.

PubMed Central

Enssle, J; Jordan, I; Mauer, B; Rethwilm, A

1996-01-01

In the foamy virus (FV) subgroup of retroviruses the pol genes are located in the +1 reading frame relative to the gag genes and possess potential ATG initiation codons in their 5' regions. This genome organization suggests either a + 1 ribosomal frameshift to generate a Gag-Pol fusion protein, similar to all other retroviruses studied so far, or new initiation of Pol translation, as used by pararetroviruses, to express the Pol protein. By using a genetic approach we have ruled out the former possibility and provide evidence for the latter. Two down-mutations (M53 and M54) of the pol ATG codon were found to abolish replication and Pol protein expression of the human FV isolate. The introduction of a new ATG in mutation M55, 3' to the down-mutated ATG of mutation M53, restored replication competence, indicating that the pol ATG functions as a translational initiation codon. Two nonsense mutants (M56 and M57), which functionally separated gag and pol with respect to potential frame-shifting sites, were also replication-competent, providing further genetic evidence that FVs express the Pol protein independently from Gag. Our results show that during a particular step of the replication cycle, FVs differ fundamentally from all other retroviruses. Images Fig. 3 PMID:8633029

Isolation and Characterization of Highly Replicable Hepatitis C Virus Genotype 1a Strain HCV-RMT

PubMed Central

Arai, Masaaki; Tokunaga, Yuko; Takagi, Asako; Tobita, Yoshimi; Hirata, Yuichi; Ishida, Yuji; Tateno, Chise; Kohara, Michinori

2013-01-01

Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient’s serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo. PMID:24358200

Isolation and characterization of highly replicable hepatitis C virus genotype 1a strain HCV-RMT.

PubMed

Arai, Masaaki; Tokunaga, Yuko; Takagi, Asako; Tobita, Yoshimi; Hirata, Yuichi; Ishida, Yuji; Tateno, Chise; Kohara, Michinori

2013-01-01

Multiple genotype 1a clones have been reported, including the very first hepatitis C virus (HCV) clone called H77. The replication ability of some of these clones has been confirmed in vitro and in vivo, although this ability is somehow compromised. We now report a newly isolated genotype 1a clone, designated HCV-RMT, which has the ability to replicate efficiently in patients, chimeric mice with humanized liver, and cultured cells. An authentic subgenomic replicon cell line was established from the HCV-RMT sequence with spontaneous introduction of three adaptive mutations, which were later confirmed to be responsible for efficient replication in HuH-7 cells as both subgenomic replicon RNA and viral genome RNA. Following transfection, the HCV-RMT RNA genome with three adaptive mutations was maintained for more than 2 months in HuH-7 cells. One clone selected from the transfected cells had a high copy number, and its supernatant could infect naïve HuH-7 cells. Direct injection of wild-type HCV-RMT RNA into the liver of chimeric mice with humanized liver resulted in vigorous replication, similar to inoculation with the parental patient's serum. A study of virus replication using HCV-RMT derivatives with various combinations of adaptive mutations revealed a clear inversely proportional relationship between in vitro and in vivo replication abilities. Thus, we suggest that HCV-RMT and its derivatives are important tools for HCV genotype 1a research and for determining the mechanism of HCV replication in vitro and in vivo.

Poliovirus Proteins Induce Membrane Association of GTPase ADP-Ribosylation Factor

PubMed Central

Belov, George A.; Fogg, Mark H.; Ehrenfeld, Ellie

2005-01-01

Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases. During normal membrane trafficking in uninfected cells, ARFs are involved in vesicle formation from different intracellular sites through interaction with numerous regulatory and coat proteins as well as in regulation of phospholipase D activity and cytoskeleton modifications. We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA. The accumulation of ARFs on membranes correlates with active replication of poliovirus RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of poliovirus 3A or 3CD proteins, and we describe mutations that abolished this activity. In infected HeLa cells, an ARF1-enhanced green fluorescent protein fusion redistributes from Golgi stacks to the perinuclear region, where poliovirus RNA replication occurs. Taken together, the data suggest an involvement of ARF in poliovirus RNA replication. PMID:15890959

A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication

PubMed Central

E, Xiaofei; Savidis, George; Chin, Christopher R.; Wang, Shixia; Lu, Shan; Brass, Abraham L.

2014-01-01

Human cytomegalovirus (HCMV) genome replication requires host DNA damage responses (DDRs) and raises the possibility that DNA repair pathways may influence viral replication. We report here that a nucleotide excision repair (NER)-associated-factor is required for efficient HCMV DNA replication. Mutations in genes encoding NER factors are associated with xeroderma pigmentosum (XP). One of the XP complementation groups, XPE, involves mutation in ddb2, which encodes DNA damage binding protein 2 (DDB2). Infectious progeny virus production was reduced by >2 logs in XPE fibroblasts compared to levels in normal fibroblasts. The levels of immediate early (IE) (IE2), early (E) (pp65), and early/late (E/L) (gB55) proteins were decreased in XPE cells. These replication defects were rescued by infection with a retrovirus expressing DDB2 cDNA. Similar patterns of reduced viral gene expression and progeny virus production were also observed in normal fibroblasts that were depleted for DDB2 by RNA interference (RNAi). Mature replication compartments (RCs) were nearly absent in XPE cells, and there were 1.5- to 2.0-log reductions in viral DNA loads in infected XPE cells relative to those in normal fibroblasts. The expression of viral genes (UL122, UL44, UL54, UL55, and UL84) affected by DDB2 status was also sensitive to a viral DNA replication inhibitor, phosphonoacetic acid (PAA), suggesting that DDB2 affects gene expression upstream of or events associated with the initiation of DNA replication. Finally, a novel, infection-associated feedback loop between DDB2 and ataxia telangiectasia mutated (ATM) was observed in infected cells. Together, these results demonstrate that DDB2 and a DDB2-ATM feedback loop influence HCMV replication. PMID:24335308

Random mutagenesis by error-prone pol plasmid replication in Escherichia coli.

PubMed

Alexander, David L; Lilly, Joshua; Hernandez, Jaime; Romsdahl, Jillian; Troll, Christopher J; Camps, Manel

2014-01-01

Directed evolution is an approach that mimics natural evolution in the laboratory with the goal of modifying existing enzymatic activities or of generating new ones. The identification of mutants with desired properties involves the generation of genetic diversity coupled with a functional selection or screen. Genetic diversity can be generated using PCR or using in vivo methods such as chemical mutagenesis or error-prone replication of the desired sequence in a mutator strain. In vivo mutagenesis methods facilitate iterative selection because they do not require cloning, but generally produce a low mutation density with mutations not restricted to specific genes or areas within a gene. For this reason, this approach is typically used to generate new biochemical properties when large numbers of mutants can be screened or selected. Here we describe protocols for an advanced in vivo mutagenesis method that is based on error-prone replication of a ColE1 plasmid bearing the gene of interest. Compared to other in vivo mutagenesis methods, this plasmid-targeted approach allows increased mutation loads and facilitates iterative selection approaches. We also describe the mutation spectrum for this mutagenesis methodology in detail, and, using cycle 3 GFP as a target for mutagenesis, we illustrate the phenotypic diversity that can be generated using our method. In sum, error-prone Pol I replication is a mutagenesis method that is ideally suited for the evolution of new biochemical activities when a functional selection is available.

Influence of Electron–Holes on DNA Sequence-Specific Mutation Rates

PubMed Central

Suárez-Villagrán, Martha Y; Azevedo, Ricardo B R; Miller, John H

2018-01-01

Abstract Biases in mutation rate can influence molecular evolution, yielding rates of evolution that vary widely in different parts of the genome and even among neighboring nucleotides. Here, we explore one possible mechanism of influence on sequence-specific mutation rates, the electron–hole, which can localize and potentially trigger a replication mismatch. A hole is a mobile site of positive charge created during one-electron oxidation by, for example, radiation, contact with a mutagenic agent, or oxidative stress. Its quantum wavelike properties cause it to localize at various sites with probabilities that vary widely, by orders of magnitude, and depend strongly on the local sequence. We find significant correlations between hole probabilities and mutation rates within base triplets, observed in published mutation accumulation experiments on four species of bacteria. We have also computed hole probability spectra for hypervariable segment I of the human mtDNA control region, which contains several mutational hotspots, and for heptanucleotides in noncoding regions of the human genome, whose polymorphism levels have recently been reported. We observe significant correlations between hole probabilities, and context-specific mutation and substitution rates. The correlation with hole probability cannot be explained entirely by CpG methylation in the heptanucleotide data. Peaks in hole probability tend to coincide with mutational hotspots, even in mtDNA where CpG methylation is rare. Our results suggest that hole-enhanced mutational mechanisms, such as oxidation-stabilized tautomerization and base deamination, contribute to molecular evolution. PMID:29617801

Novel Acylguanidine-Based Inhibitor of HIV-1

PubMed Central

Mwimanzi, Philip; Tietjen, Ian; Miller, Scott C.; Shahid, Aniqa; Cobarrubias, Kyle; Kinloch, Natalie N.; Baraki, Bemuluyigza; Richard, Jonathan; Finzi, Andrés; Fedida, David; Brumme, Zabrina L.

2016-01-01

ABSTRACT The emergence of transmissible HIV-1 strains with resistance to antiretroviral drugs highlights a continual need for new therapies. Here we describe a novel acylguanidine-containing compound, 1-(2-(azepan-1-yl)nicotinoyl)guanidine (or SM111), that inhibits in vitro replication of HIV-1, including strains resistant to licensed protease, reverse transcriptase, and integrase inhibitors, without major cellular toxicity. At inhibitory concentrations, intracellular p24Gag production was unaffected, but virion release (measured as extracellular p24Gag) was reduced and virion infectivity was substantially impaired, suggesting that SM111 acts at a late stage of viral replication. SM111-mediated inhibition of HIV-1 was partially overcome by a Vpu I17R mutation alone or a Vpu W22* truncation in combination with Env N136Y. These mutations enhanced virion infectivity and Env expression on the surface of infected cells in the absence and presence of SM111 but also impaired Vpu's ability to downregulate CD4 and BST2/tetherin. Taken together, our results support acylguanidines as a class of HIV-1 inhibitors with a distinct mechanism of action compared to that of licensed antiretrovirals. Further research on SM111 and similar compounds may help to elucidate knowledge gaps related to Vpu's role in promoting viral egress and infectivity. IMPORTANCE New inhibitors of HIV-1 replication may be useful as therapeutics to counteract drug resistance and as reagents to perform more detailed studies of viral pathogenesis. SM111 is a small molecule that blocks the replication of wild-type and drug-resistant HIV-1 strains by impairing viral release and substantially reducing virion infectivity, most likely through its ability to prevent Env expression at the infected cell surface. Partial resistance to SM111 is mediated by mutations in Vpu and/or Env, suggesting that the compound affects host/viral protein interactions that are important during viral egress. Further characterization of SM111 and similar compounds may allow more detailed pharmacological studies of HIV-1 egress and provide opportunities to develop new treatments for HIV-1. PMID:27512074

Mutation of Putative N-Glycosylation Sites on Dengue Virus NS4B Decreases RNA Replication.

PubMed

Naik, Nenavath Gopal; Wu, Huey-Nan

2015-07-01

Dengue virus (DENV) nonstructural protein 4B (NS4B) is an endoplasmic reticulum (ER) membrane-associated protein, and mutagenesis studies have revealed its significance in viral genome replication. In this work, we demonstrated that NS4B is an N-glycosylated protein in virus-infected cells as well as in recombinant protein expression. NS4B is N glycosylated at residues 58 and 62 and exists in two forms, glycosylated and unglycosylated. We manipulated full-length infectious RNA clones and subgenomic replicons to generate N58Q, N62Q, and N58QN62Q mutants. Each of the single mutants had distinct effects, but the N58QN62Q mutation resulted in dramatic reduction of viral production efficiency without affecting secretion or infectivity of the virion in mammalian and mosquito C6/36 hosts. Real-time quantitative PCR (qPCR), subgenomic replicon, and trans-complementation assays indicated that the N58QN62Q mutation affected RNA replication possibly by the loss of glycans. In addition, four intragenic mutations (S59Y, S59F, T66A, and A137T) were obtained from mammalian and/or mosquito C6/36 cell culture systems. All of these second-site mutations compensated for the replication defect of the N58QN62Q mutant without creating novel glycosylation sites. In vivo protein stability analyses revealed that the N58QN62Q mutation alone or plus a compensatory mutation did not affect the stability of NS4B. Overall, our findings indicated that mutation of putative N-glycosylation sites affected the biological function of NS4B in the viral replication complex. This is the first report to identify and reveal the biological significance of dengue virus (DENV) nonstructural protein 4B (NS4B) posttranslation N-glycosylation to the virus life cycle. The study demonstrated that NS4B is N glycosylated in virus-infected cells and in recombinant protein expression. NS4B is modified by glycans at Asn-58 and Asn-62. Functional characterization implied that DENV NS4B utilizes the glycosylation machinery in both mammalian and mosquito hosts. Four intragenic mutations were found to compensate for replication and subsequent viral production deficiencies without creating novel N-glycosylation sites or modulating the stabilities of the protein, suggesting that glycans may be involved in maintaining the NS4B protein conformation. NS4B glycans may be necessary elements of the viral life cycle, but compensatory mutations can circumvent their requirement. This novel finding may have broader implications in flaviviral biology as the most likely glycan at Asn-62 of NS4B is conserved in DENV serotypes and in some related flaviviruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Consequences of HLA-B*13-Associated Escape Mutations on HIV-1 Replication and Nef Function

PubMed Central

Shahid, Aniqa; Olvera, Alex; Anmole, Gursev; Kuang, Xiaomei T.; Cotton, Laura A.; Plana, Montserrat; Brander, Christian; Brockman, Mark A.

2015-01-01

ABSTRACT HLA-B*13 is associated with superior in vivo HIV-1 viremia control. Protection is thought to be mediated by sustained targeting of key cytotoxic T lymphocyte (CTL) epitopes and viral fitness costs of CTL escape in Gag although additional factors may contribute. We assessed the impact of 10 published B*13-associated polymorphisms in Gag, Pol, and Nef, in 23 biologically relevant combinations, on HIV-1 replication capacity and Nef-mediated reduction of cell surface CD4 and HLA class I expression. Mutations were engineered into HIV-1NL4.3, and replication capacity was measured using a green fluorescent protein (GFP) reporter T cell line. Nef-mediated CD4 and HLA-A*02 downregulation was assessed by flow cytometry, and T cell recognition of infected target cells was measured via coculture with an HIV-specific luciferase reporter cell line. When tested individually, only Gag-I147L and Gag-I437L incurred replicative costs (5% and 17%, respectively), consistent with prior reports. The Gag-I437L-mediated replication defect was rescued to wild-type levels by the adjacent K436R mutation. A novel B*13 epitope, comprising 8 residues and terminating at Gag147, was identified in p24Gag (GQMVHQAIGag140–147). No other single or combination Gag, Pol, or Nef mutant impaired viral replication. Single Nef mutations did not affect CD4 or HLA downregulation; however, the Nef double mutant E24Q-Q107R showed 40% impairment in HLA downregulation with no evidence of Nef stability defects. Moreover, target cells infected with HIV-1-NefE24Q-Q107R were recognized better by HIV-specific T cells than those infected with HIV-1NL4.3 or single Nef mutants. Our results indicate that CTL escape in Gag and Nef can be functionally costly and suggest that these effects may contribute to long-term HIV-1 control by HLA-B*13. IMPORTANCE Protective effects of HLA-B*13 on HIV-1 disease progression are mediated in part by fitness costs of CTL escape mutations in conserved Gag epitopes, but other mechanisms remain incompletely known. We extend our knowledge of the impact of B*13-driven escape on HIV-1 replication by identifying Gag-K436R as a compensatory mutation for the fitness-costly Gag-I437L. We also identify Gag-I147L, the most rapidly and commonly selected B*13-driven substitution in HIV-1, as a putative C-terminal anchor residue mutation in a novel B*13 epitope. Most notably, we identify a novel escape-driven fitness defect: B*13-driven substitutions E24Q and Q107R in Nef, when present together, substantially impair this protein's ability to downregulate HLA class I. This, in turn, increases the visibility of infected cells to HIV-specific T cells. Our results suggest that B*13-associated escape mutations impair HIV-1 replication by two distinct mechanisms, that is, by reducing Gag fitness and dampening Nef immune evasion function. PMID:26355081

The Escherichia coli Cryptic Prophage Protein YfdR Binds to DnaA and Initiation of Chromosomal Replication Is Inhibited by Overexpression of the Gene Cluster yfdQ-yfdR-yfdS-yfdT

PubMed Central

Noguchi, Yasunori; Katayama, Tsutomu

2016-01-01

The initiation of bacterial chromosomal replication is regulated by multiple pathways. To explore novel regulators, we isolated multicopy suppressors for the cold-sensitive hda-185 ΔsfiA(sulA) mutant. Hda is crucial for the negative regulation of the initiator DnaA and the hda-185 mutation causes severe replication overinitiation at the replication origin oriC. The SOS-associated division inhibitor SfiA inhibits FtsZ ring formation, an essential step for cell division regulation during the SOS response, and ΔsfiA enhances the cold sensitivity of hda-185 cells in colony formation. One of the suppressors comprised the yfdQ-yfdR-yfdS-yfdT gene cluster carried on a cryptic prophage. Increased copy numbers of yfdQRT or yfdQRS inhibited not only hda-185-dependent overinitiation, but also replication overinitiation in a hyperactive dnaA mutant, and in a mutant lacking an oriC-binding initiation-inhibitor SeqA. In addition, increasing the copy number of the gene set inhibited the growth of cells bearing specific, initiation-impairing dnaA mutations. In wild-type cells, multicopy supply of yfdQRT or yfdQRS also inhibited replication initiation and increased hydroxyurea (HU)-resistance, as seen in cells lacking DiaA, a stimulator of DnaA assembly on oriC. Deletion of the yfdQ-yfdR-yfdS-yfdT genes did not affect either HU resistance or initiation regulation. Furthermore, we found that DnaA bound specifically to YfdR in soluble protein extracts oversupplied with YfdQRST. Purified YfdR also bound to DnaA, and DnaA Phe46, an amino acid residue crucial for DnaA interactions with DiaA and DnaB replicative helicase was important for this interaction. Consistently, YfdR moderately inhibited DiaA-DnaA and DnaB-DnaA interactions. In addition, protein extracts oversupplied with YfdQRST inhibited replication initiation in vitro. Given the roles of yfdQ and yfdS in cell tolerance to specific environmental stresses, the yfdQ-yfdR-yfdS-yfdT genes might downregulate the initiator DnaA-oriC complex under specific growth conditions. PMID:26973617

The Escherichia coli Cryptic Prophage Protein YfdR Binds to DnaA and Initiation of Chromosomal Replication Is Inhibited by Overexpression of the Gene Cluster yfdQ-yfdR-yfdS-yfdT.

PubMed

Noguchi, Yasunori; Katayama, Tsutomu

2016-01-01

The initiation of bacterial chromosomal replication is regulated by multiple pathways. To explore novel regulators, we isolated multicopy suppressors for the cold-sensitive hda-185 ΔsfiA(sulA) mutant. Hda is crucial for the negative regulation of the initiator DnaA and the hda-185 mutation causes severe replication overinitiation at the replication origin oriC. The SOS-associated division inhibitor SfiA inhibits FtsZ ring formation, an essential step for cell division regulation during the SOS response, and ΔsfiA enhances the cold sensitivity of hda-185 cells in colony formation. One of the suppressors comprised the yfdQ-yfdR-yfdS-yfdT gene cluster carried on a cryptic prophage. Increased copy numbers of yfdQRT or yfdQRS inhibited not only hda-185-dependent overinitiation, but also replication overinitiation in a hyperactive dnaA mutant, and in a mutant lacking an oriC-binding initiation-inhibitor SeqA. In addition, increasing the copy number of the gene set inhibited the growth of cells bearing specific, initiation-impairing dnaA mutations. In wild-type cells, multicopy supply of yfdQRT or yfdQRS also inhibited replication initiation and increased hydroxyurea (HU)-resistance, as seen in cells lacking DiaA, a stimulator of DnaA assembly on oriC. Deletion of the yfdQ-yfdR-yfdS-yfdT genes did not affect either HU resistance or initiation regulation. Furthermore, we found that DnaA bound specifically to YfdR in soluble protein extracts oversupplied with YfdQRST. Purified YfdR also bound to DnaA, and DnaA Phe46, an amino acid residue crucial for DnaA interactions with DiaA and DnaB replicative helicase was important for this interaction. Consistently, YfdR moderately inhibited DiaA-DnaA and DnaB-DnaA interactions. In addition, protein extracts oversupplied with YfdQRST inhibited replication initiation in vitro. Given the roles of yfdQ and yfdS in cell tolerance to specific environmental stresses, the yfdQ-yfdR-yfdS-yfdT genes might downregulate the initiator DnaA-oriC complex under specific growth conditions.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ambrose, R.L.; Mackenzie, J.M., E-mail: jason.mackenzie@unimelb.edu.au

The West Nile virus strain Kunjin virus (WNV{sub KUN}) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNV{sub KUN} replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNV{sub KUN} replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion andmore » membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. - Highlights: • Mutation of Proline13 of the WNV NS4A protein is lethal to replication. • 1st TMB helix of NS4A contributes to protein stability and membrane remodelling. • Unstable mutants of NS4A can be rescued with a proteasome inhibitor. • This study (and of others) contributes to a functional mapping of the NS4A protein.« less

A Tumor-stroma Targeted Oncolytic Adenovirus Replicated in Human Ovary Cancer Samples and Inhibited Growth of Disseminated Solid Tumors in Mice

PubMed Central

Lopez, M Veronica; Rivera, Angel A; Viale, Diego L; Benedetti, Lorena; Cuneo, Nicasio; Kimball, Kristopher J; Wang, Minghui; Douglas, Joanne T; Zhu, Zeng B; Bravo, Alicia I; Gidekel, Manuel; Alvarez, Ronald D; Curiel, David T; Podhajcer, Osvaldo L

2012-01-01

Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 1010 v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15–40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression. PMID:22948673

Amino acid changes within the E protein hinge region that affect dengue virus type 2 infectivity and fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Butrapet, Siritorn; Childers, Thomas; Moss, Kelley J.

Fifteen mutant dengue viruses were engineered and used to identify AAs in the molecular hinge of the envelope protein that are critical to viral infection. Substitutions at Q52, A54, or E133 reduced infectivity in mammalian cells and altered the pH threshold of fusion. Mutations at F193, G266, I270, or G281 affected viral replication in mammalian and mosquito cells, but only I270W had reduced fusion activity. T280Y affected the pH threshold for fusion and reduced replication in C6/36 cells. Three different mutations at L135 were lethal in mammalian cells. Among them, L135G abrogated fusion and reduced replication in C6/36 cells, butmore » only slightly reduced the mosquito infection rate. Conversely, L135W replicated well in C6/36 cells, but had the lowest mosquito infection rate. Possible interactions between hinge residues 52 and 277, or among 53, 135, 170, 186, 265, and 276 required for hinge function were discovered by sequence analysis to identify compensatory mutations.« less

RPA Mediates Recruitment of MRX to Forks and Double-Strand Breaks to Hold Sister Chromatids Together.

PubMed

Seeber, Andrew; Hegnauer, Anna Maria; Hustedt, Nicole; Deshpande, Ishan; Poli, Jérôme; Eglinger, Jan; Pasero, Philippe; Gut, Heinz; Shinohara, Miki; Hopfner, Karl-Peter; Shimada, Kenji; Gasser, Susan M

2016-12-01

The Mre11-Rad50-Xrs2 (MRX) complex is related to SMC complexes that form rings capable of holding two distinct DNA strands together. MRX functions at stalled replication forks and double-strand breaks (DSBs). A mutation in the N-terminal OB fold of the 70 kDa subunit of yeast replication protein A, rfa1-t11, abrogates MRX recruitment to both types of DNA damage. The rfa1 mutation is functionally epistatic with loss of any of the MRX subunits for survival of replication fork stress or DSB recovery, although it does not compromise end-resection. High-resolution imaging shows that either the rfa1-t11 or the rad50Δ mutation lets stalled replication forks collapse and allows the separation not only of opposing ends but of sister chromatids at breaks. Given that cohesin loss does not provoke visible sister separation as long as the RPA-MRX contacts are intact, we conclude that MRX also serves as a structural linchpin holding sister chromatids together at breaks. Copyright © 2016 Elsevier Inc. All rights reserved.

Inherited Mitochondrial Diseases of DNA Replication

PubMed Central

Copeland, William C.

2007-01-01

Mitochondrial genetic diseases can result from defects in mitochondrial DNA (mtDNA) in the form of deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These mutations may be spontaneous, maternally inherited, or a result of inherited nuclear defects in genes that maintain mtDNA. This review focuses on our current understanding of nuclear gene mutations that produce mtDNA alterations and cause mitochondrial depletion syndrome (MDS), progressive external ophthalmoplegia (PEO), ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). To date, all of these etiologic nuclear genes fall into one of two categories: genes whose products function directly at the mtDNA replication fork, such as POLG, POLG2, and TWINKLE, or genes whose products supply the mitochondria with deoxynucleotide triphosphate pools needed for DNA replication, such as TK2, DGUOK, TP, SUCLA2, ANT1, and possibly the newly identified MPV17. PMID:17892433

Experimentally guided models reveal replication principles that shape the mutation distribution of RNA viruses

PubMed Central

Schulte, Michael B; Draghi, Jeremy A; Plotkin, Joshua B; Andino, Raul

2015-01-01

Life history theory posits that the sequence and timing of events in an organism's lifespan are fine-tuned by evolution to maximize the production of viable offspring. In a virus, a life history strategy is largely manifested in its replication mode. Here, we develop a stochastic mathematical model to infer the replication mode shaping the structure and mutation distribution of a poliovirus population in an intact single infected cell. We measure production of RNA and poliovirus particles through the infection cycle, and use these data to infer the parameters of our model. We find that on average the viral progeny produced from each cell are approximately five generations removed from the infecting virus. Multiple generations within a single cell infection provide opportunities for significant accumulation of mutations per viral genome and for intracellular selection. DOI: http://dx.doi.org/10.7554/eLife.03753.001 PMID:25635405

HIV-1 Vpr increases Env expression by preventing Env from endoplasmic reticulum-associated protein degradation (ERAD).

PubMed

Zhang, Xianfeng; Zhou, Tao; Frabutt, Dylan A; Zheng, Yong-Hui

2016-09-01

Vpr enhances HIV-1 replication in macrophages and dendritic cells, as well as the human CD4(+) CEM.NKR T cell line. Recently, Vpr was reported to increase HIV-1 Env expression in macrophages. Here, we report that Vpr also increases HIV-1 Env expression in dendritic cells and CEM.NKR cells. The Vpr activity depends on its N-terminal region, which was disrupted by a single A30L mutation. Env was rapidly degraded in the absence of Vpr, which was blocked by the ERAD pathway inhibitor kifunesine or the lysosome inhibitor Bafilomycin. As2O3 or PK11195, which reportedly enhances HIV-1 Env folding, also blocked the Env degradation in CEM.NKR cells. Thus, these results not only identify Env as a primary target for Vpr to boost HIV-1 replication, but also suggest that Vpr likely promotes Env folding in the ER, which is otherwise misfolded and targeted by the ERAD pathway to lysosomes for degradation. Copyright © 2016 Elsevier Inc. All rights reserved.

Lamin A/C Depletion Enhances DNA Damage-Induced Stalled Replication Fork Arrest

PubMed Central

Singh, Mayank; Hunt, Clayton R.; Pandita, Raj K.; Kumar, Rakesh; Yang, Chin-Rang; Horikoshi, Nobuo; Bachoo, Robert; Serag, Sara; Story, Michael D.; Shay, Jerry W.; Powell, Simon N.; Gupta, Arun; Jeffery, Jessie; Pandita, Shruti; Chen, Benjamin P. C.; Deckbar, Dorothee; Löbrich, Markus; Yang, Qin; Khanna, Kum Kum; Worman, Howard J.

2013-01-01

The human LMNA gene encodes the essential nuclear envelope proteins lamin A and C (lamin A/C). Mutations in LMNA result in altered nuclear morphology, but how this impacts the mechanisms that maintain genomic stability is unclear. Here, we report that lamin A/C-deficient cells have a normal response to ionizing radiation but are sensitive to agents that cause interstrand cross-links (ICLs) or replication stress. In response to treatment with ICL agents (cisplatin, camptothecin, and mitomycin), lamin A/C-deficient cells displayed normal γ-H2AX focus formation but a higher frequency of cells with delayed γ-H2AX removal, decreased recruitment of the FANCD2 repair factor, and a higher frequency of chromosome aberrations. Similarly, following hydroxyurea-induced replication stress, lamin A/C-deficient cells had an increased frequency of cells with delayed disappearance of γ-H2AX foci and defective repair factor recruitment (Mre11, CtIP, Rad51, RPA, and FANCD2). Replicative stress also resulted in a higher frequency of chromosomal aberrations as well as defective replication restart. Taken together, the data can be interpreted to suggest that lamin A/C has a role in the restart of stalled replication forks, a prerequisite for initiation of DNA damage repair by the homologous recombination pathway, which is intact in lamin A/C-deficient cells. We propose that lamin A/C is required for maintaining genomic stability following replication fork stalling, induced by either ICL damage or replicative stress, in order to facilitate fork regression prior to DNA damage repair. PMID:23319047

Mutations in Replicative Stress Response Pathways Are Associated with S Phase-specific Defects in Nucleotide Excision Repair*

PubMed Central

Bélanger, François; Angers, Jean-Philippe; Fortier, Émile; Hammond-Martel, Ian; Costantino, Santiago; Drobetsky, Elliot; Wurtele, Hugo

2016-01-01

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1–3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response. PMID:26578521

Importance of DNA repair in tumor suppression

NASA Astrophysics Data System (ADS)

Brumer, Yisroel; Shakhnovich, Eugene I.

2004-12-01

The transition from a normal to cancerous cell requires a number of highly specific mutations that affect cell cycle regulation, apoptosis, differentiation, and many other cell functions. One hallmark of cancerous genomes is genomic instability, with mutation rates far greater than those of normal cells. In microsatellite instability (MIN tumors), these are often caused by damage to mismatch repair genes, allowing further mutation of the genome and tumor progression. These mutation rates may lie near the error catastrophe found in the quasispecies model of adaptive RNA genomes, suggesting that further increasing mutation rates will destroy cancerous genomes. However, recent results have demonstrated that DNA genomes exhibit an error threshold at mutation rates far lower than their conservative counterparts. Furthermore, while the maximum viable mutation rate in conservative systems increases indefinitely with increasing master sequence fitness, the semiconservative threshold plateaus at a relatively low value. This implies a paradox, wherein inaccessible mutation rates are found in viable tumor cells. In this paper, we address this paradox, demonstrating an isomorphism between the conservatively replicating (RNA) quasispecies model and the semiconservative (DNA) model with post-methylation DNA repair mechanisms impaired. Thus, as DNA repair becomes inactivated, the maximum viable mutation rate increases smoothly to that of a conservatively replicating system on a transformed landscape, with an upper bound that is dependent on replication rates. On a specific single fitness peak landscape, the repair-free semiconservative system is shown to mimic a conservative system exactly. We postulate that inactivation of post-methylation repair mechanisms is fundamental to the progression of a tumor cell and hence these mechanisms act as a method for the prevention and destruction of cancerous genomes.

Meier–Gorlin syndrome genotype–phenotype studies: 35 individuals with pre-replication complex gene mutations and 10 without molecular diagnosis

PubMed Central

de Munnik, Sonja A; Bicknell, Louise S; Aftimos, Salim; Al-Aama, Jumana Y; van Bever, Yolande; Bober, Michael B; Clayton-Smith, Jill; Edrees, Alaa Y; Feingold, Murray; Fryer, Alan; van Hagen, Johanna M; Hennekam, Raoul C; Jansweijer, Maaike C E; Johnson, Diana; Kant, Sarina G; Opitz, John M; Ramadevi, A Radha; Reardon, Willie; Ross, Alison; Sarda, Pierre; Schrander-Stumpel, Constance T R M; Schoots, Jeroen; Temple, I Karen; Terhal, Paulien A; Toutain, Annick; Wise, Carol A; Wright, Michael; Skidmore, David L; Samuels, Mark E; Hoefsloot, Lies H; Knoers, Nine V A M; Brunner, Han G; Jackson, Andrew P; Bongers, Ernie M H F

2012-01-01

Meier–Gorlin syndrome (MGS) is an autosomal recessive disorder characterized by microtia, patellar aplasia/hypoplasia, and short stature. Recently, mutations in five genes from the pre-replication complex (ORC1, ORC4, ORC6, CDT1, and CDC6), crucial in cell-cycle progression and growth, were identified in individuals with MGS. Here, we report on genotype–phenotype studies in 45 individuals with MGS (27 females, 18 males; age 3 months–47 years). Thirty-five individuals had biallelic mutations in one of the five causative pre-replication genes. No homozygous or compound heterozygous null mutations were detected. In 10 individuals, no definitive molecular diagnosis was made. The triad of microtia, absent/hypoplastic patellae, and short stature was observed in 82% of individuals with MGS. Additional frequent clinical features were mammary hypoplasia (100%) and abnormal genitalia (42% predominantly cryptorchidism and hypoplastic labia minora/majora). One individual with ORC1 mutations only had short stature, emphasizing the highly variable clinical spectrum of MGS. Individuals with ORC1 mutations had significantly shorter stature and smaller head circumferences than individuals from other gene categories. Furthermore, compared with homozygous missense mutations, compound heterozygous mutations appeared to have a more severe effect on phenotype, causing more severe growth retardation in ORC4 and more frequently pulmonary emphysema in CDT1. A lethal phenotype was seen in four individuals with compound heterozygous ORC1 and CDT1 mutations. No other clear genotype–phenotype association was observed. Growth hormone and estrogen treatment may be of some benefit, respectively, to growth retardation and breast hypoplasia, though further studies in this patient group are needed. PMID:22333897

In vivo virulence of MHC-adapted AIDS virus serially-passaged through MHC-mismatched hosts

PubMed Central

Yamamoto, Hiroyuki; Ishii, Hiroshi; Matsuoka, Saori; Mizuta, Kazuta; Sakawaki, Hiromi; Miura, Tomoyuki; Naruse, Taeko K.; Kimura, Akinori

2017-01-01

CD8+ T-cell responses exert strong suppressive pressure on HIV replication and select for viral escape mutations. Some of these major histocompatibility complex class I (MHC-I)-associated mutations result in reduction of in vitro viral replicative capacity. While these mutations can revert after viral transmission to MHC-I-disparate hosts, recent studies have suggested that these MHC-I-associated mutations accumulate in populations and make viruses less pathogenic in vitro. Here, we directly show an increase in the in vivo virulence of an MHC-I-adapted virus serially-passaged through MHC-I-mismatched hosts in a macaque AIDS model despite a reduction in in vitro viral fitness. The first passage simian immunodeficiency virus (1pSIV) obtained 1 year after SIVmac239 infection in a macaque possessing a protective MHC-I haplotype 90-120-Ia was transmitted into 90-120-Ia- macaques, whose plasma 1 year post-infection was transmitted into other 90-120-Ia- macaques to obtain the third passage SIV (3pSIV). Most of the 90-120-Ia-associated mutations selected in 1pSIV did not revert even in 3pSIV. 3pSIV showed lower in vitro viral fitness but induced persistent viremia in 90-120-Ia- macaques. Remarkably, 3pSIV infection in 90-120-Ia+ macaques resulted in significantly higher viral loads and reduced survival compared to wild-type SIVmac239. These results indicate that MHC-I-adapted SIVs serially-transmitted through MHC-I-mismatched hosts can have higher virulence in MHC-I-matched hosts despite their lower in vitro viral fitness. This study suggests that multiply-passaged HIVs could result in loss of HIV-specific CD8+ T cell responses in human populations and the in vivo pathogenic potential of these escaped viruses may be enhanced. PMID:28931083

A novel aminoacid determinant of HIV-1 restriction in the TRIM5α variable 1 region isolated in a random mutagenic screen.

PubMed

Pham, Quang Toan; Veillette, Maxime; Brandariz-Nuñez, Alberto; Pawlica, Paulina; Thibert-Lefebvre, Caroline; Chandonnet, Nadia; Diaz-Griffero, Felipe; Berthoux, Lionel

2013-05-01

Human-derived antiretroviral transgenes are of great biomedical interest and are actively pursued. HIV-1 is efficiently inhibited at post-entry, pre-integration replication stages by point mutations in the variable region 1 (v1) of the human restriction factor TRIM5α. Here we use a mutated megaprimer approach to create a mutant library of TRIM5αHu v1 and to isolate a mutation at Gly330 (G330E) that inhibits transduction of an HIV-1 vector as efficiently as the previously described mutants at positions Arg332 and Arg335. As was the case for these other mutations, modification of the local v1 charge toward increased acidity was key to inhibiting HIV-1. G330E TRIM5αHu also disrupted replication-competent HIV-1 propagation in a human T cell line. Interestingly, G330E did not enhance restriction of HIV-1 when combined with mutations at Arg332 or Arg335. Accordingly, the triple mutant G330E-R332G-R335G bound purified recombinant HIV-1 capsid tubes less efficiently than the double mutant R332G-R335G did. In a structural model of the TRIM5αHu PRYSPRY domain, the addition of G330E to the double mutant R332G-R335G caused extensive changes to the capsid-binding surface, which may explain why the triple mutant was no more restrictive than the double mutant. The HIV-1 inhibitory potential of Gly330 mutants was not predicted by examination of natural TRIM5α orthologs that are known to strongly inhibit HIV-1. This work underlines the potential of random mutagenesis to isolate novel variants of human proteins with antiviral properties. Copyright © 2013 Elsevier B.V. All rights reserved.

Differential Function of Lip Residues in the Mechanism and Biology of an Anthrax Hemophore

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ekworomadu, MarCia T.; Poor, Catherine B.; Owens, Cedric P.

To replicate in mammalian hosts, bacterial pathogens must acquire iron. The majority of iron is coordinated to the protoporphyrin ring of heme, which is further bound to hemoglobin. Pathogenic bacteria utilize secreted hemophores to acquire heme from heme sources such as hemoglobin. Bacillus anthracis, the causative agent of anthrax disease, secretes two hemophores, IsdX1 and IsdX2, to acquire heme from host hemoglobin and enhance bacterial replication in iron-starved environments. Both proteins contain NEAr-iron Transporter (NEAT) domains, a conserved protein module that functions in heme acquisition in Gram-positive pathogens. Here, we report the structure of IsdX1, the first of a Gram-positivemore » hemophore, with and without bound heme. Overall, IsdX1 forms an immunoglobin-like fold that contains, similar to other NEAT proteins, a 3{sub 10}-helix near the heme-binding site. Because the mechanistic function of this helix in NEAT proteins is not yet defined, we focused on the contribution of this region to hemophore and NEAT protein activity, both biochemically and biologically in cultured cells. Site-directed mutagenesis of amino acids in and adjacent to the helix identified residues important for heme and hemoglobin association, with some mutations affecting both properties and other mutations affecting only heme stabilization. IsdX1 with mutations that reduced the ability to associate with hemoglobin and bind heme failed to restore the growth of a hemophore-deficient strain of B. anthracis on hemoglobin as the sole iron source. These data indicate that not only is the 3{sub 10}-helix important for NEAT protein biology, but also that the processes of hemoglobin and heme binding can be both separate as well as coupled, the latter function being necessary for maximal heme-scavenging activity. These studies enhance our understanding of NEAT domain and hemophore function and set the stage for structure-based inhibitor design to block NEAT domain interaction with upstream ligands.« less

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs.

PubMed

Chen, Shu-Chuan; Jeng, King-Song; Lai, Michael M C

2017-10-15

Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5' untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment. Copyright © 2017 American Society for Microbiology.

Zinc Finger-Containing Cellular Transcription Corepressor ZBTB25 Promotes Influenza Virus RNA Transcription and Is a Target for Zinc Ejector Drugs

PubMed Central

Chen, Shu-Chuan; Jeng, King-Song

2017-01-01

ABSTRACT Influenza A virus (IAV) replication relies on an intricate interaction between virus and host cells. How the cellular proteins are usurped for IAV replication remains largely obscure. The aim of this study was to search for novel and potential cellular factors that participate in IAV replication. ZBTB25, a transcription repressor of a variety of cellular genes, was identified by an RNA interference (RNAi) genomic library screen. Depletion of ZBTB25 significantly reduced IAV production. Conversely, overexpression of ZBTB25 enhanced it. ZBTB25 interacted with the viral RNA-dependent RNA polymerase (RdRp) protein and modulated its transcription activity. In addition, ZBTB25 also functioned as a viral RNA (vRNA)-binding protein, binding preferentially to the U-rich sequence within the 5′ untranslated region (UTR) of vRNA. Both protein-protein and protein-RNA interactions involving ZBTB25 facilitated viral RNA transcription and replication. In addition, ZBTB25 suppressed interferon production, further enhancing viral replication. ZBTB25-associated functions required an intact zinc finger domain and posttranslational SUMO-1 modification of ZBTB25. Furthermore, treatment with disulfiram (a zinc ejector) of ZBTB25-overexpressing cells showed significantly reduced IAV production as a result of reduced RNA synthesis. Our findings indicate that IAV usurps ZBTB25 for IAV RNA synthesis and serves as a novel and potential therapeutic antiviral target. IMPORTANCE IAV-induced seasonal influenza causes severe illness and death in high-risk populations. However, IAV has developed resistance to current antiviral drugs due to its high mutation rate. Therefore, development of drugs targeting cellular factors required for IAV replication is an attractive alternative for IAV therapy. Here, we discovered a cellular protein, ZBTB25, that enhances viral RdRp activity by binding to both viral RdRp and viral RNA to stimulate viral RNA synthesis. A unique feature of ZBTB25 in the regulation of viral replication is its dual transcription functions, namely, promoting viral RNA transcription through binding to the U-rich region of vRNA and suppressing cellular interferon production. ZBTB25 contains a zinc finger domain that is required for RNA-inhibitory activity by chelating zinc ions. Disulfiram treatment disrupts the zinc finger functions, effectively repressing IAV replication. Based on our findings, we demonstrate that ZBTB25 regulates IAV RNA transcription and replication and serves as a promising antiviral target for IAV treatment. PMID:28768860

Centromere replication timing determines different forms of genomic instability in Saccharomyces cerevisiae checkpoint mutants during replication stress.

PubMed

Feng, Wenyi; Bachant, Jeff; Collingwood, David; Raghuraman, M K; Brewer, Bonita J

2009-12-01

Yeast replication checkpoint mutants lose viability following transient exposure to hydroxyurea, a replication-impeding drug. In an effort to understand the basis for this lethality, we discovered that different events are responsible for inviability in checkpoint-deficient cells harboring mutations in the mec1 and rad53 genes. By monitoring genomewide replication dynamics of cells exposed to hydroxyurea, we show that cells with a checkpoint deficient allele of RAD53, rad53K227A, fail to duplicate centromeres. Following removal of the drug, however, rad53K227A cells recover substantial DNA replication, including replication through centromeres. Despite this recovery, the rad53K227A mutant fails to achieve biorientation of sister centromeres during recovery from hydroxyurea, leading to secondary activation of the spindle assembly checkpoint (SAC), aneuploidy, and lethal chromosome segregation errors. We demonstrate that cell lethality from this segregation defect could be partially remedied by reinforcing bipolar attachment. In contrast, cells with the mec1-1 sml1-1 mutations suffer from severely impaired replication resumption upon removal of hydroxyurea. mec1-1 sml1-1 cells can, however, duplicate at least some of their centromeres and achieve bipolar attachment, leading to abortive segregation and fragmentation of incompletely replicated chromosomes. Our results highlight the importance of replicating yeast centromeres early and reveal different mechanisms of cell death due to differences in replication fork progression.

Replication Proteins and Human Disease

PubMed Central

Jackson, Andrew P.; Laskey, Ronald A.; Coleman, Nicholas

2014-01-01

In this article, we discuss the significance of DNA replication proteins in human disease. There is a broad range of mutations in genes encoding replication proteins, which result in several distinct clinical disorders that share common themes. One group of replication proteins, the MCMs, has emerged as effective biomarkers for early detection of a range of common cancers. They offer practical and theoretical advantages over other replication proteins and have been developed for widespread clinical use. PMID:23881941

Fidelity of DNA Replication in Normal and Malignant Human Brest Cells.

DTIC Science & Technology

1995-08-31

cellular DNA replication machinery, we have initiated experiments that utilize a multiprotein DNA replication complex (MRC) isolated from breast cancer...gene in an in vitro DNA replication assay. By utilizing the target gene in a bacterial mutant selection assay we have begun to determine the...frequency with which mutational sequence errors occur as a result of the in vitro DNA replication mediated by the breast cancer cell MRC and the normal breast

Meier-Gorlin syndrome mutations disrupt an Orc1 CDK inhibitory domain and cause centrosome reduplication.

PubMed

Hossain, Manzar; Stillman, Bruce

2012-08-15

Like DNA replication, centrosomes are licensed to duplicate once per cell division cycle to ensure genetic stability. In addition to regulating DNA replication, the Orc1 subunit of the human origin recognition complex controls centriole and centrosome copy number. Here we report that Orc1 harbors a PACT centrosome-targeting domain and a separate domain that differentially inhibits the protein kinase activities of Cyclin E-CDK2 and Cyclin A-CDK2. A cyclin-binding motif (Cy motif) is required for Orc1 to bind Cyclin A and inhibit Cyclin A-CDK2 kinase activity but has no effect on Cyclin E-CDK2 kinase activity. In contrast, Orc1 inhibition of Cyclin E-CDK2 kinase activity occurs by a different mechanism that is affected by Orc1 mutations identified in Meier-Gorlin syndrome patients. The cyclin/CDK2 kinase inhibitory domain of Orc1, when tethered to the PACT domain, localizes to centrosomes and blocks centrosome reduplication. Meier-Gorlin syndrome mutations that disrupt Cyclin E-CDK2 kinase inhibition also allow centrosome reduplication. Thus, Orc1 contains distinct domains that control centrosome copy number and DNA replication. We suggest that the Orc1 mutations present in some Meier-Gorlin syndrome patients contribute to the pronounced microcephaly and dwarfism observed in these individuals by altering centrosome duplication in addition to DNA replication defects.

Development of drug resistance mutations in patients on highly active antiretroviral therapy: does competitive advantage drive evolution.

PubMed

Kolber, Michael A

2007-01-01

Most physicians that treat individuals with HIV-1 disease are able to successfully suppress viral replication with the pharmacologic armamentarium available today. For the majority of patients this results in immune reconstitution and improved quality of life. However, a large fraction of these patients have transient elevations in their viral burden and even persistence of low-level viremia. In fact, many individuals whose viral load is suppressed to < 50 c/ml have evidence of low-level viral replication. The impact of low-level viremia and persistent viral replication is an area of significant study and interest owing to the potential for the development of drug resistance mutations. Here the fundamental question is whether and perhaps what factors provide a venue for the development of resistant virus. The concern is clearly the eventual progression of disease with the exhaustion of treatment options. The purpose of this review is to evaluate the current literature regarding the effect of low-level viremia on the development of drug resistance mutations. Herein, we discuss the impact of different levels of viral suppression on the development of mutations. In addition, we look at the role that resistance and fitness play in determining the survival of a breakthrough mutation within the background of drug.

DNA replication error-induced extinction of diploid yeast.

PubMed

Herr, Alan J; Kennedy, Scott R; Knowels, Gary M; Schultz, Eric M; Preston, Bradley D

2014-03-01

Genetic defects in DNA polymerase accuracy, proofreading, or mismatch repair (MMR) induce mutator phenotypes that accelerate adaptation of microbes and tumor cells. Certain combinations of mutator alleles synergistically increase mutation rates to levels that drive extinction of haploid cells. The maximum tolerated mutation rate of diploid cells is unknown. Here, we define the threshold for replication error-induced extinction (EEX) of diploid Saccharomyces cerevisiae. Double-mutant pol3 alleles that carry mutations for defective DNA polymerase-δ proofreading (pol3-01) and accuracy (pol3-L612M or pol3-L612G) induce strong mutator phenotypes in heterozygous diploids (POL3/pol3-01,L612M or POL3/pol3-01,L612G). Both pol3-01,L612M and pol3-01,L612G alleles are lethal in the homozygous state; cells with pol3-01,L612M divide up to 10 times before arresting at random stages in the cell cycle. Antimutator eex mutations in the pol3 alleles suppress this lethality (pol3-01,L612M,eex or pol3-01,L612G,eex). MMR defects synergize with pol3-01,L612M,eex and pol3-01,L612G,eex alleles, increasing mutation rates and impairing growth. Conversely, inactivation of the Dun1 S-phase checkpoint kinase suppresses strong pol3-01,L612M,eex and pol3-01,L612G,eex mutator phenotypes as well as the lethal pol3-01,L612M phenotype. Our results reveal that the lethal error threshold in diploids is 10 times higher than in haploids and likely determined by homozygous inactivation of essential genes. Pronounced loss of fitness occurs at mutation rates well below the lethal threshold, suggesting that mutator-driven cancers may be susceptible to drugs that exacerbate replication errors.

Role of Occult and Post-acute Phase Replication in Protective Immunity Induced with a Novel Live Attenuated SIV Vaccine

PubMed Central

Ham, Claire; Ferguson, Deborah; Tudor, Hannah; Mattiuzzo, Giada; Klaver, Bep; Page, Mark; Stebbings, Richard; Das, Atze T.; Berkhout, Ben; Almond, Neil; Cranage, Martin P.

2016-01-01

In order to evaluate the role of persisting virus replication during occult phase immunisation in the live attenuated SIV vaccine model, a novel SIVmac239Δnef variant (SIVrtTA) genetically engineered to replicate in the presence of doxycycline was evaluated for its ability to protect against wild-type SIVmac239. Indian rhesus macaques were vaccinated either with SIVrtTA or with SIVmac239Δnef. Doxycycline was withdrawn from 4 of 8 SIVrtTA vaccinates before challenge with wild-type virus. Unvaccinated challenge controls exhibited ~107 peak plasma viral RNA copies/ml persisting beyond the acute phase. Six vaccinates, four SIVmac239Δnef and two SIVrtTA vaccinates exhibited complete protection, defined by lack of wild-type viraemia post-challenge and virus-specific PCR analysis of tissues recovered post-mortem, whereas six SIVrtTA vaccinates were protected from high levels of viraemia. Critically, the complete protection in two SIVrtTA vaccinates was associated with enhanced SIVrtTA replication in the immediate post-acute vaccination period but was independent of doxycycline status at the time of challenge. Mutations were identified in the LTR promoter region and rtTA gene that do not affect doxycycline-control but were associated with enhanced post-acute phase replication in protected vaccinates. High frequencies of total circulating CD8+T effector memory cells and a higher total frequency of SIV-specific CD8+ mono and polyfunctional T cells on the day of wild-type challenge were associated with complete protection but these parameters were not predictive of outcome when assessed 130 days after challenge. Moreover, challenge virus-specific Nef CD8+ polyfunctional T cell responses and antigen were detected in tissues post mortem in completely-protected macaques indicating post-challenge control of infection. Within the parameters of the study design, on-going occult-phase replication may not be absolutely required for protective immunity. PMID:28002473

Random codon re-encoding induces stable reduction of replicative fitness of Chikungunya virus in primate and mosquito cells.

PubMed

Nougairede, Antoine; De Fabritus, Lauriane; Aubry, Fabien; Gould, Ernest A; Holmes, Edward C; de Lamballerie, Xavier

2013-02-01

Large-scale codon re-encoding represents a powerful method of attenuating viruses to generate safe and cost-effective vaccines. In contrast to specific approaches of codon re-encoding which modify genome-scale properties, we evaluated the effects of random codon re-encoding on the re-emerging human pathogen Chikungunya virus (CHIKV), and assessed the stability of the resultant viruses during serial in cellulo passage. Using different combinations of three 1.4 kb randomly re-encoded regions located throughout the CHIKV genome six codon re-encoded viruses were obtained. Introducing a large number of slightly deleterious synonymous mutations reduced the replicative fitness of CHIKV in both primate and arthropod cells, demonstrating the impact of synonymous mutations on fitness. Decrease of replicative fitness correlated with the extent of re-encoding, an observation that may assist in the modulation of viral attenuation. The wild-type and two re-encoded viruses were passaged 50 times either in primate or insect cells, or in each cell line alternately. These viruses were analyzed using detailed fitness assays, complete genome sequences and the analysis of intra-population genetic diversity. The response to codon re-encoding and adaptation to culture conditions occurred simultaneously, resulting in significant replicative fitness increases for both re-encoded and wild type viruses. Importantly, however, the most re-encoded virus failed to recover its replicative fitness. Evolution of these viruses in response to codon re-encoding was largely characterized by the emergence of both synonymous and non-synonymous mutations, sometimes located in genomic regions other than those involving re-encoding, and multiple convergent and compensatory mutations. However, there was a striking absence of codon reversion (<0.4%). Finally, multiple mutations were rapidly fixed in primate cells, whereas mosquito cells acted as a brake on evolution. In conclusion, random codon re-encoding provides important information on the evolution and genetic stability of CHIKV viruses and could be exploited to develop a safe, live attenuated CHIKV vaccine.

The infectivities of turnip yellow mosaic virus genomes with altered tRNA mimicry are not dependent on compensating mutations in the viral replication protein.

PubMed

Filichkin, S A; Bransom, K L; Goodwin, J B; Dreher, T W

2000-09-01

Five highly infectious turnip yellow mosaic virus (TYMV) genomes with sequence changes in their 3'-terminal regions that result in altered aminoacylation and eEF1A binding have been studied. These genomes were derived from cloned parental RNAs of low infectivity by sequential passaging in plants. Three of these genomes that are incapable of aminoacylation have been reported previously (J. B. Goodwin, J. M. Skuzeski, and T. W. Dreher, Virology 230:113-124, 1997). We now demonstrate by subcloning the 3' untranslated regions into wild-type TYMV RNA that the high infectivities and replication rates of these genomes compared to their progenitors are mostly due to a small number of mutations acquired in the 3' tRNA-like structure during passaging. Mutations in other parts of the genome, including the replication protein coding region, are not required for high infectivity but probably do play a role in optimizing viral amplification and spread in plants. Two other TYMV RNA variants of suboptimal infectivities, one that accepts methionine instead of the usual valine and one that interacts less tightly with eEF1A, were sequentially passaged to produce highly infectious genomes. The improved infectivities of these RNAs were not associated with increased replication in protoplasts, and no mutations were acquired in their 3' tRNA-like structures. Complete sequencing of one genome identified two mutations that result in amino acid changes in the movement protein gene, suggesting that improved infectivity may be a function of improved viral dissemination in plants. Our results show that the wild-type TYMV replication proteins are able to amplify genomes with 3' termini of variable sequence and tRNA mimicry. These and previous results have led to a model in which the binding of eEF1A to the 3' end to antagonize minus-strand initiation is a major role of the tRNA-like structure.

Changes in protein domains outside the catalytic site of the bacteriophage Qβ replicase reduce the mutagenic effect of 5-azacytidine.

PubMed

Cabanillas, Laura; Sanjuán, Rafael; Lázaro, Ester

2014-09-01

The high genetic heterogeneity and great adaptability of RNA viruses are ultimately caused by the low replication fidelity of their polymerases. However, single amino acid substitutions that modify replication fidelity can evolve in response to mutagenic treatments with nucleoside analogues. Here, we investigated how two independent mutants of the bacteriophage Qβ replicase (Thr210Ala and Tyr410His) reduce sensitivity to the nucleoside analogue 5-azacytidine (AZC). Despite being located outside the catalytic site, both mutants reduced the mutation frequency in the presence of the drug. However, they did not modify the type of AZC-induced substitutions, which was mediated mainly by ambiguous base pairing of the analogue with purines. Furthermore, the Thr210Ala and Tyr410His substitutions had little or no effect on replication fidelity in untreated viruses. Also, both substitutions were costly in the absence of AZC or when the action of the drug was suppressed by adding an excess of natural pyrimidines (uridine or cytosine). Overall, the phenotypic properties of these two mutants were highly convergent, despite the mutations being located in different domains of the Qβ replicase. This suggests that treatment with a given nucleoside analogue tends to select for a unique functional response in the viral replicase. In the last years, artificial increase of the replication error rate has been proposed as an antiviral therapy. In this study, we investigated the mechanisms by which two substitutions in the Qβ replicase confer partial resistance to the mutagenic nucleoside analogue AZC. As opposed to previous work with animal viruses, where different mutations selected sequentially conferred nucleoside analogue resistance through different mechanisms, our results suggest that there are few or no alternative AZC resistance phenotypes in Qβ. Also, despite resistance mutations being highly costly in the absence of the drug, there was no sequential fixation of secondary mutations. Bacteriophage Qβ is the virus with the highest reported mutation rate, which should make it particularly sensitive to nucleoside analogue treatments, probably favoring resistance mutations even if they incur high costs. The results are also relevant for understanding the possible pathways by which fidelity of the replication machinery can be modified. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Experimentally Guided Models Reveal Replication Principles that Shape the Mutation Distribution of RNA Viruses

DTIC Science & Technology

2015-01-30

intracel- lular replication. Two classic replication modes have been described for single-stranded RNA viruses: the ‘stamping machine’ mode ( Stent ...Journal of Theoretical Biology 218:309–321. doi: 10.1006/jtbi.2002.3078. Stent GS. 1963. Molecular Biology of Bacterial Viruses. San Francisco, Calif: W H

Low-fidelity Venezuelan equine encephalitis virus polymerase mutants to improve live-attenuated vaccine safety and efficacy

PubMed Central

Kautz, Tiffany F; Guerbois, Mathilde; Khanipov, Kamil; Yun, Ruimei; Warmbrod, Kelsey L; Fofanov, Yuriy; Weaver, Scott C; Forrester, Naomi L

2018-01-01

Abstract During RNA virus replication, there is the potential to incorporate mutations that affect virulence or pathogenesis. For live-attenuated vaccines, this has implications for stability, as replication may result in mutations that either restore the wild-type phenotype via reversion or compensate for the attenuating mutations by increasing virulence (pseudoreversion). Recent studies have demonstrated that altering the mutation rate of an RNA virus is an effective attenuation tool. To validate the safety of low-fidelity mutations to increase vaccine attenuation, several mutations in the RNA-dependent RNA-polymerase (RdRp) were tested in the live-attenuated Venezuelan equine encephalitis virus vaccine strain, TC-83. Next generation sequencing after passage in the presence of mutagens revealed a mutant containing three mutations in the RdRp, TC-83 3x, to have decreased replication fidelity, while a second mutant, TC-83 4x displayed no change in fidelity, but shared many phenotypic characteristics with TC-83 3x. Both mutants exhibited increased, albeit inconsistent attenuation in an infant mouse model, as well as increased immunogenicity and complete protection against lethal challenge of an adult murine model compared with the parent TC-83. During serial passaging in a highly permissive model, the mutants increased in virulence but remained less virulent than the parent TC-83. These results suggest that the incorporation of low-fidelity mutations into the RdRp of live-attenuated vaccines for RNA viruses can confer increased immunogenicity whilst showing some evidence of increased attenuation. However, while in theory such constructs may result in more effective vaccines, the instability of the vaccine phenotype decreases the likelihood of this being an effective vaccine strategy. PMID:29593882

Quinacrine promotes replication and conformational mutation of chronic wasting disease prions.

PubMed

Bian, Jifeng; Kang, Hae-Eun; Telling, Glenn C

2014-04-22

Quinacrine's ability to reduce levels of pathogenic prion protein (PrP(Sc)) in mouse cells infected with experimentally adapted prions led to several unsuccessful clinical studies in patients with prion diseases, a 10-y investment to understand its mechanism of action, and the production of related compounds with expectations of greater efficacy. We show here, in stark contrast to this reported inhibitory effect, that quinacrine enhances deer and elk PrP(Sc) accumulation and promotes propagation of prions causing chronic wasting disease (CWD), a fatal, transmissible, neurodegenerative disorder of cervids of uncertain zoonotic potential. Surprisingly, despite increased prion titers in quinacrine-treated cells, transmission of the resulting prions produced prolonged incubation times and altered PrP(Sc) deposition patterns in the brains of diseased transgenic mice. This unexpected outcome is consistent with quinacrine affecting the intrinsic properties of the CWD prion. Accordingly, quinacrine-treated CWD prions were comprised of an altered PrP(Sc) conformation. Our findings provide convincing evidence for drug-induced conformational mutation of prions without the prerequisite of generating drug-resistant variants of the original strain. More specifically, they show that a drug capable of restraining prions in one species/strain setting, and consequently used to treat human prion diseases, improves replicative ability in another and therefore force reconsideration of current strategies to screen antiprion compounds.

Quinacrine promotes replication and conformational mutation of chronic wasting disease prions

PubMed Central

Bian, Jifeng; Kang, Hae-Eun; Telling, Glenn C.

2014-01-01

Quinacrine’s ability to reduce levels of pathogenic prion protein (PrPSc) in mouse cells infected with experimentally adapted prions led to several unsuccessful clinical studies in patients with prion diseases, a 10-y investment to understand its mechanism of action, and the production of related compounds with expectations of greater efficacy. We show here, in stark contrast to this reported inhibitory effect, that quinacrine enhances deer and elk PrPSc accumulation and promotes propagation of prions causing chronic wasting disease (CWD), a fatal, transmissible, neurodegenerative disorder of cervids of uncertain zoonotic potential. Surprisingly, despite increased prion titers in quinacrine-treated cells, transmission of the resulting prions produced prolonged incubation times and altered PrPSc deposition patterns in the brains of diseased transgenic mice. This unexpected outcome is consistent with quinacrine affecting the intrinsic properties of the CWD prion. Accordingly, quinacrine-treated CWD prions were comprised of an altered PrPSc conformation. Our findings provide convincing evidence for drug-induced conformational mutation of prions without the prerequisite of generating drug-resistant variants of the original strain. More specifically, they show that a drug capable of restraining prions in one species/strain setting, and consequently used to treat human prion diseases, improves replicative ability in another and therefore force reconsideration of current strategies to screen antiprion compounds. PMID:24711410

ERASE-Seq: Leveraging replicate measurements to enhance ultralow frequency variant detection in NGS data

PubMed Central

Kamps-Hughes, Nick; McUsic, Andrew; Kurihara, Laurie; Harkins, Timothy T.; Pal, Prithwish; Ray, Claire

2018-01-01

The accurate detection of ultralow allele frequency variants in DNA samples is of interest in both research and medical settings, particularly in liquid biopsies where cancer mutational status is monitored from circulating DNA. Next-generation sequencing (NGS) technologies employing molecular barcoding have shown promise but significant sensitivity and specificity improvements are still needed to detect mutations in a majority of patients before the metastatic stage. To address this we present analytical validation data for ERASE-Seq (Elimination of Recurrent Artifacts and Stochastic Errors), a method for accurate and sensitive detection of ultralow frequency DNA variants in NGS data. ERASE-Seq differs from previous methods by creating a robust statistical framework to utilize technical replicates in conjunction with background error modeling, providing a 10 to 100-fold reduction in false positive rates compared to published molecular barcoding methods. ERASE-Seq was tested using spiked human DNA mixtures with clinically realistic DNA input quantities to detect SNVs and indels between 0.05% and 1% allele frequency, the range commonly found in liquid biopsy samples. Variants were detected with greater than 90% sensitivity and a false positive rate below 0.1 calls per 10,000 possible variants. The approach represents a significant performance improvement compared to molecular barcoding methods and does not require changing molecular reagents. PMID:29630678

HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR.

PubMed

Vivarini, Áislan de Carvalho; Pereira, Renata de Meirelles Santos; Barreto-de-Souza, Victor; Temerozo, Jairo Ramos; Soares, Deivid C; Saraiva, Elvira M; Saliba, Alessandra Mattos; Bou-Habib, Dumith Chequer; Lopes, Ulisses Gazos

2015-11-26

HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49-57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling.

Structure and Dynamics of Replication-Mutation Systems

NASA Astrophysics Data System (ADS)

Schuster, Peter

1987-03-01

The kinetic equations of polynucleotide replication can be brought into fairly simple form provided certain environmental conditions are fulfilled. Two flow reactors, the continuously stirred tank reactor (CSTR) and a special dialysis reactor are particularly suitable for the analysis of replication kinetics. An experimental setup to study the chemical reaction network of RNA synthesis was derived from the bacteriophage Qβ. It consists of a virus specific RNA polymerase, Qβ replicase, the activated ribonucleosides GTP, ATP, CTP and UTP as well as a template suitable for replication. The ordinary differential equations for replication and mutation under the conditions of the flow reactors were analysed by the qualitative methods of bifurcation theory as well as by numerical integration. The various kinetic equations are classified according to their dynamical properties: we distinguish "quasilinear systems" which have uniquely stable point attractors and "nonlinear systems" with inherent nonlinearities which lead to multiple steady states, Hopf bifuractions, Feigenbaum-like sequences and chaotic dynamics for certain parameter ranges. Some examples which are relevant in molecular evolution and population genetics are discussed in detail.

DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos

PubMed Central

Jacob, Vinitha; Chernyavskaya, Yelena; Chen, Xintong; Tan, Poh Seng; Kent, Brandon; Hoshida, Yujin; Sadler, Kirsten C.

2015-01-01

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the re-replicating and arrested hepatocytes. Importantly, the DNA re-replication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis. PMID:25564650

DNA hypomethylation induces a DNA replication-associated cell cycle arrest to block hepatic outgrowth in uhrf1 mutant zebrafish embryos.

PubMed

Jacob, Vinitha; Chernyavskaya, Yelena; Chen, Xintong; Tan, Poh Seng; Kent, Brandon; Hoshida, Yujin; Sadler, Kirsten C

2015-02-01

UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 to hemimethylated DNA during replication and is essential for maintaining DNA methylation. uhrf1 mutant zebrafish have global DNA hypomethylation and display embryonic defects, including a small liver, and they die as larvae. We make the surprising finding that, despite their reduced organ size, uhrf1 mutants express high levels of genes controlling S-phase and have many more cells undergoing DNA replication, as measured by BrdU incorporation. In contrast to wild-type hepatocytes, which are continually dividing during hepatic outgrowth and thus dilute the BrdU label, uhrf1 mutant hepatocytes retain BrdU throughout outgrowth, reflecting cell cycle arrest. Pulse-chase-pulse experiments with BrdU and EdU, and DNA content analysis indicate that uhrf1 mutant cells undergo DNA re-replication and that apoptosis is the fate of many of the re-replicating and arrested hepatocytes. Importantly, the DNA re-replication phenotype and hepatic outgrowth failure are preceded by global loss of DNA methylation. Moreover, uhrf1 mutants are phenocopied by mutation of dnmt1, and Dnmt1 knockdown in uhrf1 mutants enhances their small liver phenotype. Together, these data indicate that unscheduled DNA replication and failed cell cycle progression leading to apoptosis are the mechanisms by which DNA hypomethylation prevents organ expansion in uhrf1 mutants. We propose that cell cycle arrest leading to apoptosis is a strategy that restricts propagation of epigenetically damaged cells during embryogenesis. © 2015. Published by The Company of Biologists Ltd.

Fidelity of DNA Replication in Normal and Malignant Human Breast Cells.

DTIC Science & Technology

1996-08-01

In order to better understand the extent to which the intact DNA replication machinery contributes to the overall mutation frequencies observed in...normal and malignant breast cells, I have designed experiments to examine the degree of fidelity exhibited during the DNA replication process in both...normal and cancerous breast cells. To accomplish this goal I have isolated a multiprotein DNA replication complex (which we have designated the DNA

Stabilisation of p53 enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation.

PubMed

Pan, D; Pan, L-Z; Hill, R; Marcato, P; Shmulevitz, M; Vassilev, L T; Lee, P W K

2011-09-27

Naturally oncolytic reovirus preferentially kills cancer cells, making it a promising cancer therapeutic. Mutations in tumour suppressor p53 are prevalent in cancers, yet the role of p53 in reovirus oncolysis is relatively unexplored. Human cancer cell lines were exposed to Nutlin-3a, reovirus or a combination of the two and cells were processed for reovirus titration, western blot, real-time PCR and apoptosis assay using Annexin V and 7-AAD staining. Confocal microscopy was used to determine translocation of the NF-κB p65 subunit. We show that despite similar reovirus replication in p53(+/+) and p53(-/-) cells, stabilisation of p53 by Nutlin-3a significantly enhanced reovirus-induced apoptosis and hence virus release and dissemination while having no direct effect on virus replication. Enhanced apoptosis by Nutlin-3a was not observed in p53(-/-) or p53 knockdown cells; however, increased expression of Bax and p21 are required. Moreover, elevated NF-κB activation in reovirus-infected cells following Nutlin-3a treatment was necessary for enhanced reovirus-induced apoptosis, as synergistic cytotoxicity was overcome by specific NF-κB inhibitors. Nutlin-3a treatment enhances reovirus-induced apoptosis and virus spread through p53-dependent NF-κB activation, and combination of reovirus and Nutlin-3a might represent an improved therapy against cancers harbouring wild-type p53.

Loss of Hda activity stimulates replication initiation from I-box, but not R4 mutant origins in Escherichia coli.

PubMed

Riber, Leise; Fujimitsu, Kazuyuki; Katayama, Tsutomu; Løbner-Olesen, Anders

2009-01-01

Initiation of chromosome replication in Escherichia coli is limited by the initiator protein DnaA associated with ATP. Within the replication origin, binding sites for DnaA associated with ATP or ADP (R boxes) and the DnaA(ATP) specific sites (I-boxes, tau-boxes and 6-mer sites) are found. We analysed chromosome replication of cells carrying mutations in conserved regions of oriC. Cells carrying mutations in DnaA-boxes I2, I3, R2, R3 and R5 as well as FIS and IHF binding sites resembled wild-type cells with respect to origin concentration. Initiation of replication in these mutants occurred in synchrony or with slight asynchrony only. Furthermore, lack of Hda stimulated initiation in all these mutants. The DnaA(ATP) containing complex that leads to initiation can therefore be formed in the absence of several of the origin DnaA binding sites including both DnaA(ATP) specific I-boxes. However, competition between I-box mutant and wild-type origins, revealed a positive role of I-boxes on initiation. On the other hand, mutations affecting DnaA-box R4 were found to be compromised for initiation and could not be augmented by an increase in cellular DnaA(ATP)/DnaA(ADP) ratio. Compared with the sites tested here, R4 therefore seems to contribute to initiation most critically.

Concerted activities of Mcm4, Sld3, and Dbf4 in control of origin activation and DNA replication fork progression

PubMed Central

Sheu, Yi-Jun; Kinney, Justin B.; Stillman, Bruce

2016-01-01

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins in a temporally specific manner during S phase. The replicative helicase Mcm2-7 functions in both initiation and fork progression and thus is an important target of regulation. Mcm4, a helicase subunit, possesses an unstructured regulatory domain that mediates control from multiple kinase signaling pathways, including the Dbf4-dependent Cdc7 kinase (DDK). Following replication stress in S phase, Dbf4 and Sld3, an initiation factor and essential target of Cyclin-Dependent Kinase (CDK), are targets of the checkpoint kinase Rad53 for inhibition of initiation from origins that have yet to be activated, so-called late origins. Here, whole-genome DNA replication profile analysis is used to access under various conditions the effect of mutations that alter the Mcm4 regulatory domain and the Rad53 targets, Sld3 and Dbf4. Late origin firing occurs under genotoxic stress when the controls on Mcm4, Sld3, and Dbf4 are simultaneously eliminated. The regulatory domain of Mcm4 plays an important role in the timing of late origin firing, both in an unperturbed S phase and in dNTP limitation. Furthermore, checkpoint control of Sld3 impacts fork progression under replication stress. This effect is parallel to the role of the Mcm4 regulatory domain in monitoring fork progression. Hypomorph mutations in sld3 are suppressed by a mcm4 regulatory domain mutation. Thus, in response to cellular conditions, the functions executed by Sld3, Dbf4, and the regulatory domain of Mcm4 intersect to control origin firing and replication fork progression, thereby ensuring genome stability. PMID:26733669

Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins.

PubMed

Rangarajan, Savithri; Woodgate, Roger; Goodman, Myron F

2002-02-01

In Escherichia coli, UV-irradiated cells resume DNA synthesis after a transient inhibition by a process called replication restart. To elucidate the role of several key proteins involved in this process, we have analysed the time dependence of replication restart in strains carrying a combination of mutations in lexA, recA, polB (pol II), umuDC (pol V), priA, dnaC, recF, recO or recR. We find that both pol II and the origin-independent primosome-assembling function of PriA are essential for the immediate recovery of DNA synthesis after UV irradiation. In their absence, translesion replication or 'replication readthrough' occurs approximately 50 min after UV and is pol V-dependent. In a wild-type, lexA+ background, mutations in recF, recO or recR block both pathways. Similar results were obtained with a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def) recR strains, although unable to facilitate PriA-pol II-dependent restart, were able to perform pol V-dependent readthrough. The defects in restart attributed to mutations in recF, recO or recR were suppressed in a recA730 lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest that in a wild-type background, RecF, O and R are important for the induction of the SOS response and the formation of RecA*-dependent recombination intermediates necessary for PriA/Pol II-dependent replication restart. In con-trast, only RecF is required for the activation of RecA that leads to the formation of pol V (UmuD'2C) and facilitates replication readthrough.

Oncolytic Herpes Simplex Virus Vectors Fully Retargeted to Tumor- Associated Antigens.

PubMed

Uchida, Hiroaki; Hamada, Hirofumi; Nakano, Kenji; Kwon, Heechung; Tahara, Hideaki; Cohen, Justus B; Glorioso, Joseph C

2018-01-01

Oncolytic virotherapy is a novel therapeutic modality for malignant diseases that exploits selective viral replication in cancer cells. Herpes simplex virus (HSV) is a promising agent for oncolytic virotherapy due to its broad cell tropism and the identification of mutations that favor its replication in tumor over normal cells. However, these attenuating mutations also tend to limit the potency of current oncolytic HSV vectors that have entered clinical studies. As an alternative, vector retargeting to novel entry receptors has the potential to achieve tumor specificity at the stage of virus entry, eliminating the need for replication-attenuating mutations. Here, we summarize the molecular mechanism of HSV entry and recent advances in the development of fully retargeted HSV vectors for oncolytic virotherapy. Retargeted HSV vectors offer an attractive platform for the creation of a new generation of oncolytic HSV with improved efficacy and specificity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.

PubMed

Audry, Julien; Maestroni, Laetitia; Delagoutte, Emmanuelle; Gauthier, Tiphaine; Nakamura, Toru M; Gachet, Yannick; Saintomé, Carole; Géli, Vincent; Coulon, Stéphane

2015-07-14

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in DNA replication, recombination, and repair. In fission yeast, the Rpa1-D223Y mutation provokes telomere shortening. Here, we show that this mutation impairs lagging-strand telomere replication and leads to the accumulation of secondary structures and recruitment of the homologous recombination factor Rad52. The presence of these secondary DNA structures correlates with reduced association of shelterin subunits Pot1 and Ccq1 at telomeres. Strikingly, heterologous expression of the budding yeast Pif1 known to efficiently unwind G-quadruplex rescues all the telomeric defects of the D223Y cells. Furthermore, in vitro data show that the identical D to Y mutation in human RPA specifically affects its ability to bind G-quadruplex. We propose that RPA prevents the formation of G-quadruplex structures at lagging-strand telomeres to promote shelterin association and facilitate telomerase action at telomeres. © 2015 The Authors.

A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses

PubMed Central

Pauly, Matthew D; Procario, Megan C; Lauring, Adam S

2017-01-01

Influenza virus’ low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the influenza virus mutation rate is 2.7 × 10–6 - 3.0 × 10–5 substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal fitness impacts and fluctuation tests typically investigate only a subset of all possible single nucleotide mutations. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins, which allowed us to measure the rates of selectively neutral mutations representative of the twelve different mutation types. We measured an overall mutation rate of 1.8 × 10–4 s/n/r for PR8 (H1N1) and 2.5 × 10–4 s/n/r for Hong Kong 2014 (H3N2) and a transitional bias of 2.7–3.6. Our data suggest that each replicated genome will have an average of 2–3 mutations and highlight the importance of mutational load in influenza virus evolution. DOI: http://dx.doi.org/10.7554/eLife.26437.001 PMID:28598328

Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roehrig, John T., E-mail: jtr1@cdc.gov; Butrapet, Siritorn; Liss, Nathan M.

Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cellsmore » and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.« less

Evaluation of chromosome 6p22 as a breast cancer risk modifier locus in a follow-up study of BRCA2 mutation carriers

PubMed Central

Stevens, Kristen N.; Wang, Xianshu; Fredericksen, Zachary; Pankratz, Vernon S.; Greene, Mark H.; Andrulis, Irene L.; Thomassen, Mads; Caligo, Maria; Nathanson, Katherine L.; Jakubowska, Anna; Osorio, Ana; Hamann, Ute; Godwin, Andrew K.; Stoppa-Lyonnet, Dominique; Southey, Melissa; Buys, Saundra S.; Singer, Christian F.; Hansen, Thomas V.O.; Arason, Adalgeir; Offit, Kenneth; Piedmonte, Marion; Montagna, Marco; Imyanitov, Evgeny; Tihomirova, Laima; Sucheston, Lara; Beattie, Mary; Neuhausen, Susan L.; Szabo, Csilla I.; Simard, Jacques; Spurdle, Amanda B.; Healey, Sue; Chen, Xiaoqing; Rebbeck, Timothy R.; Easton, Douglas F.; Chenevix-Trench, Georgia; Antoniou, Antonis C; Couch, Fergus J.

2012-01-01

Several common germline variants identified through genome-wide association studies of breast cancer risk in the general population have recently been shown to be associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. When combined, these variants can identify marked differences in the absolute risk of developing breast cancer for mutation carriers, suggesting that additional modifier loci may further enhance individual risk assessment for BRCA1 and BRCA2 mutation carriers. Recently, a common variant on 6p22 (rs9393597) was found to be associated with increased breast cancer risk for BRCA2 mutation carriers [Hazard ratio (HR)=1.55, 95% CI 1.25–1.92, p=6.0×10−5]. This observation was based on data from GWAS studies in which, despite statistical correction for multiple comparisons, the possibility of false discovery remains a concern. Here we report on an analysis of this variant in an additional 6,165 BRCA1 and 3,900 BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). In this replication analysis, rs9393597 was not associated with breast cancer risk for BRCA2 mutation carriers [HR=1.09, 95% CI 0.96–1.24, p=0.18]. No association with ovarian cancer risk for BRCA1 or BRCA2 mutation carriers or with breast cancer risk for BRCA1 mutation carriers was observed. This follow-up study suggests that, contrary to our initial report, this variant is not associated with breast cancer risk among individuals with germline BRCA2 mutations. PMID:23011509

DNA replication stress as a hallmark of cancer.

PubMed

Macheret, Morgane; Halazonetis, Thanos D

2015-01-01

Human cancers share properties referred to as hallmarks, among which sustained proliferation, escape from apoptosis, and genomic instability are the most pervasive. The sustained proliferation hallmark can be explained by mutations in oncogenes and tumor suppressors that regulate cell growth, whereas the escape from apoptosis hallmark can be explained by mutations in the TP53, ATM, or MDM2 genes. A model to explain the presence of the three hallmarks listed above, as well as the patterns of genomic instability observed in human cancers, proposes that the genes driving cell proliferation induce DNA replication stress, which, in turn, generates genomic instability and selects for escape from apoptosis. Here, we review the data that support this model, as well as the mechanisms by which oncogenes induce replication stress. Further, we argue that DNA replication stress should be considered as a hallmark of cancer because it likely drives cancer development and is very prevalent.

Role of the mitochondrial DNA replication machinery in mitochondrial DNA mutagenesis, aging and age-related diseases

PubMed Central

DeBalsi, Karen L.; Hoff, Kirsten E.; Copeland, William C.

2016-01-01

As regulators of bioenergetics in the cell and the primary source of endogenous reactive oxygen species (ROS), dysfunctional mitochondria have been implicated for decades in the process of aging and age-related diseases. Mitochondrial DNA (mtDNA) is replicated and repaired by nuclear-encoded mtDNA polymerase γ (Pol γ) and several other associated proteins, which compose the mtDNA replication machinery. Here, we review evidence that errors caused by this replication machinery and failure to repair these mtDNA errors results in mtDNA mutations. Clonal expansion of mtDNA mutations results in mitochondrial dysfunction, such as decreased electron transport chain (ETC) enzyme activity and impaired cellular respiration. We address the literature that mitochondrial dysfunction, in conjunction with altered mitochondrial dynamics, is a major driving force behind aging and age-related diseases. Additionally, interventions to improve mitochondrial function and attenuate the symptoms of aging are examined. PMID:27143693

Incorporation of the catalytic domain of a hammerhead ribozyme into antisense RNA enhances its inhibitory effect on the replication of human immunodeficiency virus type 1.

PubMed Central

Homann, M; Tzortzakaki, S; Rittner, K; Sczakiel, G; Tabler, M

1993-01-01

The catalytic domain of a hammerhead ribozyme was incorporated into a 413 nucleotides long antisense RNA directed against the 5'-leader/gag region of the human immunodeficiency virus type 1 (HIV-1) (pos. +222 to +634). The resulting catalytic antisense RNA was shown to cleave its target RNA in vitro specifically at physiological ion strength and temperature. We compared the antiviral effectiveness of this catalytic antisense RNA with that of the corresponding unmodified antisense RNA and with a mutated catalytic antisense RNA, which did not cleave the substrate RNA in vitro. Each of these RNAs was co-transfected into human SW480 cells together with infectious complete proviral HIV-1 DNA, followed by analysis of HIV-1 replication. The presence of the catalytically active domain resulted in 4 to 7 fold stronger inhibition of HIV-1 replication as compared to the parental antisense RNA and the inactive mutant. Kinetic and structural studies performed in vitro indicated that the ability for double strand formation was not changed in catalytic antisense RNA versus parental antisense RNA. Together, these data suggest that the ability to cleave target RNA is a crucial prerequisite for the observed increase of inhibition of the replication of HIV-1. Images PMID:8332489

Recombination in Enteroviruses Is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length ‘Imprecise’ Intermediates

PubMed Central

Lowry, Kym; Woodman, Andrew; Cook, Jonathan; Evans, David J.

2014-01-01

Recombination in enteroviruses provides an evolutionary mechanism for acquiring extensive regions of novel sequence, is suggested to have a role in genotype diversity and is known to have been key to the emergence of novel neuropathogenic variants of poliovirus. Despite the importance of this evolutionary mechanism, the recombination process remains relatively poorly understood. We investigated heterologous recombination using a novel reverse genetic approach that resulted in the isolation of intermediate chimeric intertypic polioviruses bearing genomes with extensive duplicated sequences at the recombination junction. Serial passage of viruses exhibiting such imprecise junctions yielded progeny with increased fitness which had lost the duplicated sequences. Mutations or inhibitors that changed polymerase fidelity or the coalescence of replication complexes markedly altered the yield of recombinants (but did not influence non-replicative recombination) indicating both that the process is replicative and that it may be possible to enhance or reduce recombination-mediated viral evolution if required. We propose that extant recombinants result from a biphasic process in which an initial recombination event is followed by a process of resolution, deleting extraneous sequences and optimizing viral fitness. This process has implications for our wider understanding of ‘evolution by duplication’ in the positive-strand RNA viruses. PMID:24945141

Reduction of Werner Syndrome Protein Enhances G:C → A:T Transition by O6-Methylguanine in Human Cells.

PubMed

Suzuki, Tetsuya; Kuramoto, Yoshie; Kamiya, Hiroyuki

2018-05-21

O 6 -Methylguanine ( O 6 -MeG) is a damaged base produced by methylating reagents. The Werner syndrome protein (WRN) is a cancer-related human DNA helicase. The effects of WRN reduction on O 6 -MeG-caused mutagenesis were assessed by an siRNA-mediated knockdown in human U2OS cells, using a shuttle plasmid with a single O 6 -MeG base in the supF gene. The plasmid DNA was replicated in the cells, isolated, and electroporated into an Escherichia coli indicator strain. The lowered amount of WRN increased the frequency of mutations induced by O 6 -MeG, mainly G:C → A:T substitution. The increased mutation rate suggested that the cancer-related WRN suppresses the G:C → A:T substitution by O 6 -MeG in human cells.

HIV-1 protease inhibitor mutations affect the development of HIV-1 resistance to the maturation inhibitor bevirimat.

PubMed

Fun, Axel; van Maarseveen, Noortje M; Pokorná, Jana; Maas, Renée Em; Schipper, Pauline J; Konvalinka, Jan; Nijhuis, Monique

2011-08-24

Maturation inhibitors are an experimental class of antiretrovirals that inhibit Human Immunodeficiency Virus (HIV) particle maturation, the structural rearrangement required to form infectious virus particles. This rearrangement is triggered by the ordered cleavage of the precursor Gag polyproteins into their functional counterparts by the viral enzyme protease. In contrast to protease inhibitors, maturation inhibitors impede particle maturation by targeting the substrate of protease (Gag) instead of the protease enzyme itself. Direct cross-resistance between protease and maturation inhibitors may seem unlikely, but the co-evolution of protease and its substrate, Gag, during protease inhibitor therapy, could potentially affect future maturation inhibitor therapy. Previous studies showed that there might also be an effect of protease inhibitor resistance mutations on the development of maturation inhibitor resistance, but the exact mechanism remains unclear. We used wild-type and protease inhibitor resistant viruses to determine the impact of protease inhibitor resistance mutations on the development of maturation inhibitor resistance. Our resistance selection studies demonstrated that the resistance profiles for the maturation inhibitor bevirimat are more diverse for viruses with a mutated protease compared to viruses with a wild-type protease. Viral replication did not appear to be a major factor during emergence of bevirimat resistance. In all in vitro selections, one of four mutations was selected: Gag V362I, A364V, S368N or V370A. The impact of these mutations on maturation inhibitor resistance and viral replication was analyzed in different protease backgrounds. The data suggest that the protease background affects development of HIV-1 resistance to bevirimat and the replication profiles of bevirimat-selected HIV-1. The protease-dependent bevirimat resistance and replication levels can be explained by differences in CA/p2 cleavage processing by the different proteases. These findings highlight the complicated interactions between the viral protease and its substrate. By providing a better understanding of these interactions, we aim to help guide the development of second generation maturation inhibitors.

POLD1: central mediator of DNA replication and repair, and implication in cancer and other pathologies

PubMed Central

Nicolas, Emmanuelle; Golemis, Erica A.; Arora, Sanjeevani

2016-01-01

The evolutionarily conserved human polymerase delta (POLD1) gene encodes the large p125 subunit which provides the essential catalytic activities of polymerase δ (Polδ), mediated by 5’–3’ DNA polymerase and 3’–5’ exonuclease moieties. POLD1 associates with three smaller subunits (POLD2, POLD3, POLD4), which together with Replication Factor C and Proliferating Nuclear Cell Antigen constitute the polymerase holoenzyme. Polδ function is essential for replication, with a primary role as the replicase for the lagging strand. Polδ also has an important proofreading ability conferred by the exonuclease activity, which is critical for ensuring replicative fidelity, but also serves to repair DNA lesions arising as a result of exposure to mutagens. Polδ has been shown to be important for multiple forms of DNA repair, including nucleotide excision repair, double strand break repair, base excision repair, and mismatch repair. A growing number of studies in the past decade have linked germline and sporadic mutations in POLD1 and the other subunits of Polδ with human pathologies. Mutations in Polδ in mice and humans lead to genomic instability, mutator phenotype and tumorigenesis. The advent of genome sequencing techniques has identified damaging mutations in the proofreading domain of POLD1 as the underlying cause of some inherited cancers, and suggested that mutations in POLD1 may influence therapeutic management. In addition, mutations in POLD1 have been identified in the developmental disorders of mandibular hypoplasia, deafness, progeroid features and lipodystrophy and atypical Werner syndrome, while changes in expression or activity of POLD1 have been linked to senescence and aging. Intriguingly, some recent evidence suggests POLD1 function may also be altered in diabetes. We provide an overview of critical Polδ activities in the context of these pathologic conditions. PMID:27320729

Recessive Twinkle mutations in early onset encephalopathy with mtDNA depletion.

PubMed

Hakonen, Anna H; Isohanni, Pirjo; Paetau, Anders; Herva, Riitta; Suomalainen, Anu; Lönnqvist, Tuula

2007-11-01

Twinkle is a mitochondrial replicative helicase, the mutations of which have been associated with autosomal dominant progressive external ophthalmoplegia (adPEO), and recessively inherited infantile onset spinocerebellar ataxia (IOSCA). We report here a new phenotype in two siblings with compound heterozygous Twinkle mutations (A318T and Y508C), characterized by severe early onset encephalopathy and signs of liver involvement. The clinical manifestations included hypotonia, athetosis, sensory neuropathy, ataxia, hearing deficit, ophthalmoplegia, intractable epilepsy and elevation of serum transaminases. The liver showed mtDNA depletion, whereas the muscle mtDNA was only slightly affected. Alpers-Huttenlocher syndrome has previously been associated with mutations of polymerase gamma, a replicative polymerase of mtDNA. We show here that recessive mutations of the close functional partner of the polymerase, the Twinkle helicase, can also manifest as early encephalopathy with liver involvement, a phenotype reminiscent of Alpers syndrome, and are a new genetic cause underlying tissue-specific mtDNA depletion.

Local neutral networks help maintain inaccurately replicating ribozymes.

PubMed

Szilágyi, András; Kun, Ádám; Szathmáry, Eörs

2014-01-01

The error threshold of replication limits the selectively maintainable genome size against recurrent deleterious mutations for most fitness landscapes. In the context of RNA replication a distinction between the genotypic and the phenotypic error threshold has been made; where the latter concerns the maintenance of secondary structure rather than sequence. RNA secondary structure is treated as a proxy for function. The phenotypic error threshold allows higher per digit mutation rates than its genotypic counterpart, and is known to increase with the frequency of neutral mutations in sequence space. Here we show that the degree of neutrality, i.e. the frequency of nearest-neighbour (one-step) neutral mutants is a remarkably accurate proxy for the overall frequency of such mutants in an experimentally verifiable formula for the phenotypic error threshold; this we achieve by the full numerical solution for the concentration of all sequences in mutation-selection balance up to length 16. We reinforce our previous result that currently known ribozymes could be selectively maintained by the accuracy known from the best available polymerase ribozymes. Furthermore, we show that in silico stabilizing selection can increase the mutational robustness of ribozymes due to the fact that they were produced by artificial directional selection in the first place. Our finding offers a better understanding of the error threshold and provides further insight into the plausibility of an ancient RNA world.

Muver, a computational framework for accurately calling accumulated mutations.

PubMed

Burkholder, Adam B; Lujan, Scott A; Lavender, Christopher A; Grimm, Sara A; Kunkel, Thomas A; Fargo, David C

2018-05-09

Identification of mutations from next-generation sequencing data typically requires a balance between sensitivity and accuracy. This is particularly true of DNA insertions and deletions (indels), that can impart significant phenotypic consequences on cells but are harder to call than substitution mutations from whole genome mutation accumulation experiments. To overcome these difficulties, we present muver, a computational framework that integrates established bioinformatics tools with novel analytical methods to generate mutation calls with the extremely low false positive rates and high sensitivity required for accurate mutation rate determination and comparison. Muver uses statistical comparison of ancestral and descendant allelic frequencies to identify variant loci and assigns genotypes with models that include per-sample assessments of sequencing errors by mutation type and repeat context. Muver identifies maximally parsimonious mutation pathways that connect these genotypes, differentiating potential allelic conversion events and delineating ambiguities in mutation location, type, and size. Benchmarking with a human gold standard father-son pair demonstrates muver's sensitivity and low false positive rates. In DNA mismatch repair (MMR) deficient Saccharomyces cerevisiae, muver detects multi-base deletions in homopolymers longer than the replicative polymerase footprint at rates greater than predicted for sequential single-base deletions, implying a novel multi-repeat-unit slippage mechanism. Benchmarking results demonstrate the high accuracy and sensitivity achieved with muver, particularly for indels, relative to available tools. Applied to an MMR-deficient Saccharomyces cerevisiae system, muver mutation calls facilitate mechanistic insights into DNA replication fidelity.

The interaction between cytosine methylation and processes of DNA replication and repair shape the mutational landscape of cancer genomes.

PubMed

Poulos, Rebecca C; Olivier, Jake; Wong, Jason W H

2017-07-27

Methylated cytosines (5mCs) are frequently mutated in the genome. However, no studies have yet comprehensively analysed mutation-methylation associations across cancer types. Here we analyse 916 cancer genomes, together with tissue type-specific methylation and replication timing data. We describe a strong mutation-methylation association across colorectal cancer subtypes, most interestingly in samples with microsatellite instability (MSI) or Polymerase epsilon (POLE) exonuclease domain mutations. By analysing genomic regions with differential mismatch repair (MMR) efficiency, we suggest a possible role for MMR in the correction of 5mC deamination events, potentially accounting for the high rate of 5mC mutation accumulation in MSI tumours. Additionally, we propose that mutant POLE asserts a mutator phenotype specifically at 5mCs, and we find coding mutation hotspots in POLE-mutant cancers at highly-methylated CpGs in the tumour-suppressor genes APC and TP53. Finally, using multivariable regression models, we demonstrate that different cancers exhibit distinct mutation-methylation associations, with DNA repair influencing such associations in certain cancer genomes. Taken together, we find differential associations with methylation that are vital for accurately predicting expected mutation loads across cancer types. Our findings reveal links between methylation and common mutation and repair processes, with these mechanisms defining a key part of the mutational landscape of cancer genomes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli.

PubMed

Gibbs, P E; Kilbey, B J; Banerjee, S K; Lawrence, C W

1993-05-01

We have compared the mutagenic properties of a T-T cyclobutane dimer in baker's yeast, Saccharomyces cerevisiae, with those in Escherichia coli by transforming each of these species with the same single-stranded shuttle vector carrying either the cis-syn or the trans-syn isomer of this UV photoproduct at a unique site. The mutagenic properties investigated were the frequency of replicational bypass of the photoproduct, the error rate of bypass, and the mutation spectrum. In SOS-induced E. coli, the cis-syn dimer was bypassed in approximately 16% of the vector molecules, and 7.6% of the bypass products had targeted mutations. In S. cerevisiae, however, bypass occurred in about 80% of these molecules, and the bypass was at least 19-fold more accurate (approximately 0.4% targeted mutations). Each of these yeast mutations was a single unique event, and none were like those in E. coli, suggesting that in fact the difference in error rate is much greater. Bypass of the trans-syn dimer occurred in about 17% of the vector molecules in both species, but with this isomer the error rate was higher in S. cerevisiae (21 to 36% targeted mutations) than in E. coli (13%). However, the spectra of mutations induced by the latter photoproduct were virtually identical in the two organisms. We conclude that bypass and error frequencies are determined both by the structure of the photoproduct-containing template and by the particular replication proteins concerned but that the types of mutations induced depend predominantly on the structure of the template. Unlike E. coli, bypass in S. cerevisiae did not require UV-induced functions.

Characterization of miR-122-independent propagation of HCV

PubMed Central

Motooka, Daisuke; Nakamura, Shota; Yamamoto, Satomi; Mori, Hiroyuki; Sato, Asuka; Uemura, Kentaro; Fauzyah, Yuzy; Suda, Takahiro; Nishio, Akira; Hmwe, Su Su; Okamoto, Toru; Tatsumi, Tomohide; Takehara, Tetsuo; Chayama, Kazuaki; Wakita, Takaji; Koike, Kazuhiko

2017-01-01

miR-122, a liver-specific microRNA, is one of the determinants for liver tropism of hepatitis C virus (HCV) infection. Although miR-122 is required for efficient propagation of HCV, we have previously shown that HCV replicates at a low rate in miR-122-deficient cells, suggesting that HCV-RNA is capable of propagating in an miR-122-independent manner. We herein investigated the roles of miR-122 in both the replication of HCV-RNA and the production of infectious particles by using miR-122-knockout Huh7 (Huh7-122KO) cells. A slight increase of intracellular HCV-RNA levels and infectious titers in the culture supernatants was observed in Huh7-122KO cells upon infection with HCV. Moreover, after serial passages of HCV in miR-122-knockout Huh7.5.1 cells, we obtained an adaptive mutant, HCV122KO, possessing G28A substitution in the 5’UTR of the HCV genotype 2a JFH1 genome, and this mutant may help to enhance replication complex formation, a possibility supported by polysome analysis. We also found the introduction of adaptive mutation around miR-122 binding site in the genotype 1b/2a chimeric virus, which originally had an adenine at the nucleotide position 29. HCV122KO exhibited efficient RNA replication in miR-122-knockout cells and non-hepatic cells without exogenous expression of miR-122. Competition assay revealed that the G28A mutant was dominant in the absence of miR-122, but its effects were equivalent to those of the wild type in the presence of miR-122, suggesting that the G28A mutation does not confer an advantage for propagation in miR-122-rich hepatocytes. These observations may explain the clinical finding that the positive rate of G28A mutation was higher in miR-122-deficient PBMCs than in the patient serum, which mainly included the hepatocyte-derived virus from HCV-genotype-2a patients. These results suggest that the emergence of HCV mutants that can propagate in non-hepatic cells in an miR-122-independent manner may participate in the induction of extrahepatic manifestations in chronic hepatitis C patients. PMID:28494029

Application of multi-objective optimization to pooled experiments of next generation sequencing for detection of rare mutations.

PubMed

Zilinskas, Julius; Lančinskas, Algirdas; Guarracino, Mario Rosario

2014-01-01

In this paper we propose some mathematical models to plan a Next Generation Sequencing experiment to detect rare mutations in pools of patients. A mathematical optimization problem is formulated for optimal pooling, with respect to minimization of the experiment cost. Then, two different strategies to replicate patients in pools are proposed, which have the advantage to decrease the overall costs. Finally, a multi-objective optimization formulation is proposed, where the trade-off between the probability to detect a mutation and overall costs is taken into account. The proposed solutions are devised in pursuance of the following advantages: (i) the solution guarantees mutations are detectable in the experimental setting, and (ii) the cost of the NGS experiment and its biological validation using Sanger sequencing is minimized. Simulations show replicating pools can decrease overall experimental cost, thus making pooling an interesting option.

Back to the future: revisiting HIV-1 lethal mutagenesis

PubMed Central

Dapp, Michael J.; Patterson, Steven E.; Mansky, Louis M.

2012-01-01

The concept of eliminating HIV-1 infectivity by elevating the viral mutation rate was first proposed over a decade ago, even though the general concept had been conceived earlier for RNA viruses. Lethal mutagenesis was originally viewed as a novel chemotherapeutic approach for treating HIV-1 infection in which use of a viral mutagen would over multiple rounds of replication lead to the lethal accumulation of mutations, rendering the virus population non infectious – known as the slow mutation accumulation model. There have been limitations in obtaining good efficacy data with drug leads, leaving some doubt into clinical translation. More recent studies of the APOBEC3 proteins as well as new progress in the use of nucleoside analogs for inducing lethal mutagenesis have helped to refocus attention on rapid induction of HIV-1 lethal mutagenesis in a single or limited number of replication cycles leading to a rapid mutation accumulation model. PMID:23195922

Human Adenovirus Core Protein V Is Targeted by the Host SUMOylation Machinery To Limit Essential Viral Functions.

PubMed

Freudenberger, Nora; Meyer, Tina; Groitl, Peter; Dobner, Thomas; Schreiner, Sabrina

2018-02-15

Human adenoviruses (HAdV) are nonenveloped viruses containing a linear, double-stranded DNA genome surrounded by an icosahedral capsid. To allow proper viral replication, the genome is imported through the nuclear pore complex associated with viral core proteins. Until now, the role of these incoming virion proteins during the early phase of infection was poorly understood. The core protein V is speculated to bridge the core and the surrounding capsid. It binds the genome in a sequence-independent manner and localizes in the nucleus of infected cells, accumulating at nucleoli. Here, we show that protein V contains conserved SUMO conjugation motifs (SCMs). Mutation of these consensus motifs resulted in reduced SUMOylation of the protein; thus, protein V represents a novel target of the host SUMOylation machinery. To understand the role of protein V SUMO posttranslational modification during productive HAdV infection, we generated a replication-competent HAdV with SCM mutations within the protein V coding sequence. Phenotypic analyses revealed that these SCM mutations are beneficial for adenoviral replication. Blocking protein V SUMOylation at specific sites shifts the onset of viral DNA replication to earlier time points during infection and promotes viral gene expression. Simultaneously, the altered kinetics within the viral life cycle are accompanied by more efficient proteasomal degradation of host determinants and increased virus progeny production than that observed during wild-type infection. Taken together, our studies show that protein V SUMOylation reduces virus growth; hence, protein V SUMOylation represents an important novel aspect of the host antiviral strategy to limit virus replication and thereby points to potential intervention strategies. IMPORTANCE Many decades of research have revealed that HAdV structural proteins promote viral entry and mainly physical stability of the viral genome in the capsid. Our work over the last years showed that this concept needs expansion as the functions are more diverse. We showed that capsid protein VI regulates the antiviral response by modulation of the transcription factor Daxx during infection. Moreover, core protein VII interacts with SPOC1 restriction factor, which is beneficial for efficient viral gene expression. Here, we were able to show that core protein V also represents a novel substrate of the host SUMOylation machinery and contains several conserved SCMs; mutation of these consensus motifs reduced SUMOylation of the protein. Unexpectedly, we observed that introducing these mutations into HAdV promotes adenoviral replication. In conclusion, we offer novel insights into adenovirus core proteins and provide evidence that SUMOylation of HAdV factors regulates replication efficiency. Copyright © 2018 American Society for Microbiology.

Novel mutation in the replication focus targeting sequence domain of DNMT1 causes hereditary sensory and autonomic neuropathy IE.

PubMed

Yuan, Junhui; Higuchi, Yujiro; Nagado, Tatsui; Nozuma, Satoshi; Nakamura, Tomonori; Matsuura, Eiji; Hashiguchi, Akihiro; Sakiyama, Yusuke; Yoshimura, Akiko; Takashima, Hiroshi

2013-03-01

DNMT1, encoding DNA methyltransferase 1 (Dnmt1), is a critical enzyme which is mainly responsible for conversion of unmethylated DNA into hemimethylated DNA. To date, two phenotypes produced by DNMT1 mutations have been reported, including hereditary sensory and autonomic neuropathy (HSAN) type IE with mutations in exon 20, and autosomal dominant cerebellar ataxia, deafness, and narcolepsy caused by mutations in exon 21. We report a sporadic case in a Japanese patient with loss of pain and vibration sense, chronic osteomyelitis, autonomic system dysfunctions, hearing loss, and mild dementia, but without definite cerebellar ataxia. Electrophysiological studies revealed absent sensory nerve action potential with nearly normal motor nerve conduction studies. Brain magnetic resonance imaging revealed mild diffuse cerebral and cerebellar atrophy. Using a next-generation sequencing system, 16 candidate genes were analyzed and a novel missense mutation, c.1706A>G (p.His569Arg), was identified in exon 21 of DNMT1. Our findings suggest that mutation in exon 21 of DNMT1 may also produce a HSAN phenotype. Because all reported mutations of DNMT1 are concentrated in exons 20 and 21, which encode the replication focus targeting sequence (RFTS) domain of Dnmt1, the RFTS domain could be a mutation hot spot. © 2013 Peripheral Nerve Society.

Conserved amino acids within the N-terminus of the West Nile virus NS4A protein contribute to virus replication, protein stability and membrane proliferation.

PubMed

Ambrose, R L; Mackenzie, J M

2015-07-01

The West Nile virus strain Kunjin virus (WNVKUN) NS4A protein is a multifunctional protein involved in many aspects of the virus life-cycle and is a major component of the WNVKUN replication complex (RC). Previously we identified a conserved region in the C-terminus of NS4A regulating proteolytic processing and RC assembly, and now investigate key conserved residues in the N-terminus of NS4A and their contribution to WNVKUN replication. Mutation of P13 completely ablated replication, whereas, mutation of P48 and D49, near the first transmembrane helix, and G66 within the helix, showed variable defects in replication, virion secretion and membrane proliferation. Intriguingly, the P48 and G66 NS4A mutants resulted in specific proteasome depletion of NS4A that could in part be rescued with a proteasome inhibitor. Our results suggest that the N-terminus of NS4A contributes to correct folding and stability, essential for facilitating the essential roles of NS4A during replication. Copyright © 2015 Elsevier Inc. All rights reserved.

New Views on Strand Asymmetry in Insect Mitochondrial Genomes

PubMed Central

Wei, Shu-Jun; Shi, Min; Chen, Xue-Xin; Sharkey, Michael J.; van Achterberg, Cornelis; Ye, Gong-Yin; He, Jun-Hua

2010-01-01

Strand asymmetry in nucleotide composition is a remarkable feature of animal mitochondrial genomes. Understanding the mutation processes that shape strand asymmetry is essential for comprehensive knowledge of genome evolution, demographical population history and accurate phylogenetic inference. Previous studies found that the relative contributions of different substitution types to strand asymmetry are associated with replication alone or both replication and transcription. However, the relative contributions of replication and transcription to strand asymmetry remain unclear. Here we conducted a broad survey of strand asymmetry across 120 insect mitochondrial genomes, with special reference to the correlation between the signs of skew values and replication orientation/gene direction. The results show that the sign of GC skew on entire mitochondrial genomes is reversed in all species of three distantly related families of insects, Philopteridae (Phthiraptera), Aleyrodidae (Hemiptera) and Braconidae (Hymenoptera); the replication-related elements in the A+T-rich regions of these species are inverted, confirming that reversal of strand asymmetry (GC skew) was caused by inversion of replication origin; and finally, the sign of GC skew value is associated with replication orientation but not with gene direction, while that of AT skew value varies with gene direction, replication and codon positions used in analyses. These findings show that deaminations during replication and other mutations contribute more than selection on amino acid sequences to strand compositions of G and C, and that the replication process has a stronger affect on A and T content than does transcription. Our results may contribute to genome-wide studies of replication and transcription mechanisms. PMID:20856815

Identification of Respiratory Syncytial Virus Nonstructural Protein 2 Residues Essential for Exploitation of the Host Ubiquitin System and Inhibition of Innate Immune Responses.

PubMed

Whelan, Jillian N; Tran, Kim C; van Rossum, Damian B; Teng, Michael N

2016-07-15

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in young children worldwide. The RSV nonstructural protein 2 (NS2) is a multifunctional protein that primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. We investigated the structural determinants of NS2 important for interaction with the host ubiquitin system to degrade STAT2 during infection. We found that NS2 expression enhances ubiquitination of host proteins. Bioinformatics analysis provided a platform for identification of specific residues that limit NS2-induced ubiquitination. Combinations of multiple mutations displayed an additive effect on reducing NS2-induced ubiquitination. Using a reverse genetics system, we generated recombinant RSV (rRSV) containing NS2 ubiquitin mutations, which maintained their effect on ubiquitin expression during infection. Interestingly, STAT2 degradation activity was ablated in the NS2 ubiquitin mutant rRSV. In addition, NS2 ubiquitin mutations decreased rRSV replication, indicating a correlation between NS2's ubiquitin function and antagonism of innate immune signaling to enhance viral replication. Our approach of targeting NS2 residues required for NS2 inhibition of immune responses provides a mechanism for attenuating RSV for vaccine development. RSV has been circulating globally for more than 60 years, causing severe respiratory disease in pediatric, elderly, and immunocompromised populations. Production of a safe, effective vaccine against RSV is a public health priority. The NS2 protein is an effective target for prevention and treatment of RSV due to its antagonistic activity against the innate immune system. However, NS2-deleted RSV vaccine candidates rendered RSV overattenuated or poorly immunogenic. Alternatively, we can modify essential NS2 structural features to marginally limit viral growth while maintaining immune responses, providing the necessary balance between antigenicity and safety required for an effective vaccine. We coupled bioinformatics analysis with reverse genetics to introduce mutations into RSV's negative-sense genome. In this way we constructed rRSV NS2 ubiquitin mutants that limited NS2's ability to antagonize the innate immune system, thereby attenuating rRSV growth and increasing innate immune responses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

DNA reactivity as a mode of action and its relevance to cancer risk assessment.

PubMed

Preston, R Julian

2013-02-01

The ability of a chemical to induce mutations has long been a driver in the cancer risk assessment process. The default strategy has been that mutagenic chemicals demonstrate linear cancer dose responses, especially at low exposure levels. In the absence of additional confounding information, this is a reasonable approach, because risk assessment is appropriately considered as being protective of human health. The concept of mode of action has allowed for an opportunity to move off this default position; mutagenicity is now not considered as the driver but rather the mode of action is. In a more precise way, it is the set of key events that define a mode of action that is fundamental in defining the shape of a cancer dose response. A key event is an informative bioindicator of the cancer response and as such should be predictive of the tumor response, at least in a qualitative way. A clear example of the use of key events in cancer risk assessment is for DNA reactive chemicals. A series of such key events is initiated by the production of DNA damage in target cells from direct interaction of the chemical with DNA leading to the production of mutations by misreplication that results in enhanced cell replication. This enhanced cell replication eventually leads to the development of preneoplastic cells and ultimately overt neoplasms. The response of each of these key events to dose of the chemical can inform the cancer dose-response curve shape. Thus, the dose-response curve for any DNA-reactive chemical can be predicted from knowledge of its mode of action and the behavior of the induced key events.

Multiple Natural Substitutions in Avian Influenza A Virus PB2 Facilitate Efficient Replication in Human Cells.

PubMed

Mänz, Benjamin; de Graaf, Miranda; Mögling, Ramona; Richard, Mathilde; Bestebroer, Theo M; Rimmelzwaan, Guus F; Fouchier, Ron A M

2016-07-01

A strong restriction of the avian influenza A virus polymerase in mammalian cells generally limits viral host-range switching. Although substitutions like E627K in the PB2 polymerase subunit can facilitate polymerase activity to allow replication in mammals, many human H5N1 and H7N9 viruses lack this adaptive substitution. Here, several previously unknown, naturally occurring, adaptive substitutions in PB2 were identified by bioinformatics, and their enhancing activity was verified using in vitro assays. Adaptive substitutions enhanced polymerase activity and virus replication in mammalian cells for avian H5N1 and H7N9 viruses but not for a partially human-adapted H5N1 virus. Adaptive substitutions toward basic amino acids were frequent and were mostly clustered in a putative RNA exit channel in a polymerase crystal structure. Phylogenetic analysis demonstrated divergent dependency of influenza viruses on adaptive substitutions. The novel adaptive substitutions found in this study increase basic understanding of influenza virus host adaptation and will help in surveillance efforts. Influenza viruses from birds jump the species barrier into humans relatively frequently. Such influenza virus zoonoses may pose public health risks if the virus adapts to humans and becomes a pandemic threat. Relatively few amino acid substitutions-most notably in the receptor binding site of hemagglutinin and at positions 591 and 627 in the polymerase protein PB2-have been identified in pandemic influenza virus strains as determinants of host adaptation, to facilitate efficient virus replication and transmission in humans. Here, we show that substantial numbers of amino acid substitutions are functionally compensating for the lack of the above-mentioned mutations in PB2 and could facilitate influenza virus emergence in humans. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Torchia, B.S.; Call, L.M.; Migeon, B.R.

The relationship between the transcriptional state of a locus and the time when it replicates during DNA synthesis is increasingly apparent. Active autosomal genes tend to replicate early, whereas inactive ones are more permissive and frequently replicate later. Although the inactive X chromosome replicates later than its active homologue, little is known about the replication of X-linked genes. The authors have used FISH to examine the replication of loci on the active X chromosome that are not transcribed, either because the tissue analyzed was not the expressing tissue (F8C), because the locus is silent on all active X chromosomes (XIST),more » or because it has been mutated by expansion and methylation of a CpG island (FMR1). In this assay, an unreplicated locus is characterized by a single hybridization signal, and a replicated locus is characterized by a doublet hybridization signal. The percentage of doublets is used as a measure of relative time of replication in S phase. The results show that the FMR1 gene replicates relatively later in fragile X(fraX) males with the full mutation than in normal males, irrespective of the probe used. The F8C locus is late replicating in both normal and fraX males and replicates at nearly the same time on active and inactive X in females. The XIST locus replicates late in all the males studied and asynchronously in female cells. From the late replication of the locus on the active X in males, the authors deduce that the locus on the active X is the later replicating locus in female cells. They conclude that (1) the expansion of the FMR1 locus leads to late replication, (2) silence of the XIST gene in males is associated with late replication of the locus, and (3) this assay will be useful for further studies of the relationship between transcription and replication. 32 refs., 2 figs., 5 tabs.« less

Emergence of mammalian cell-adapted vesicular stomatitis virus from persistent infections of insect vector cells.

PubMed

Novella, Isabel S; Ebendick-Corpus, Bonnie E; Zárate, Selene; Miller, Eric L

2007-06-01

Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.

MCM5: a new actor in the link between DNA replication and Meier-Gorlin syndrome.

PubMed

Vetro, Annalisa; Savasta, Salvatore; Russo Raucci, Annalisa; Cerqua, Cristina; Sartori, Geppo; Limongelli, Ivan; Forlino, Antonella; Maruelli, Silvia; Perucca, Paola; Vergani, Debora; Mazzini, Giuliano; Mattevi, Andrea; Stivala, Lucia Anna; Salviati, Leonardo; Zuffardi, Orsetta

2017-05-01

Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.

Variation, "evolution", immortality and genetic instabilities in tumour cells.

PubMed

Bignold, L P

2007-08-18

The pathological characteristics of tumour cells often include variation of their histopathological features (i.e. "degrees of de-differentiation") between cases of the same tumour type and between different foci within individual tumours. Usually, only a few cell lines from tumours are immortal. Currently, somatic mutation, replicative infidelity of DNA and aneuploidy are suggested as alternative mechanisms of genomic disturbance underlying tumours. Nevertheless, apart from Hansemann's ideas of "anaplasia" and "de-differentiation" (proposed in the 1890s), and supposed "evolutionary themes" in cancer cell biology, little has been published concerning how histopathologic variation and immortality in tumour cells might arise. This paper reviews applications of the concepts of "variation" to tumours, including concepts of "evolution" and "cellular Darwinism". It is proposed that combinations of somatic mutation, DNA replicative infidelity and aneuploidy may explain the variabilities in tumours, and provide immortality in occasional tumour cells. A possible model involves (i) an initial somatic mutation causing reduced replicative fidelity of DNA, which could be variable in intensity, and thus give rise to variations between cases; (ii) a phase of replicative infidelity of DNA causing daughter cells lines to develop various abnormalities to different degrees, and hence provide for variation between areas of the same tumour. As a last event (iii) occasional asymmetric chromosomal distributions (aneuploidy) might "refresh" the ability of a daughter cell to replicate DNA faithfully causing them to become immortal. Thus extensively mutant and variable, hyperploid, and occasionally immortal cells might arise.

Genetic Manipulation of Glycogen Allocation Affects Replicative Lifespan in E. coli

PubMed Central

Röösli, Thomas; Bigosch, Colette; Ackermann, Martin

2016-01-01

In bacteria, replicative aging manifests as a difference in growth or survival between the two cells emerging from division. One cell can be regarded as an aging mother with a decreased potential for future survival and division, the other as a rejuvenated daughter. Here, we aimed at investigating some of the processes involved in aging in the bacterium Escherichia coli, where the two types of cells can be distinguished by the age of their cell poles. We found that certain changes in the regulation of the carbohydrate metabolism can affect aging. A mutation in the carbon storage regulator gene, csrA, leads to a dramatically shorter replicative lifespan; csrA mutants stop dividing once their pole exceeds an age of about five divisions. These old-pole cells accumulate glycogen at their old cell poles; after their last division, they do not contain a chromosome, presumably because of spatial exclusion by the glycogen aggregates. The new-pole daughters produced by these aging mothers are born young; they only express the deleterious phenotype once their pole is old. These results demonstrate how manipulations of nutrient allocation can lead to the exclusion of the chromosome and limit replicative lifespan in E. coli, and illustrate how mutations can have phenotypic effects that are specific for cells with old poles. This raises the question how bacteria can avoid the accumulation of such mutations in their genomes over evolutionary times, and how they can achieve the long replicative lifespans that have recently been reported. PMID:27093302

The interaction between cytosine methylation and processes of DNA replication and repair shape the mutational landscape of cancer genomes

PubMed Central

Poulos, Rebecca C.

2017-01-01

Abstract Methylated cytosines (5mCs) are frequently mutated in the genome. However, no studies have yet comprehensively analysed mutation–methylation associations across cancer types. Here we analyse 916 cancer genomes, together with tissue type-specific methylation and replication timing data. We describe a strong mutation–methylation association across colorectal cancer subtypes, most interestingly in samples with microsatellite instability (MSI) or Polymerase epsilon (POLE) exonuclease domain mutations. By analysing genomic regions with differential mismatch repair (MMR) efficiency, we suggest a possible role for MMR in the correction of 5mC deamination events, potentially accounting for the high rate of 5mC mutation accumulation in MSI tumours. Additionally, we propose that mutant POLE asserts a mutator phenotype specifically at 5mCs, and we find coding mutation hotspots in POLE-mutant cancers at highly-methylated CpGs in the tumour-suppressor genes APC and TP53. Finally, using multivariable regression models, we demonstrate that different cancers exhibit distinct mutation–methylation associations, with DNA repair influencing such associations in certain cancer genomes. Taken together, we find differential associations with methylation that are vital for accurately predicting expected mutation loads across cancer types. Our findings reveal links between methylation and common mutation and repair processes, with these mechanisms defining a key part of the mutational landscape of cancer genomes. PMID:28531315

Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang Shurong; Risques, Rosa Ana; Martin, George M.

2008-01-01

LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. Tomore » our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectable WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes.« less

The Infectivities of Turnip Yellow Mosaic Virus Genomes with Altered tRNA Mimicry Are Not Dependent on Compensating Mutations in the Viral Replication Protein†

PubMed Central

Filichkin, Sergei A.; Bransom, Kay L.; Goodwin, Joel B.; Dreher, Theo W.

2000-01-01

Five highly infectious turnip yellow mosaic virus (TYMV) genomes with sequence changes in their 3′-terminal regions that result in altered aminoacylation and eEF1A binding have been studied. These genomes were derived from cloned parental RNAs of low infectivity by sequential passaging in plants. Three of these genomes that are incapable of aminoacylation have been reported previously (J. B. Goodwin, J. M. Skuzeski, and T. W. Dreher, Virology 230:113–124, 1997). We now demonstrate by subcloning the 3′ untranslated regions into wild-type TYMV RNA that the high infectivities and replication rates of these genomes compared to their progenitors are mostly due to a small number of mutations acquired in the 3′ tRNA-like structure during passaging. Mutations in other parts of the genome, including the replication protein coding region, are not required for high infectivity but probably do play a role in optimizing viral amplification and spread in plants. Two other TYMV RNA variants of suboptimal infectivities, one that accepts methionine instead of the usual valine and one that interacts less tightly with eEF1A, were sequentially passaged to produce highly infectious genomes. The improved infectivities of these RNAs were not associated with increased replication in protoplasts, and no mutations were acquired in their 3′ tRNA-like structures. Complete sequencing of one genome identified two mutations that result in amino acid changes in the movement protein gene, suggesting that improved infectivity may be a function of improved viral dissemination in plants. Our results show that the wild-type TYMV replication proteins are able to amplify genomes with 3′ termini of variable sequence and tRNA mimicry. These and previous results have led to a model in which the binding of eEF1A to the 3′ end to antagonize minus-strand initiation is a major role of the tRNA-like structure. PMID:10954536

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment

PubMed Central

Moreno, Elena; Gallego, Isabel; Gregori, Josep; Lucía-Sanz, Adriana; Soria, María Eugenia; Castro, Victoria; Beach, Nathan M.; Manrubia, Susanna; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M.; Gómez, Jordi; Gastaminza, Pablo

2017-01-01

ABSTRACT Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment. IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments. PMID:28275194

Mutant POLG2 Disrupts DNA Polymerase γ Subunits and Causes Progressive External Ophthalmoplegia

PubMed Central

Longley, Matthew J.; Clark, Susanna; Yu Wai Man, Cynthia; Hudson, Gavin; Durham, Steve E.; Taylor, Robert W.; Nightingale, Simon; Turnbull, Douglass M.; Copeland, William C.; Chinnery, Patrick F.

2006-01-01

DNA polymerase γ (pol γ) is required to maintain the genetic integrity of the 16,569-bp human mitochondrial genome (mtDNA). Mutation of the nuclear gene for the catalytic subunit of pol γ (POLG) has been linked to a wide range of mitochondrial diseases involving mutation, deletion, and depletion of mtDNA. We describe a heterozygous dominant mutation (c.1352G→A/p.G451E) in POLG2, the gene encoding the p55 accessory subunit of pol γ, that causes progressive external ophthalmoplegia with multiple mtDNA deletions and cytochrome c oxidase (COX)–deficient muscle fibers. Biochemical characterization of purified, recombinant G451E-substituted p55 protein in vitro revealed incomplete stimulation of the catalytic subunit due to compromised subunit interaction. Although G451E p55 retains a wild-type ability to bind DNA, it fails to enhance the DNA-binding strength of the p140-p55 complex. In vivo, the disease most likely arises through haplotype insufficiency or heterodimerization of the mutated and wild-type proteins, which promote mtDNA deletions by stalling the DNA replication fork. The progressive accumulation of mtDNA deletions causes COX deficiency in muscle fibers and results in the clinical phenotype. PMID:16685652

Identification and characterization of mutant clones with enhanced propagation rates from phage-displayed peptide libraries.

PubMed

Nguyen, Kieu T H; Adamkiewicz, Marta A; Hebert, Lauren E; Zygiel, Emily M; Boyle, Holly R; Martone, Christina M; Meléndez-Ríos, Carola B; Noren, Karen A; Noren, Christopher J; Hall, Marilena Fitzsimons

2014-10-01

A target-unrelated peptide (TUP) can arise in phage display selection experiments as a result of a propagation advantage exhibited by the phage clone displaying the peptide. We previously characterized HAIYPRH, from the M13-based Ph.D.-7 phage display library, as a propagation-related TUP resulting from a G→A mutation in the Shine-Dalgarno sequence of gene II. This mutant was shown to propagate in Escherichia coli at a dramatically faster rate than phage bearing the wild-type Shine-Dalgarno sequence. We now report 27 additional fast-propagating clones displaying 24 different peptides and carrying 14 unique mutations. Most of these mutations are found either in or upstream of the gene II Shine-Dalgarno sequence, but still within the mRNA transcript of gene II. All 27 clones propagate at significantly higher rates than normal library phage, most within experimental error of wild-type M13 propagation, suggesting that mutations arise to compensate for the reduced virulence caused by the insertion of a lacZα cassette proximal to the replication origin of the phage used to construct the library. We also describe an efficient and convenient assay to diagnose propagation-related TUPS among peptide sequences selected by phage display. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Erosion of functional independence early in the evolution of a microbial mutualism

PubMed Central

Hillesland, Kristina L.; Lim, Sujung; Flowers, Jason J.; Turkarslan, Serdar; Pinel, Nicolas; Zane, Grant M.; Elliott, Nicholas; Qin, Yujia; Wu, Liyou; Baliga, Nitin S.; Zhou, Jizhong; Wall, Judy D.; Stahl, David A.

2014-01-01

Many species have evolved to function as specialized mutualists, often to the detriment of their ability to survive independently. However, there are few, if any, well-controlled observations of the evolutionary processes underlying the genesis of new mutualisms. Here, we show that within the first 1,000 generations of initiating independent syntrophic interactions between a sulfate reducer (Desulfovibrio vulgaris) and a hydrogenotrophic methanogen (Methanococcus maripaludis), D. vulgaris frequently lost the capacity to grow by sulfate respiration, thus losing the primary physiological attribute of the genus. The loss of sulfate respiration was a consequence of mutations in one or more of three key genes in the pathway for sulfate respiration, required for sulfate activation (sat) and sulfate reduction to sulfite (apsA or apsB). Because loss-of-function mutations arose rapidly and independently in replicated experiments, and because these mutations were correlated with enhanced growth rate and productivity, gene loss could be attributed to natural selection, even though these mutations should significantly restrict the independence of the evolved D. vulgaris. Together, these data present an empirical demonstration that specialization for a mutualistic interaction can evolve by natural selection shortly after its origin. They also demonstrate that a sulfate-reducing bacterium can readily evolve to become a specialized syntroph, a situation that may have often occurred in nature. PMID:25267659

In vivo analysis of replication and immunogenicity of proviral clones of human T-lymphotropic virus type 1 with selective envelope surface-unit mutations

PubMed Central

Silverman, Lee R.; Phipps, Andrew J.; Montgomery, Andy; Fernandez, Soledad; Tsukahara, Tomonori; Ratner, Lee; Lairmore, Michael D.

2005-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell lymphoma/leukemia (ATL). The HTLV-1 envelope gene exhibits limited variability when examined from infected individuals, but has not been tested using infectious clones of the virus in animal models. In vitro assays indicate that HTLV-1 envelope (Env) Ser75Ile, Asn95Asp, and Asn195Asp surface unit (SU) mutants are able to replicate in and immortalize lymphocytes. Herein, we examined the effects of these Env mutants in rabbits inoculated with HTLV-1 immortalized ACH.75, ACH.95, or ACH.195 cell lines (expressing full-length molecular clones with the SU mutations) or the ACH.1 cell line (expressing wild-type SU). All rabbits became infected, and the fidelity of the mutations was maintained throughout the 8-week study. However, SU point mutations resulted in decreased antibody responses to viral group-associated antigen (Gag) and Env antigens. ACH.195 rabbits had a selective decreased antibody response to SU, and one ACH.195 rabbit had an antibody response to both HTLV-1 and HTLV-2 SUs. Some mutant inoculation groups had altered proviral loads. However, peripheral-blood mononuclear cell (PBMC) proviral loads did not correlate with antibody responses. Our data are the first to demonstrate that mutations in critical determinants of HTLV-1 Env SU altered antibody responses and proviral loads, but do not prevent viral replication in vivo. PMID:16046523

Human hepatocytes apoptosis induced by replication of hepatitis B virus subgenotypes F1b and F4: Role of basal core promoter and preCore mutations.

PubMed

Elizalde, María Mercedes; Sevic, Ina; González López Ledesma, María Mora; Campos, Rodolfo Héctor; Barbini, Luciana; Flichman, Diego Martin

2018-01-01

In the context of pathogenesis of HBV infection, HBV genotypes and mutants have been shown to affect the natural course of chronic infection and treatment outcomes. In this work, we studied the induction of apoptosis by the replication of HBV subgenotypes F1b and F4, and the naturally occurring mutants BCP and preCore. Both subgenotypes F1b and F4 HBV genome transfections induced cell death by apoptosis in human hepatocytes. The BCPdm (A1762T/G1764A) and preCore (G1896A) mutants induced higher levels of apoptosis than the wt virus. This increase in apoptosis was not associated with the enhanced viral replication of the variants. HBV-mediated apoptosis was independent of viral subgenotypes, and associated with the modulation of members of the regulatory Bcl-2 family proteins expression in the mitochondrial apoptotic pathway. Finally, the apoptosis induction increase observed for the preCore mutants suggests that HBeAg might have an anti-apoptotic effect in human hepatocytes. Copyright © 2017 Elsevier Inc. All rights reserved.

Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

NASA Astrophysics Data System (ADS)

Gaspar, Miguel; Shenk, Thomas

2006-02-01

The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

Replication and Adaptive Mutations of Low Pathogenic Avian Influenza Viruses in Tracheal Organ Cultures of Different Avian Species

PubMed Central

Petersen, Henning; Matrosovich, Mikhail; Pleschka, Stephan; Rautenschlein, Silke

2012-01-01

Transmission of avian influenza viruses (AIV) between different avian species may require genome mutations that allow efficient virus replication in a new species and could increase virulence. To study the role of domestic poultry in the evolution of AIV we compared replication of low pathogenic (LP) AIV of subtypes H9N2, H7N7 and H6N8 in tracheal organ cultures (TOC) and primary embryo fibroblast cultures of chicken, turkey, Pekin duck and homing pigeon. Virus strain-dependent and avian species-related differences between LPAIV were observed in growth kinetics and induction of ciliostasis in TOC. In particular, our data demonstrate high susceptibility to LPAIV of turkey TOC contrasted with low susceptibility of homing pigeon TOC. Serial virus passages in the cells of heterologous host species resulted in adaptive mutations in the AIV genome, especially in the receptor-binding site and protease cleavage site of the hemagglutinin. Our data highlight differences in susceptibility of different birds to AIV viruses and emphasizes potential role of poultry in the emergence of new virus variants. PMID:22912693

The timing of UV mutagenesis in yeast: a pedigree analysis of induced recessive mutation.

PubMed

James, A P; Kilbey, B J

1977-10-01

The mechanism of UV-induced mutation in eukaryotes was studied in individual yeast cells by a procedure that combined pedigree analysis and tetrad analysis. The technique involved the induction of recessive lethals and semilethals in G1 diploid cells. Induced frequencies were 25 and 61 percent at survival levels of 90 and 77 percent, respectively. No evidence of gross chromosome aberrations was detected. Recessive mutations that affect only one strand or that affect both strands of the DNA molecule are induced much at random among a population of cells, and both types can occur within the same cell. However, the data confirm that two-strand mutations are in the majority after a low level of irradiation. The simplest explanation involves a mechanism whereby most mutations are fixed in both strands prior to the first round of post-irradiation DNA replication. The recessive mutational consequences of irradiation are exhausted at the conclusion of the first post-irradiation cell division, although dominant-lethal sectoring continues at a high level through the second post-irradiation division. It is concluded that pyrimidine dimers that persist to the second round of DNA replication are rare or ineffective.

New paradigms in the repair of oxidative damage in human genome: mechanisms ensuring repair of mutagenic base lesions during replication and involvement of accessory proteins.

PubMed

Dutta, Arijit; Yang, Chunying; Sengupta, Shiladitya; Mitra, Sankar; Hegde, Muralidhar L

2015-05-01

Oxidized bases in the mammalian genome, which are invariably mutagenic due to their mispairing property, are continuously induced by endogenous reactive oxygen species and more abundantly after oxidative stress. Unlike bulky base adducts induced by UV and other environmental mutagens in the genome that block replicative DNA polymerases, oxidatively damaged bases such as 5-hydroxyuracil, produced by oxidative deamination of cytosine in the template strand, do not block replicative polymerases and thus need to be repaired prior to replication to prevent mutation. Following up our earlier studies, which showed that the Nei endonuclease VIII like 1 (NEIL1) DNA glycosylase, one of the five base excision repair (BER)-initiating enzymes in mammalian cells, has enhanced expression during the S-phase and higher affinity for replication fork-mimicking single-stranded (ss) DNA substrates, we recently provided direct experimental evidence for NEIL1's role in replicating template strand repair. The key requirement for this event, which we named as the 'cow-catcher' mechanism of pre-replicative BER, is NEIL1's non-productive binding (substrate binding without product formation) to the lesion base in ss DNA template to stall DNA synthesis, causing fork regression. Repair of the lesion in reannealed duplex is then carried out by NEIL1 in association with the DNA replication proteins. NEIL1 (and other BER-initiating enzymes) also interact with several accessory and non-canonical proteins including the heterogeneous nuclear ribonucleoprotein U and Y-box-binding protein 1 as well as high mobility group box 1 protein, whose precise roles in BER are still obscure. In this review, we have discussed the recent advances in our understanding of oxidative genome damage repair pathways with particular focus on the pre-replicative template strand repair and the role of scaffold factors like X-ray repairs cross-complementing protein 1 and poly (ADP-ribose) polymerase 1 and other accessory proteins guiding distinct BER sub-pathways.

Novel Insights for Inhibiting Mutant Heterodimer IDH1wt-R132H in Cancer: An In-Silico Approach.

PubMed

Juritz, Ezequiel Iván; Bascur, Juan Pablo; Almonacid, Daniel Eduardo; González-Nilo, Fernando Danilo

2018-06-01

Isocitrate dehydrogenase 1 (IDH1) is a dimeric enzyme responsible for supplying the cell's nicotinamide adenine dinucleotide phosphate (NADPH) reserves via dehydrogenation of isocitrate (ICT) and reduction of NADP+. Mutations in position R132 trigger cancer by enabling IDH1 to produce D-2-hydroxyglutarate (2-HG) and reduce inhibition by ICT. Mutant IDH1 can be found as a homodimer or a heterodimer. We propose a novel strategy to inhibit IDH1 R132 variants as a means not to decrease the concentration of 2-HG but to provoke a cytotoxic effect, as the cell malignancy at this point no longer depends on 2-HG. We aim to inhibit the activity of the mutant heterodimer to block the wild-type subunit. Limiting the NADPH reserves in a cancerous cell will enhance its susceptibility to the oxidative stress provoked by chemotherapy. We performed a virtual screening using all US FDA-approved drugs to replicate the loss of inhibition of mutant IDH1 by ICT. We characterized our results based on molecular interactions and correlated them with the described phenotypes. We replicated the loss of inhibition by ICT in mutant IDH1. We identified 20 drugs with the potential to inhibit the heterodimeric isoform. Six of them are used in cancer treatment. We present 20 FDA-approved drugs with the potential to inhibit IDH1 wild-type activity in mutated cells. We believe this work may provide important insights into current and new approaches to dealing with IDH1 mutations. In addition, it may be used as a basis for additional studies centered on drugs presenting differential sensitivities to different IDH1 isoforms.

Emerging complexities of APOBEC3G action on immunity and viral fitness during HIV infection and treatment.

PubMed

Monajemi, Mahdis; Woodworth, Claire F; Benkaroun, Jessica; Grant, Michael; Larijani, Mani

2012-04-30

The enzyme APOBEC3G (A3G) mutates the human immunodeficiency virus (HIV) genome by converting deoxycytidine (dC) to deoxyuridine (dU) on minus strand viral DNA during reverse transcription. A3G restricts viral propagation by degrading or incapacitating the coding ability of the HIV genome. Thus, this enzyme has been perceived as an innate immune barrier to viral replication whilst adaptive immunity responses escalate to effective levels. The discovery of A3G less than a decade ago led to the promise of new anti-viral therapies based on manipulation of its cellular expression and/or activity. The rationale for therapeutic approaches has been solidified by demonstration of the effectiveness of A3G in diminishing viral replication in cell culture systems of HIV infection, reports of its mutational footprint in virions from patients, and recognition of its unusually robust enzymatic potential in biochemical studies in vitro. Despite its effectiveness in various experimental systems, numerous recent studies have shown that the ability of A3G to combat HIV in the physiological setting is severely limited. In fact, it has become apparent that its mutational activity may actually enhance viral fitness by accelerating HIV evolution towards the evasion of both anti-viral drugs and the immune system. This body of work suggests that the role of A3G in HIV infection is more complex than heretofore appreciated and supports the hypothesis that HIV has evolved to exploit the action of this host factor. Here we present an overview of recent data that bring to light historical overestimation of A3G's standing as a strictly anti-viral agent. We discuss the limitations of experimental systems used to assess its activities as well as caveats in data interpretation.

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dai Heqiao; Liu Jianying; Malkas, Linda H.

2009-04-15

Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediatedmore » by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC{sub 50} of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases {alpha}, {delta} and {epsilon} is 15, 45 and 125 {mu}M, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC{sub 50} = 88 {mu}M), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2-13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr(III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication correlates with the genotoxic and cytotoxic effects of this metal ion; and promotes cell killing via inhibition of the DNA polymerase activity mediating the DNA replication and repair processes utilized by human cells.« less

Chromium reduces the in vitro activity and fidelity of DNA replication mediated by the human cell DNA synthesome

PubMed Central

Dai, Heqiao; Liu, Jianying; Malkas, Linda H.; Catalano, Jennifer; Alagharu, Srilakshmi; Hickey, Robert J.

2009-01-01

Hexavalent chromium Cr(VI) is known to be a carcinogenic metal ion, with a complicated mechanism of action. It can be found within our environment in soil and water contaminated by manufacturing processes. Cr(VI) ion is readily taken up by cells, and is recognized to be both genotoxic and cytotoxic; following its reduction to the stable trivalent form of the ion, chromium(Cr(III)), within cells. This form of the ion is known to impede the activity of cellular DNA polymerase and polymerase-mediated DNA replication. Here, we report the effects of chromium on the activity and fidelity of the DNA replication process mediated by the human cell DNA synthesome. The DNA synthesome is a functional multiprotein complex that is fully competent to carry-out each phase of the DNA replication process. The IC50 of Cr(III) toward the activity of DNA synthesome-associated DNA polymerases α, δ and ε is 15, 45 and 125 μM, respectively. Cr(III) inhibits synthesome-mediated DNA synthesis (IC50 =88 μM), and significantly reduces the fidelity of synthesome-mediated DNA replication. The mutation frequency induced by the different concentrations of Cr(III) ion used in our assays ranges from 2–13 fold higher than that which occurs spontaneously, and the types of mutations include single nucleotide substitutions, insertions, and deletions. Single nucleotide substitutions are the predominant type of mutation, and they occur primarily at GC base-pairs. Cr(III) ion produces a lower number of transition and a higher number of transversion mutations than occur spontaneously. Unlike Cr (III), Cr(VI) ion has little effect on the in vitro DNA synthetic activity and fidelity of the DNA synthesome, but does significantly inhibit DNA synthesis in intact cells. Cell growth and proliferation is also arrested by increasing concentrations of Cr(VI) ion. Our studies provide evidence indicating that the chromium ion induced decrease in the fidelity and activity of synthesome mediated DNA replication correlates with the genotoxic and cytotoxic effects of this metal ion; and promotes cell killing via inhibition of the DNA polymerase activity mediating the DNA replication and repair processes utilized by human cells. PMID:19371627

Important role of N108 residue in binding of bovine foamy virus transactivator Tas to viral promoters.

PubMed

Bing, Tiejun; Zhang, Suzhen; Liu, Xiaojuan; Liang, Zhibin; Shao, Peng; Zhang, Song; Qiao, Wentao; Tan, Juan

2016-06-30

Bovine foamy virus (BFV) encodes the transactivator BTas, which enhances viral gene transcription by binding to the long terminal repeat promoter and the internal promoter. In this study, we investigated the different replication capacities of two similar BFV full-length DNA clones, pBS-BFV-Y and pBS-BFV-B. Here, functional analysis of several chimeric clones revealed a major role for the C-terminal region of the viral genome in causing this difference. Furthermore, BTas-B, which is located in this C-terminal region, exhibited a 20-fold higher transactivation activity than BTas-Y. Sequence alignment showed that these two sequences differ only at amino acid 108, with BTas-B containing N108 and BTas-Y containing D108 at this position. Results of mutagenesis studies demonstrated that residue N108 is important for BTas binding to viral promoters. In addition, the N108D mutation in pBS-BFV-B reduced the viral replication capacity by about 1.5-fold. Our results suggest that residue N108 is important for BTas binding to BFV promoters and has a major role in BFV replication. These findings not only advances our understanding of the transactivation mechanism of BTas, but they also highlight the importance of certain sequence polymorphisms in modulating the replication capacity of isolated BFV clones.

A Case Study of the De Novo Evolution of a Complex Odometric Behavior in Digital Organisms

PubMed Central

Grabowski, Laura M.; Bryson, David M.; Dyer, Fred C.; Pennock, Robert T.; Ofria, Charles

2013-01-01

Investigating the evolution of animal behavior is difficult. The fossil record leaves few clues that would allow us to recapitulate the path that evolution took to build a complex behavior, and the large population sizes and long time scales required prevent us from re-evolving such behaviors in a laboratory setting. We present results of a study in which digital organisms–self-replicating computer programs that are subject to mutations and selection–evolved in different environments that required information about past experience for fitness-enhancing behavioral decisions. One population evolved a mechanism for step-counting, a surprisingly complex odometric behavior that was only indirectly related to enhancing fitness. We examine in detail the operation of the evolved mechanism and the evolutionary transitions that produced this striking example of a complex behavior. PMID:23577113

Selective Bottlenecks Shape Evolutionary Pathways Taken during Mammalian Adaptation of a 1918-like Avian Influenza Virus.

PubMed

Moncla, Louise H; Zhong, Gongxun; Nelson, Chase W; Dinis, Jorge M; Mutschler, James; Hughes, Austin L; Watanabe, Tokiko; Kawaoka, Yoshihiro; Friedrich, Thomas C

2016-02-10

Avian influenza virus reassortants resembling the 1918 human pandemic virus can become transmissible among mammals by acquiring mutations in hemagglutinin (HA) and polymerase. Using the ferret model, we trace the evolutionary pathway by which an avian-like virus evolves the capacity for mammalian replication and airborne transmission. During initial infection, within-host HA diversity increased drastically. Then, airborne transmission fixed two polymerase mutations that do not confer a detectable replication advantage. In later transmissions, selection fixed advantageous HA1 variants. Transmission initially involved a "loose" bottleneck, which became strongly selective after additional HA mutations emerged. The stringency and evolutionary forces governing between-host bottlenecks may therefore change throughout host adaptation. Mutations occurred in multiple combinations in transmitted viruses, suggesting that mammalian transmissibility can evolve through multiple genetic pathways despite phenotypic constraints. Our data provide a glimpse into avian influenza virus adaptation in mammals, with broad implications for surveillance on potentially zoonotic viruses. Copyright © 2016 Elsevier Inc. All rights reserved.

Developing Novel Oncolytic Adenoviruses through Bioselection

PubMed Central

Yan, Wen; Kitzes, Galila; Dormishian, Farid; Hawkins, Lynda; Sampson-Johannes, Adam; Watanabe, Josh; Holt, Jenny; Lee, Vivian; Dubensky, Thomas; Fattaey, Ali; Hermiston, Terry; Balmain, Allan; Shen, Yuqiao

2003-01-01

Mutants of human adenovirus 5 (Ad5) with enhanced oncolytic activity were isolated by using a procedure termed bioselection. Two mutants, ONYX-201 and ONYX-203, were plaque purified from a pool of randomly mutagenized Ad5 that was repeatedly passaged in the human colorectal cancer cell line HT29, and they were subsequently characterized. ONYX-201 and ONYX-203 replicated more rapidly in HT29 cells than wild-type Ad5, and they lysed HT29 cells up to 1,000-fold more efficiently. The difference was most profound when cells were infected at a relatively low multiplicity of infection, presumably due to the compounding effects of multiple rounds of infection. This enhanced cytolytic activity was observed not only in HT29 cells but also in many other human cancer cell lines tested. In contrast, the cytotoxicity of the bioselected mutants in a number of normal primary human cells was similar to that of wild-type Ad5, thus enhancing the therapeutic index (cytotoxicity in tumor cells versus that in normal cells) of these oncolytic agents. Both ONYX-201 and -203 contain seven single-base-pair mutations when compared with Ad5, four of which were common between ONYX-201 and -203. The mutation at nucleotide 8350, shared by both mutant viruses, was shown to be essential for the observed phenotypes. This mutation was mapped to the i-leader region of the major late transcription unit, resulting in the truncation of 21 amino acids from the C terminus of the i-leader protein. This work demonstrates that bioselection is a powerful tool for developing novel tumor-selective oncolytic viruses. Other potential applications of this technology are discussed. PMID:12552003

Tyrosine phosphorylation of histone H2A by CK2 regulates transcriptional elongation

PubMed Central

Basnet, Harihar; Bessie Su, Xue; Tan, Yuliang; Meisenhelder, Jill; Merkurjev, Daria; Ohgi, Kenneth A.; Hunter, Tony; Pillus, Lorraine; Rosenfeld, Michael G.

2014-01-01

Post-translational histone modifications play critical roles in regulating transcription, the cell cycle, DNA replication and DNA damage repair1. The identification of new histone modifications critical for transcriptional regulation at initiation, elongation, or termination is of particular interest. Here, we report a new layer of regulation in transcriptional elongation that is conserved from yeast to mammals, based on a phosphorylation of a highly-conserved tyrosine residue, Y57, in histone H2A that is mediated by an unsuspected tyrosine kinase activity of casein kinase 2 (CK2). Mutation of H2A-Y57 in yeast or inhibition of CK2 activity impairs transcriptional elongation in yeast as well as in mammalian cells. Genome-wide binding analysis reveals that CK2α, the catalytic subunit of CK2, binds across RNA polymerase II-transcribed coding genes and active enhancers. Mutation of Y57 causes a loss of H2B mono-ubiquitylation as well as H3K4me3 and H3K79me3, histone marks associated with active transcription. Mechanistically, both CK2 inhibition and H2A-Y57F mutation enhance the H2B deubiquitylation activity of the SAGA complex, suggesting a critical role of this phosphorylation in coordinating the activity of the SAGA during transcription. Together, these results identify a new component of regulation in transcriptional elongation based on CK2-dependent tyrosine phosphorylation of the globular domain of H2A. PMID:25252977

Meiotic recombination counteracts male-biased mutation (male-driven evolution).

PubMed

Mawaribuchi, Shuuji; Ito, Michihiko; Ogata, Mitsuaki; Oota, Hiroki; Katsumura, Takafumi; Takamatsu, Nobuhiko; Miura, Ikuo

2016-01-27

Meiotic recombination is believed to produce greater genetic variation despite the fact that deoxyribonucleic acid (DNA)-replication errors are a major source of mutations. In some vertebrates, mutation rates are higher in males than in females, which developed the theory of male-driven evolution (male-biased mutation). However, there is little molecular evidence regarding the relationships between meiotic recombination and male-biased mutation. Here we tested the theory using the frog Rana rugosa, which has both XX/XY- and ZZ/ZW-type sex-determining systems within the species. The male-to-female mutation-rate ratio (α) was calculated from homologous sequences on the X/Y or Z/W sex chromosomes, which supported male-driven evolution. Surprisingly, each α value was notably higher in the XX/XY-type group than in the ZZ/ZW-type group, although α should have similar values within a species. Interestingly, meiotic recombination between homologous chromosomes did not occur except at terminal regions in males of this species. Then, by subdividing α into two new factors, a replication-based male-to-female mutation-rate ratio (β) and a meiotic recombination-based XX-to-XY/ZZ-to-ZW mutation-rate ratio (γ), we constructed a formula describing the relationship among a nucleotide-substitution rate and the two factors, β and γ. Intriguingly, the β- and γ-values were larger and smaller than 1, respectively, indicating that meiotic recombination might reduce male-biased mutations. © 2016 The Author(s).

Nonlinear dynamics of the rock-paper-scissors game with mutations.

PubMed

Toupo, Danielle F P; Strogatz, Steven H

2015-05-01

We analyze the replicator-mutator equations for the rock-paper-scissors game. Various graph-theoretic patterns of mutation are considered, ranging from a single unidirectional mutation pathway between two of the species, to global bidirectional mutation among all the species. Our main result is that the coexistence state, in which all three species exist in equilibrium, can be destabilized by arbitrarily small mutation rates. After it loses stability, the coexistence state gives birth to a stable limit cycle solution created in a supercritical Hopf bifurcation. This attracting periodic solution exists for all the mutation patterns considered, and persists arbitrarily close to the limit of zero mutation rate and a zero-sum game.

Inhibition of DNA2 nuclease as a therapeutic strategy targeting replication stress in cancer cells.

PubMed

Kumar, S; Peng, X; Daley, J; Yang, L; Shen, J; Nguyen, N; Bae, G; Niu, H; Peng, Y; Hsieh, H-J; Wang, L; Rao, C; Stephan, C C; Sung, P; Ira, G; Peng, G

2017-04-17

Replication stress is a characteristic feature of cancer cells, which is resulted from sustained proliferative signaling induced by activation of oncogenes or loss of tumor suppressors. In cancer cells, oncogene-induced replication stress manifests as replication-associated lesions, predominantly double-strand DNA breaks (DSBs). An essential mechanism utilized by cells to repair replication-associated DSBs is homologous recombination (HR). In order to overcome replication stress and survive, cancer cells often require enhanced HR repair capacity. Therefore, the key link between HR repair and cellular tolerance to replication-associated DSBs provides us with a mechanistic rationale for exploiting synthetic lethality between HR repair inhibition and replication stress. DNA2 nuclease is an evolutionarily conserved essential enzyme in replication and HR repair. Here we demonstrate that DNA2 is overexpressed in pancreatic cancers, one of the deadliest and more aggressive forms of human cancers, where mutations in the KRAS are present in 90-95% of cases. In addition, depletion of DNA2 significantly reduces pancreatic cancer cell survival and xenograft tumor growth, suggesting the therapeutic potential of DNA2 inhibition. Finally, we develop a robust high-throughput biochemistry assay to screen for inhibitors of the DNA2 nuclease activity. The top inhibitors were shown to be efficacious against both yeast Dna2 and human DNA2. Treatment of cancer cells with DNA2 inhibitors recapitulates phenotypes observed upon DNA2 depletion, including decreased DNA double strand break end resection and attenuation of HR repair. Similar to genetic ablation of DNA2, chemical inhibition of DNA2 selectively attenuates the growth of various cancer cells with oncogene-induced replication stress. Taken together, our findings open a new avenue to develop a new class of anticancer drugs by targeting druggable nuclease DNA2. We propose DNA2 inhibition as new strategy in cancer therapy by targeting replication stress, a molecular property of cancer cells that is acquired as a result of oncogene activation instead of targeting currently undruggable oncoprotein itself such as KRAS.

5-Azacytidine Can Induce Lethal Mutagenesis in Human Immunodeficiency Virus Type 1▿ †

PubMed Central

Dapp, Michael J.; Clouser, Christine L.; Patterson, Steven; Mansky, Louis M.

2009-01-01

Ribonucleosides inhibit human immunodeficiency virus type 1 (HIV-1) replication by mechanisms that have not been fully elucidated. Here, we report the antiviral mechanism for the ribonucleoside analog 5-azacytidine (5-AZC). We hypothesized that the anti-HIV-1 activity of 5-AZC was due to an increase in the HIV-1 mutation rate following its incorporation into viral RNA during transcription. However, we demonstrate that 5-AZC's primary antiviral activity can be attributed to its effect on the early phase of HIV-1 replication. Furthermore, the antiviral activity was associated with an increase in the frequency of viral mutants, suggesting that 5-AZC's primary target is reverse transcription. Sequencing analysis showed an enrichment in G-to-C transversion mutations and further supports the idea that reverse transcription is an antiviral target of 5-AZC. These results indicate that 5-AZC is incorporated into viral DNA following reduction to 5-aza-2′-deoxycytidine. Incorporation into the viral DNA leads to an increase in mutant frequency that is consistent with lethal mutagenesis during reverse transcription as the primary antiviral mechanism of 5-AZC. Antiviral activity and increased mutation frequency were also associated with the late phase of HIV-1 replication; however, 5-AZC's effect on the late phase was less robust. These results reveal that the primary antiviral mechanism of 5-AZC can be attributed to its ability to increase the HIV-1 mutation frequency through viral-DNA incorporation during reverse transcription. Our observations indicate that 5-AZC can affect two steps in HIV-1 replication (i.e., transcription and reverse transcription) but that its primary antiviral activity is due to incorporation during reverse transcription. PMID:19726509

Evidence That BRCA1- or BRCA2-Associated Cancers Are Not Inevitable

PubMed Central

Levin, Bess; Lech, Denise; Friedenson, Bernard

2012-01-01

Inheriting a BRCA1 or BRCA2 gene mutation can cause a deficiency in repairing complex DNA damage. This step leads to genomic instability and probably contributes to an inherited predisposition to breast and ovarian cancer. Complex DNA damage has been viewed as an integral part of DNA replication before cell division. It causes temporary replication blocks, replication fork collapse, chromosome breaks and sister chromatid exchanges (SCEs). Chemical modification of DNA may also occur spontaneously as a byproduct of normal processes. Pathways containing BRCA1 and BRCA2 gene products are essential to repair spontaneous complex DNA damage or to carry out SCEs if repair is not possible. This scenario creates a theoretical limit that effectively means there are spontaneous BRCA1/2-associated cancers that cannot be prevented or delayed. However, much evidence for high rates of spontaneous DNA mutation is based on measuring SCEs by using bromodeoxyuridine (BrdU). Here we find that the routine use of BrdU has probably led to overestimating spontaneous DNA damage and SCEs because BrdU is itself a mutagen. Evidence based on spontaneous chromosome abnormalities and epidemiologic data indicates strong effects from exogenous mutagens and does not support the inevitability of cancer in all BRCA1/2 mutation carriers. We therefore remove a theoretical argument that has limited efforts to develop chemoprevention strategies to delay or prevent cancers in BRCA1/2 mutation carriers. PMID:22972572

Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation.

PubMed

Mostafa, Heba H; Thompson, Thornton W; Konen, Adam J; Haenchen, Steve D; Hilliard, Joshua G; Macdonald, Stuart J; Morrison, Lynda A; Davido, David J

2018-04-01

In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39 , which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes in vivo , we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39 mut ), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection. IMPORTANCE HSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in greater detail the events that modulate HSV-1 replication and pathogenesis. In the current study, we identified a neuroattenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also known as ICP6). This mutant form of ICP6 was responsible for the attenuation of KOS-NA in vivo and resulted in diminished ICP6 protein levels and antiapoptotic effect. Thus, we have determined that subtle alteration of the UL39 gene regulates expression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially making KOS-NA a promising vaccine candidate against HSV-1. Copyright © 2018 American Society for Microbiology.

Comparative genome analysis of a thermotolerant Escherichia coli obtained by Genome Replication Engineering Assisted Continuous Evolution (GREACE) and its parent strain provides new understanding of microbial heat tolerance.

PubMed

Luan, Guodong; Bao, Guanhui; Lin, Zhao; Li, Yang; Chen, Zugen; Li, Yin; Cai, Zhen

2015-12-25

Heat tolerance of microbes is of great importance for efficient biorefinery and bioconversion. However, engineering and understanding of microbial heat tolerance are difficult and insufficient because it is a complex physiological trait which probably correlates with all gene functions, genetic regulations, and cellular metabolisms and activities. In this work, a novel strain engineering approach named Genome Replication Engineering Assisted Continuous Evolution (GREACE) was employed to improve the heat tolerance of Escherichia coli. When the E. coli strain carrying a mutator was cultivated under gradually increasing temperature, genome-wide mutations were continuously generated during genome replication and the mutated strains with improved thermotolerance were autonomously selected. A thermotolerant strain HR50 capable of growing at 50°C on LB agar plate was obtained within two months, demonstrating the efficiency of GREACE in improving such a complex physiological trait. To understand the improved heat tolerance, genomes of HR50 and its wildtype strain DH5α were sequenced. Evenly distributed 361 mutations covering all mutation types were found in HR50. Closed material transportations, loose genome conformation, and possibly altered cell wall structure and transcription pattern were the main differences of HR50 compared with DH5α, which were speculated to be responsible for the improved heat tolerance. This work not only expanding our understanding of microbial heat tolerance, but also emphasizing that the in vivo continuous genome mutagenesis method, GREACE, is efficient in improving microbial complex physiological trait. Copyright © 2015 Elsevier B.V. All rights reserved.

Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice.

PubMed

Kon, Ayana; Yamazaki, Satoshi; Nannya, Yasuhito; Kataoka, Keisuke; Ota, Yasunori; Nakagawa, Masahiro Marshall; Yoshida, Kenichi; Shiozawa, Yusuke; Morita, Maiko; Yoshizato, Tetsuichi; Sanada, Masashi; Nakayama, Manabu; Koseki, Haruhiko; Nakauchi, Hiromitsu; Ogawa, Seishi

2018-02-08

Splicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre -mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2 -mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2 -mutated mice. RNA sequencing of the Srsf2 -mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r , Fyn , Gnas , Nsd1 , Hnrnpa2b1 , and Trp53bp1 Among the mutant Srsf2 -associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults. © 2018 by The American Society of Hematology.

Competence in Streptococcus pneumoniae Is a Response to an Increasing Mutational Burden

PubMed Central

Gagne, Alyssa L.; Stevens, Kathleen E.; Cassone, Marco; Pujari, Amit; Abiola, Olufunke E.; Chang, Diana J.; Sebert, Michael E.

2013-01-01

Competence for genetic transformation in Streptococcus pneumoniae has previously been described as a quorum-sensing trait regulated by a secreted peptide pheromone. Recently we demonstrated that competence is also activated by reduction in the accuracy of protein biosynthesis. We have now investigated whether errors upstream of translation in the form of random genomic mutations can provide a similar stimulus. Here we show that generation of a mutator phenotype in S. pneumoniae through deletions of mutX, hexA or hexB enhanced the expression of competence. Similarly, chemical mutagenesis with the nucleotide analog dPTP promoted development of competence. To investigate the relationship between mutational load and the activation of competence, replicate lineages of the mutX strain were serially passaged under conditions of relaxed selection allowing random accumulation of secondary mutations. Competence increased with propagation in these lineages but not in control lineages having wild-type mutX. Resequencing of these derived strains revealed between 1 and 9 single nucleotide polymorphisms (SNPs) per lineage, which were broadly distributed across the genome and did not involve known regulators of competence. Notably, the frequency of competence development among the sequenced strains correlated significantly with the number of nonsynonymous mutations that had been acquired. Together, these observations provide support for the hypothesis that competence in S. pneumoniae is regulated in response to the accumulated burden of coding mutations in the bacterial genome. In contrast to previously described DNA damage response systems that are activated by physical lesions in the chromosome, this pneumococcal pathway may represent a unique stress response system that monitors the coding integrity of the genome. PMID:23967325

An interferon regulatory factor binding site in the U5 region of the bovine leukemia virus long terminal repeat stimulates Tax-independent gene expression.

PubMed

Kiermer, V; Van Lint, C; Briclet, D; Vanhulle, C; Kettmann, R; Verdin, E; Burny, A; Droogmans, L

1998-07-01

Bovine leukemia virus (BLV) replication is controlled by both cis- and trans-acting elements. The virus-encoded transactivator, Tax, is necessary for efficient transcription from the BLV promoter, although it is not present during the early stages of infection. Therefore, sequences that control Tax-independent transcription must play an important role in the initiation of viral gene expression. This study demonstrates that the R-U5 sequence of BLV stimulates Tax-independent reporter gene expression directed by the BLV promoter. R-U5 was also stimulatory when inserted immediately downstream from the transcription initiation site of a heterologous promoter. Progressive deletion analysis of this region revealed that a 46-bp element corresponding to the 5' half of U5 is principally responsible for the stimulation. This element exhibited enhancer activity when inserted upstream or downstream from the herpes simplex virus thymidine kinase promoter. This enhancer contains a binding site for the interferon regulatory factors IRF-1 and IRF-2. A 3-bp mutation that destroys the IRF recognition site caused a twofold decrease in Tax-independent BLV long terminal repeat-driven gene expression. These observations suggest that the IRF binding site in the U5 region of BLV plays a role in the initiation of virus replication.

Adenovirus sequences required for replication in vivo.

PubMed Central

Wang, K; Pearson, G D

1985-01-01

We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occupies the first 18 to 21 bp and includes sequences conserved between all adenovirus serotypes. The adjacent auxillary region extends past nucleotide 36 but not past nucleotide 67 and contains the binding site for nuclear factor I. Images PMID:2991857

Alterations of the three short open reading frames in the Rous sarcoma virus leader RNA modulate viral replication and gene expression.

PubMed Central

Moustakas, A; Sonstegard, T S; Hackett, P B

1993-01-01

The Rous sarcoma virus (RSV) leader RNA has three short open reading frames (ORF1 to ORF3) which are conserved in all avian sarcoma-leukosis retroviruses. Effects on virus propagation were determined following three types of alterations in the ORFs: (i) replacement of AUG initiation codons in order to prohibit ORF translation, (ii) alterations of the codon context around the AUG initiation codon to enhance translation of the normally silent ORF3, and (iii) elongation of the ORF coding sequences. Mutagenesis of the AUG codons for ORF1 and ORF2 (AUG1 and AUG2) singly or together delayed the onset of viral replication and cell transformation. In contrast, mutagenesis of AUG3 almost completely suppressed these viral activities. Mutagenesis of ORF3 to enhance its translation inhibited viral propagation. When the mutant ORF3 included an additional frameshift mutation which extended the ORF beyond the initiation site for the gag, gag-pol, and env proteins, host cells were initially transformed but died soon thereafter. Elongation of ORF1 from 7 to 62 codons led to the accumulation of transformation-defective virus with a delayed onset of replication. In contrast, viruses with elongation of ORF1 from 7 to 30 codons, ORF2 from 16 to 48 codons, or ORF3 from 9 to 64 codons, without any alterations in the AUG context, exhibited wild-type phenotypes. These results are consistent with a model that translation of the ORFs is necessary to facilitate virus production. Images PMID:7685415

Clinical Management and Tumor Surveillance Recommendations of Inherited Mismatch Repair Deficiency in Childhood.

PubMed

Tabori, Uri; Hansford, Jordan R; Achatz, Maria Isabel; Kratz, Christian P; Plon, Sharon E; Frebourg, Thierry; Brugières, Laurence

2017-06-01

Replication proofreading is crucial to avoid mutation accumulation in dividing cells. In humans, proofreading and replication repair is maintained by the exonuclease domains of DNA polymerases and the mismatch repair system. Individuals harboring germline mutations in genes involved in this process are at increased risk of early cancers from multiple organs. Biallelic mutations in any of the four mismatch repair genes MSH2, MSH6, MLH1 , and PMS2 result in one of the most aggressive childhood cancer predisposition syndromes, termed constitutional mismatch repair deficiency or constitutional mismatch repair deficiency syndrome (CMMRD). Data gathered in the last decade allow us to better define the clinical manifestations, tumor spectrum, and diagnostic algorithms for CMMRD. In this article, we summarize this information and present a comprehensive consensus surveillance protocol for these individuals. Ongoing research will allow for further definition of replication repair-deficient cancer syndromes, assessing the cost-effectiveness of such surveillance protocols and potential therapeutic interventions for these children and families. Clin Cancer Res; 23(11); e32-e37. ©2017 AACR See all articles in the online-only CCR Pediatric Oncology Series. ©2017 American Association for Cancer Research.

Limited copy number - high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies

PubMed Central

Do, Hongdo; Dobrovic, Alexander

2009-01-01

Background Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations. We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns. Results Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions. LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification. Conclusion LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations. PMID:19811662

Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies.

PubMed

Do, Hongdo; Dobrovic, Alexander

2009-10-08

Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations.We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns. Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions.LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification. LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations.

PTEN in the maintenance of genome integrity: From DNA replication to chromosome segregation.

PubMed

Hou, Sheng-Qi; Ouyang, Meng; Brandmaier, Andrew; Hao, Hongbo; Shen, Wen H

2017-10-01

Faithful DNA replication and accurate chromosome segregation are the key machineries of genetic transmission. Disruption of these processes represents a hallmark of cancer and often results from loss of tumor suppressors. PTEN is an important tumor suppressor that is frequently mutated or deleted in human cancer. Loss of PTEN has been associated with aneuploidy and poor prognosis in cancer patients. In mice, Pten deletion or mutation drives genomic instability and tumor development. PTEN deficiency induces DNA replication stress, confers stress tolerance, and disrupts mitotic spindle architecture, leading to accumulation of structural and numerical chromosome instability. Therefore, PTEN guards the genome by controlling multiple processes of chromosome inheritance. Here, we summarize current understanding of the PTEN function in promoting high-fidelity transmission of genetic information. We also discuss the PTEN pathways of genome maintenance and highlight potential targets for cancer treatment. © 2017 WILEY Periodicals, Inc.

Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA

PubMed Central

Ashenberg, Orr; Padmakumar, Jai

2017-01-01

The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors. PMID:28346537

Computational DNA hole spectroscopy: A new tool to predict mutation hotspots, critical base pairs, and disease ‘driver’ mutations

PubMed Central

Suárez, Martha Y.; Villagrán; Miller, John H.

2015-01-01

We report on a new technique, computational DNA hole spectroscopy, which creates spectra of electron hole probabilities vs. nucleotide position. A hole is a site of positive charge created when an electron is removed. Peaks in the hole spectrum depict sites where holes tend to localize and potentially trigger a base pair mismatch during replication. Our studies of mitochondrial DNA reveal a correlation between L-strand hole spectrum peaks and spikes in the human mutation spectrum. Importantly, we also find that hole peak positions that do not coincide with large variant frequencies often coincide with disease-implicated mutations and/or (for coding DNA) encoded conserved amino acids. This enables combining hole spectra with variant data to identify critical base pairs and potential disease ‘driver’ mutations. Such integration of DNA hole and variance spectra could ultimately prove invaluable for pinpointing critical regions of the vast non-protein-coding genome. An observed asymmetry in correlations, between the spectrum of human mtDNA variations and the L- and H-strand hole spectra, is attributed to asymmetric DNA replication processes that occur for the leading and lagging strands. PMID:26310834

Computational DNA hole spectroscopy: A new tool to predict mutation hotspots, critical base pairs, and disease 'driver' mutations.

PubMed

Villagrán, Martha Y Suárez; Miller, John H

2015-08-27

We report on a new technique, computational DNA hole spectroscopy, which creates spectra of electron hole probabilities vs. nucleotide position. A hole is a site of positive charge created when an electron is removed. Peaks in the hole spectrum depict sites where holes tend to localize and potentially trigger a base pair mismatch during replication. Our studies of mitochondrial DNA reveal a correlation between L-strand hole spectrum peaks and spikes in the human mutation spectrum. Importantly, we also find that hole peak positions that do not coincide with large variant frequencies often coincide with disease-implicated mutations and/or (for coding DNA) encoded conserved amino acids. This enables combining hole spectra with variant data to identify critical base pairs and potential disease 'driver' mutations. Such integration of DNA hole and variance spectra could ultimately prove invaluable for pinpointing critical regions of the vast non-protein-coding genome. An observed asymmetry in correlations, between the spectrum of human mtDNA variations and the L- and H-strand hole spectra, is attributed to asymmetric DNA replication processes that occur for the leading and lagging strands.

Natural selection underlies apparent stress-induced mutagenesis in a bacteriophage infection model.

PubMed

Yosef, Ido; Edgar, Rotem; Levy, Asaf; Amitai, Gil; Sorek, Rotem; Munitz, Ariel; Qimron, Udi

2016-04-18

The emergence of mutations following growth-limiting conditions underlies bacterial drug resistance, viral escape from the immune system and fundamental evolution-driven events. Intriguingly, whether mutations are induced by growth limitation conditions or are randomly generated during growth and then selected by growth limitation conditions remains an open question(1). Here, we show that bacteriophage T7 undergoes apparent stress-induced mutagenesis when selected for improved recognition of its host's receptor. In our unique experimental set-up, the growth limitation condition is physically and temporally separated from mutagenesis: growth limitation occurs while phage DNA is outside the host, and spontaneous mutations occur during phage DNA replication inside the host. We show that the selected beneficial mutations are not pre-existing and that the initial slow phage growth is enabled by the phage particle's low-efficiency DNA injection into the host. Thus, the phage particle allows phage populations to initially extend their host range without mutagenesis by virtue of residual recognition of the host receptor. Mutations appear during non-selective intracellular replication, and the frequency of mutant phages increases by natural selection acting on free phages, which are not capable of mutagenesis.

Phosphorylation at the Homotypic Interface Regulates Nucleoprotein Oligomerization and Assembly of the Influenza Virus Replication Machinery

PubMed Central

Mondal, Arindam; Potts, Gregory K.; Dawson, Anthony R.; Coon, Joshua J.; Mehle, Andrew

2015-01-01

Negative-sense RNA viruses assemble large ribonucleoprotein (RNP) complexes that direct replication and transcription of the viral genome. Influenza virus RNPs contain the polymerase, genomic RNA and multiple copies of nucleoprotein (NP). During RNP assembly, monomeric NP oligomerizes along the length of the genomic RNA. Regulated assembly of the RNP is essential for virus replication, but how NP is maintained as a monomer that subsequently oligomerizes to form RNPs is poorly understood. Here we elucidate a mechanism whereby NP phosphorylation regulates oligomerization. We identified new evolutionarily conserved phosphorylation sites on NP and demonstrated that phosphorylation of NP decreased formation of higher-order complexes. Two phosphorylation sites were located on opposite sides of the NP:NP interface. In both influenza A and B virus, mutating or mimicking phosphorylation at these residues blocked homotypic interactions and drove NP towards a monomeric form. Highlighting the central role of this process during infection, these mutations impaired RNP formation, polymerase activity and virus replication. Thus, dynamic phosphorylation of NP regulates RNP assembly and modulates progression through the viral life cycle. PMID:25867750

Adaptations of an RNA virus to increasing thermal stress

PubMed Central

Singhal, Sonia; Leon Guerrero, Cierra M.; Whang, Stella G.; McClure, Erin M.; Busch, Hannah G.; Kerr, Benjamin

2017-01-01

Environments can change in incremental fashions, where a shift from one state to another occurs over multiple organismal generations. The rate of the environmental change is expected to influence how and how well populations adapt to the final environmental state. We used a model system, the lytic RNA bacteriophage Φ6, to investigate this question empirically. We evolved viruses for thermostability by exposing them to heat shocks that increased to a maximum temperature at different rates. We observed increases in the ability of many heat-shocked populations to survive high temperature heat shocks. On their first exposure to the highest temperature, populations that experienced a gradual increase in temperature had higher average survival than populations that experienced a rapid temperature increase. However, at the end of the experiment, neither the survival of populations at the highest temperature nor the number of mutations per population varied significantly according to the rate of thermal change. We also evaluated mutations from the endpoint populations for their effects on viral thermostability and growth. As expected, some mutations did increase viral thermostability. However, other mutations decreased thermostability but increased growth rate, suggesting that benefits of an increased replication rate may have sometimes outweighed the benefits of enhanced thermostability. Our study highlights the importance of considering the effects of multiple selective pressures, even in environments where a single factor changes. PMID:29267297

Impact of Genetic Variations in HIV-1 Tat on LTR-Mediated Transcription via TAR RNA Interaction.

PubMed

Ronsard, Larance; Ganguli, Nilanjana; Singh, Vivek K; Mohankumar, Kumaravel; Rai, Tripti; Sridharan, Subhashree; Pajaniradje, Sankar; Kumar, Binod; Rai, Devesh; Chaudhuri, Suhnrita; Coumar, Mohane S; Ramachandran, Vishnampettai G; Banerjea, Akhil C

2017-01-01

HIV-1 evades host defense through mutations and recombination events, generating numerous variants in an infected patient. These variants with an undiminished virulence can multiply rapidly in order to progress to AIDS. One of the targets to intervene in HIV-1 replication is the trans -activator of transcription (Tat), a major regulatory protein that transactivates the long terminal repeat promoter through its interaction with trans -activation response (TAR) RNA. In this study, HIV-1 infected patients ( n = 120) from North India revealed Ser46Phe (20%) and Ser61Arg (2%) mutations in the Tat variants with a strong interaction toward TAR leading to enhanced transactivation activities. Molecular dynamics simulation data verified that the variants with this mutation had a higher binding affinity for TAR than both the wild-type Tat and other variants that lacked Ser46Phe and Ser61Arg. Other mutations in Tat conferred varying affinities for TAR interaction leading to differential transactivation abilities. This is the first report from North India with a clinical validation of CD4 counts to demonstrate the influence of Tat genetic variations affecting the stability of Tat and its interaction with TAR. This study highlights the co-evolution pattern of Tat and predominant nucleotides for Tat activity, facilitating the identification of genetic determinants for the attenuation of viral gene expression.

Roles of Rev1, Pol ζ, Pol32 and Pol η in the bypass of chromosomal abasic sites in Saccharomyces cerevisiae

PubMed Central

Auerbach, Paul A.; Demple, Bruce

2010-01-01

Translesion synthesis (TLS) on DNA is a process by which potentially cytotoxic replication-blocking lesions are bypassed, but at the risk of increased mutagenesis. The exact in vivo role of the individual TLS enzymes in Saccharomyces cerevisiae has been difficult to determine from previous studies due to differing results from the variety of systems used. We have generated a series of S.cerevisiae strains in which each of the TLS-related genes REV1, REV3, REV7, RAD30 and POL32 was deleted, and in which chromosomal apyrimidinic sites were generated during normal cell growth by the activity of altered forms of human uracil-DNA glycosylase that remove undamaged cytosines or thymines. Deletion of REV1, REV3 or REV7 resulted in slower growth dependent on (rev3Δ and rev7Δ) or enhanced by (rev1Δ) expression of the mutator glycosylases and a nearly complete abolition of glycosylase-induced mutagenesis. Deletion of POL32 resulted in cell death when the mutator glycosylases were expressed and, in their absence, diminished spontaneous mutagenesis. RAD30 appeared to be unnecessary for mutagenesis in response to abasic sites, as deleting this gene caused no significant change in either the mutation rates or the mutational spectra due to glycosylase expression. PMID:19901007

The Hepatitis C Virus NS4B Protein Can trans-Complement Viral RNA Replication and Modulates Production of Infectious Virus▿

PubMed Central

Jones, Daniel M.; Patel, Arvind H.; Targett-Adams, Paul; McLauchlan, John

2009-01-01

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release. PMID:19073716

[The prevalence and clinical significance of precore and core promoter mutations in Korean patients with chronic hepatitis B virus infection].

PubMed

Kim, Hyung Joon; Yoo, Byung Chul

2002-06-01

Precore and core promoter mutations of hepatitis B virus (HBV) have been reported in Korea but their prevalence and clinical significance have not been determined. The aims of this study were to determine the prevalence of precore and core promoter mutations and their relationships to hepatitis B e antigen (HBeAg) status, viral replication level, and severity of liver disease in Korea. Among the patients who visited the Liver Diseases Clinics (Chung Ang University Hospital) between December 1998 and August 1999, 150 patients were randomly selected: 50 HBeAg-positive HBV-DNA positive patients by a branched DNA (bDNA) assay, 50 HBeAg-negative bDNA-positive patients, and 50 HBeAg-negative bDNA-negative patients. Serum HBV-DNA was amplified by a polymerase chain reaction (PCR) in these patients and the core promoter/precore HBV sequence was determined in 135 of the patients whose sera were positive for HBV-DNA by PCR. All of the 135 determined HBV-DNA sequences had HBV genotype with T at nucleotide 1858. Precore mutation (A1896) was detected in 95.7% of HBeAg-negative bDNA-positive patients and 94.9% of HBeAg-negative bDNA-negative patients. In HBeAg-positive patients 88% had wild type and 12% had mixture of wild type and A1896 mutant. Core promoter TA mutation (T1762/A1764) was detected in 93.5% of HBeAg-negative bDNA-positive patients, 94.9% of HBeAg-negative bDNA-negative patients and 74% of HBeAg-positive patients. No correlation was found between the presence of precore/core promoter mutations and liver disease severity or HBV-DNA levels. Precore stop codon mutation occurred almost invariably, along with HBeAg seroconversion, irrespective of subsequent viral replication levels or disease severity. Core promoter TA mutation was frequent both in the HBeAg-positive patients and HBeAg-negative patients irrespective of viral replication levels or disease severity.

Molecular analysis of mutations in DNA polymerase η in xeroderma pigmentosum-variant patients

PubMed Central

Broughton, Bernard C.; Cordonnier, Agnes; Kleijer, Wim J.; Jaspers, Nicolaas G. J.; Fawcett, Heather; Raams, Anja; Garritsen, Victor H.; Stary, Anne; Avril, Marie-Françoise; Boudsocq, François; Masutani, Chikahide; Hanaoka, Fumio; Fuchs, Robert P.; Sarasin, Alain; Lehmann, Alan R.

2002-01-01

Xeroderma pigmentosum variant (XP-V) cells are deficient in their ability to synthesize intact daughter DNA strands after UV irradiation. This deficiency results from mutations in the gene encoding DNA polymerase η, which is required for effecting translesion synthesis (TLS) past UV photoproducts. We have developed a simple cellular procedure to identify XP-V cell strains, and have subsequently analyzed the mutations in 21 patients with XP-V. The 16 mutations that we have identified fall into three categories. Many of them result in severe truncations of the protein and are effectively null alleles. However, we have also identified five missense mutations located in the conserved catalytic domain of the protein. Extracts of cells falling into these two categories are defective in the ability to carry out TLS past sites of DNA damage. Three mutations cause truncations at the C terminus such that the catalytic domains are intact, and extracts from these cells are able to carry out TLS. From our previous work, however, we anticipate that protein in these cells will not be localized in the nucleus nor will it be relocalized into replication foci during DNA replication. The spectrum of both missense and truncating mutations is markedly skewed toward the N-terminal half of the protein. Two of the missense mutations are predicted to affect the interaction with DNA, the others are likely to disrupt the three-dimensional structure of the protein. There is a wide variability in clinical features among patients, which is not obviously related to the site or type of mutation. PMID:11773631

Evidence and age-related distribution of mtDNA D-loop point mutations in skeletal muscle from healthy subjects and mitochondrial patients.

PubMed

Del Bo, Roberto; Bordoni, Andreina; Martinelli Boneschi, Filippo; Crimi, Marco; Sciacco, Monica; Bresolin, Nereo; Scarlato, Guglielmo; Comi, Giacomo Pietri

2002-10-15

The progressive accumulation of mitochondrial DNA (mtDNA) alterations, ranging from single mutations to large-scale deletions, in both the normal ageing process and pathological conditions is a relevant phenomenon in terms of frequency and heteroplasmic degree. Recently, two point mutations (A189G and T408A) within the Displacement loop (D-loop) region, the control region for mtDNA replication, were shown to occur in skeletal muscles from aged individuals. We evaluated the presence and the heteroplasmy levels of these two mutations in muscle biopsies from 91 unrelated individuals of different ages (21 healthy subjects and 70 patients affected by mitochondrial encephalomyopathies). Overall, both mutations significantly accumulate with age. However, a different relationship was discovered among the different subgroups of patients: a higher number of A189G positive subjects younger than 53 years was detected in the subgroup of multiple-deleted patients; furthermore, a trend towards an increased risk for the mutations was evidenced among patients carrying multiple deletions when compared to healthy controls. These findings support the idea that a common biological mechanism determines the accumulation of somatic point mutations in the D-loop region, both in healthy subjects and in mitochondrial myopathy patients. At the same time, it appears that disorders caused by mutations of nuclear genes controlling mtDNA replication (the "mtDNA multiple deletions" syndromes) present a temporal advantage to mutate in the D-loop region. This observation may be relevant to the definition of the molecular pathogenesis of these latter syndromes. Copyright 2002 Elsevier Science B.V.

Multiplex Preamplification of Serum DNA to Facilitate Reliable Detection of Extremely Rare Cancer Mutations in Circulating DNA by Digital PCR.

PubMed

Jackson, Jennifer B; Choi, Daniel S; Luketich, James D; Pennathur, Arjun; Ståhlberg, Anders; Godfrey, Tony E

2016-03-01

Tumor-specific mutations can be identified in circulating, cell-free DNA in plasma or serum and may serve as a clinically relevant alternative to biopsy. Detection of tumor-specific mutations in the plasma, however, is technically challenging. First, mutant allele fractions are typically low in a large background of wild-type circulating, cell-free DNA. Second, the amount of circulating, cell-free DNA acquired from plasma is also low. Even when using digital PCR (dPCR), rare mutation detection is challenging because there is not enough circulating, cell-free DNA to run technical replicates and assay or instrument noise does not easily allow for mutation detection <0.1%. This study was undertaken to improve on the robustness of dPCR for mutation detection. A multiplexed, preamplification step using a high-fidelity polymerase before dPCR was developed to increase total DNA and the number of targets and technical replicates that can be assayed from a single sample. We were able to detect multiple cancer-relevant mutations within tumor-derived samples down to 0.01%. Importantly, the signal/noise ratio was improved for all preamplified targets, allowing for easier discrimination of low-abundance mutations against false-positive signal. Furthermore, we used this protocol on clinical samples to detect known, tumor-specific mutations in patient sera. This study provides a protocol for robust, sensitive detection of circulating tumor DNA for future clinical applications. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Insights into mutagenesis using Escherichia coli chromosomal lacZ strains that enable detection of a wide spectrum of mutational events.

PubMed

Seier, Tracey; Padgett, Dana R; Zilberberg, Gal; Sutera, Vincent A; Toha, Noor; Lovett, Susan T

2011-06-01

Strand misalignments at DNA repeats during replication are implicated in mutational hotspots. To study these events, we have generated strains carrying mutations in the Escherichia coli chromosomal lacZ gene that revert via deletion of a short duplicated sequence or by template switching within imperfect inverted repeat (quasipalindrome, QP) sequences. Using these strains, we demonstrate that mutation of the distal repeat of a quasipalindrome, with respect to replication fork movement, is about 10-fold higher than the proximal repeat, consistent with more common template switching on the leading strand. The leading strand bias was lost in the absence of exonucleases I and VII, suggesting that it results from more efficient suppression of template switching by 3' exonucleases targeted to the lagging strand. The loss of 3' exonucleases has no effect on strand misalignment at direct repeats to produce deletion. To compare these events to other mutations, we have reengineered reporters (designed by Cupples and Miller 1989) that detect specific base substitutions or frameshifts in lacZ with the reverting lacZ locus on the chromosome rather than an F' element. This set allows rapid screening of potential mutagens, environmental conditions, or genetic loci for effects on a broad set of mutational events. We found that hydroxyurea (HU), which depletes dNTP pools, slightly elevated templated mutations at inverted repeats but had no effect on deletions, simple frameshifts, or base substitutions. Mutations in nucleotide diphosphate kinase, ndk, significantly elevated simple mutations but had little effect on the templated class. Zebularine, a cytosine analog, elevated all classes.

Characterization of a Novel MMS-Sensitive Allele of Schizosaccharomyces pombe mcm4+

PubMed Central

Ranatunga, Nimna S.; Forsburg, Susan L.

2016-01-01

The minichromosome maintenance (MCM) complex is the conserved helicase motor of the eukaryotic replication fork. Mutations in the Mcm4 subunit are associated with replication stress and double strand breaks in multiple systems. In this work, we characterize a new temperature-sensitive allele of Schizosaccharomyces pombe mcm4+. Uniquely among known mcm4 alleles, this mutation causes sensitivity to the alkylation damaging agent methyl methanesulfonate (MMS). Even in the absence of treatment or temperature shift, mcm4-c106 cells show increased repair foci of RPA and Rad52, and require the damage checkpoint for viability, indicating genome stress. The mcm4-c106 mutant is synthetically lethal with mutations disrupting fork protection complex (FPC) proteins Swi1 and Swi3. Surprisingly, we found that the deletion of rif1+ suppressed the MMS-sensitive phenotype without affecting temperature sensitivity. Together, these data suggest that mcm4-c106 destabilizes replisome structure. PMID:27473316

Adventures in hepatocarcinogenesis.

PubMed

Pitot, Henry C

2007-01-01

Neoplasia is a heritably altered, relatively autonomous growth of tissue. Hepatocarcinogenesis, the pathogenesis of neoplasia in liver, as modeled in the rat exhibits three distinct, quantifiable stages: initiation, promotion, and progression. Simple mutations and/or epigenetic alterations may result in the irreversible stage of initiation. The stage of promotion results from selective enhancement of cell replication and selective inhibition of cellular apoptosis of initiated cells dependent on the genetic and/or epigenetic alterations of the latter. The irreversible stage of progression results from initial karyotypic alterations that evolve into greater degrees of genomic instability. The initial genomic alteration in the transition from promotion to progression may involve primarily epigenetic mechanisms driven by epigenetic and genetic alterations fixed during the stage of promotion.

Selective Inhibitor of Nuclear Export (SINE) Compounds Alter New World Alphavirus Capsid Localization and Reduce Viral Replication in Mammalian Cells.

PubMed

Lundberg, Lindsay; Pinkham, Chelsea; de la Fuente, Cynthia; Brahms, Ashwini; Shafagati, Nazly; Wagstaff, Kylie M; Jans, David A; Tamir, Sharon; Kehn-Hall, Kylene

2016-11-01

The capsid structural protein of the New World alphavirus, Venezuelan equine encephalitis virus (VEEV), interacts with the host nuclear transport proteins importin α/β1 and CRM1. Novel selective inhibitor of nuclear export (SINE) compounds, KPT-185, KPT-335 (verdinexor), and KPT-350, target the host's primary nuclear export protein, CRM1, in a manner similar to the archetypical inhibitor Leptomycin B. One major limitation of Leptomycin B is its irreversible binding to CRM1; which SINE compounds alleviate because they are slowly reversible. Chemically inhibiting CRM1 with these compounds enhanced capsid localization to the nucleus compared to the inactive compound KPT-301, as indicated by immunofluorescent confocal microscopy. Differences in extracellular versus intracellular viral RNA, as well as decreased capsid in cell free supernatants, indicated the inhibitors affected viral assembly, which led to a decrease in viral titers. The decrease in viral replication was confirmed using a luciferase-tagged virus and through plaque assays. SINE compounds had no effect on VEEV TC83_Cm, which encodes a mutated form of capsid that is unable to enter the nucleus. Serially passaging VEEV in the presence of KPT-185 resulted in mutations within the nuclear localization and nuclear export signals of capsid. Finally, SINE compound treatment also reduced the viral titers of the related eastern and western equine encephalitis viruses, suggesting that CRM1 maintains a common interaction with capsid proteins across the New World alphavirus genus.

Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

PubMed Central

Mielech, Anna M.; Deng, Xufang; Chen, Yafang; Kindler, Eveline; Wheeler, Dorthea L.; Mesecar, Andrew D.; Thiel, Volker; Perlman, Stanley

2015-01-01

ABSTRACT Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered the viral protease activity or affected viral replication or pathogenesis. Our studies using purified wild-type and Ubl mutant proteases revealed that mutations in the viral Ubl domain destabilize and inactivate the adjacent viral protease. Furthermore, we show that a CoV encoding the mutant Ubl domain is unable to replicate at high temperature or cause lethal disease in mice. Our results identify the coronavirus Ubl domain as a novel modulator of viral protease stability and reveal manipulating the Ubl domain as a new approach for attenuating coronavirus replication and pathogenesis. PMID:25694594

Sequencing-based breast cancer diagnostics as an alternative to routine biomarkers.

PubMed

Rantalainen, Mattias; Klevebring, Daniel; Lindberg, Johan; Ivansson, Emma; Rosin, Gustaf; Kis, Lorand; Celebioglu, Fuat; Fredriksson, Irma; Czene, Kamila; Frisell, Jan; Hartman, Johan; Bergh, Jonas; Grönberg, Henrik

2016-11-30

Sequencing-based breast cancer diagnostics have the potential to replace routine biomarkers and provide molecular characterization that enable personalized precision medicine. Here we investigate the concordance between sequencing-based and routine diagnostic biomarkers and to what extent tumor sequencing contributes clinically actionable information. We applied DNA- and RNA-sequencing to characterize tumors from 307 breast cancer patients with replication in up to 739 patients. We developed models to predict status of routine biomarkers (ER, HER2,Ki-67, histological grade) from sequencing data. Non-routine biomarkers, including mutations in BRCA1, BRCA2 and ERBB2(HER2), and additional clinically actionable somatic alterations were also investigated. Concordance with routine diagnostic biomarkers was high for ER status (AUC = 0.95;AUC(replication) = 0.97) and HER2 status (AUC = 0.97;AUC(replication) = 0.92). The transcriptomic grade model enabled classification of histological grade 1 and histological grade 3 tumors with high accuracy (AUC = 0.98;AUC(replication) = 0.94). Clinically actionable mutations in BRCA1, BRCA2 and ERBB2(HER2) were detected in 5.5% of patients, while 53% had genomic alterations matching ongoing or concluded breast cancer studies. Sequencing-based molecular profiling can be applied as an alternative to histopathology to determine ER and HER2 status, in addition to providing improved tumor grading and clinically actionable mutations and molecular subtypes. Our results suggest that sequencing-based breast cancer diagnostics in a near future can replace routine biomarkers.

The Fanconi anemia pathway limits the severity of mutagenesis.

PubMed

Hinz, John M; Nham, Peter B; Salazar, Edmund P; Thompson, Larry H

2006-08-13

Fanconi anemia (FA) is a developmental and cancer predisposition disorder in which key, yet unknown, physiological events promoting chromosome stability are compromised. FA cells exhibit excess metaphase chromatid breaks and are universally hypersensitive to DNA interstrand crosslinking agents. Published mutagenesis data from single-gene mutation assays show both increased and decreased mutation frequencies in FA cells. In this review we discuss the data from the literature and from our isogenic fancg knockout hamster CHO cells, and interpret these data within the framework of a molecular model that accommodates these seemingly divergent observations. In FA cells, reduced rates of recovery of viable X-linked hypoxanthine phosphoribosyltransferase (hprt) mutants are characteristically observed for diverse mutagenic agents, but also in untreated cultures, indicating the relevance of the FA pathway for processing assorted DNA lesions. We ascribe these reductions to: (1) impaired mutagenic translesion synthesis within hprt during DNA replication and (2) lethality of mutant cells following replication fork breakage on the X chromosome, caused by unrepaired double-strand breaks or large deletions/translocations encompassing essential genes flanking hprt. These findings, along with studies showing increased spontaneous mutability of FA cells at two autosomal loci, support a model in which FA proteins promote both translesion synthesis at replication-blocking lesions and repair of broken replication forks by homologous recombination and DNA end joining. The essence of this model is that the FANC protein pathway serves to restrict the severity of mutational outcome by favoring base substitutions and small deletions over larger deletions and chromosomal rearrangements.

Designing herpes viruses as oncolytics

PubMed Central

Peters, Cole; Rabkin, Samuel D

2015-01-01

Oncolytic herpes simplex virus (oHSV) was one of the first genetically-engineered oncolytic viruses. Because HSV is a natural human pathogen that can cause serious disease, it is incumbent that it can be genetically-engineered or significantly attenuated for safety. Here, we present a detailed explanation of the functions of HSV-1 genes frequently mutated to endow oncolytic activity. These genes are nonessential for growth in tissue culture cells but are important for growth in postmitotic cells, interfering with intrinsic antiviral and innate immune responses or causing pathology, functions dispensable for replication in cancer cells. Understanding the function of these genes leads to informed creation of new oHSVs with better therapeutic efficacy. Virus infection and replication can also be directed to cancer cells through tumor-selective receptor binding and transcriptional- or post-transcriptional miRNA-targeting, respectively. In addition to the direct effects of oHSV on infected cancer cells and tumors, oHSV can be “armed” with transgenes that are: reporters, to track virus replication and spread; cytotoxic, to kill uninfected tumor cells; immune modulatory, to stimulate antitumor immunity; or tumor microenvironment altering, to enhance virus spread or to inhibit tumor growth. In addition to HSV-1, other alphaherpesviruses are also discussed for their oncolytic activity. PMID:26462293

Imperfect Symmetry of Sp1 and Core Promoter Sequences Regulates Early and Late Virus Gene Expression of the Bidirectional BK Polyomavirus Noncoding Control Region.

PubMed

Bethge, Tobias; Ajuh, Elvis; Hirsch, Hans H

2016-11-15

Rearrangements or point mutations in the noncoding control region (NCCR) of BK polyomavirus (BKPyV) have been associated with higher viral loads and more pronounced organ pathology in immunocompromised patients. The respective alterations affect a multitude of transcription factor binding sites (TFBS) but consistently cause increased expression of the early viral gene region (EVGR) at the expense of late viral gene region (LVGR) expression. By mutating TFBS, we identified three phenotypic groups leading to strong, intermediate, or impaired EVGR expression and corresponding BKPyV replication. Unexpectedly, Sp1 TFBS mutants either activated or inhibited EVGR expression when located proximal to the LVGR (sp1-4) or the EVGR (sp1-2), respectively. We now demonstrate that the bidirectional balance of EVGR and LVGR expression is dependent on affinity, strand orientation, and the number of Sp1 sites. Swapping the LVGR-proximal high-affinity SP1-4 with the EVGR-proximal low-affinity SP1-2 in site strand flipping or inserting an additional SP1-2 site caused a rearranged NCCR phenotype of increased EVGR expression and faster BKPyV replication. The 5' rapid amplification of cDNA ends revealed an imperfect symmetry between the EVGR- and LVGR-proximal parts of the NCCR, consisting of TATA and TATA-like elements, initiator elements, and downstream promoter elements. Mutation or deletion of the archetypal LVGR promoter, which is found in activated NCCR variants, abrogated LVGR expression, which could be restored by providing large T antigen (LTag) in trans Thus, whereas Sp1 sites control the initial EVGR-LVGR expression balance, LTag expression can override inactivation of the LVGR promoter and acts as a key driver of LVGR expression independently of the Sp1 sites and core promoter elements. Polyomaviridae currently comprise more than 70 members, including 13 human polyomaviruses (PyVs), all of which share a bidirectional genome organization mediated by the NCCR, which determines species and host cell specificity, persistence, replication, and virulence. Here, we demonstrate that the BKPyV NCCR is fine-tuned by an imperfect symmetry of core promoter elements centered around TATA and TATA-like sequences close to the EVGR and LVGR, respectively, which are governed by the directionality and affinity of two Sp1 sites. The data indicated that the BKPyV NCCR is poised toward EVGR expression, which can be readily unlatched by a simple switch affecting Sp1 binding. The resulting LTag, which is the major EVGR protein, drives viral genome replication, renders subsequent LVGR expression independently of archetypal promoter elements, and can overcome enhancer/promoter mutations and deletions. The data are pivotal for understanding how human PyV NCCRs mediate secondary host cell specificity, reactivation, and virulence in their natural hosts. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Replicative DNA Polymerase δ but Not ε Proofreads Errors in Cis and in Trans

PubMed Central

Flood, Carrie L.; Rodriguez, Gina P.; Bao, Gaobin; Shockley, Arthur H.; Kow, Yoke Wah; Crouse, Gray F.

2015-01-01

It is now well established that in yeast, and likely most eukaryotic organisms, initial DNA replication of the leading strand is by DNA polymerase ε and of the lagging strand by DNA polymerase δ. However, the role of Pol δ in replication of the leading strand is uncertain. In this work, we use a reporter system in Saccharomyces cerevisiae to measure mutation rates at specific base pairs in order to determine the effect of heterozygous or homozygous proofreading-defective mutants of either Pol ε or Pol δ in diploid strains. We find that wild-type Pol ε molecules cannot proofread errors created by proofreading-defective Pol ε molecules, whereas Pol δ can not only proofread errors created by proofreading-defective Pol δ molecules, but can also proofread errors created by Pol ε-defective molecules. These results suggest that any interruption in DNA synthesis on the leading strand is likely to result in completion by Pol δ and also explain the higher mutation rates observed in Pol δ-proofreading mutants compared to Pol ε-proofreading defective mutants. For strains reverting via AT→GC, TA→GC, CG→AT, and GC→AT mutations, we find in addition a strong effect of gene orientation on mutation rate in proofreading-defective strains and demonstrate that much of this orientation dependence is due to differential efficiencies of mispair elongation. We also find that a 3′-terminal 8 oxoG, unlike a 3′-terminal G, is efficiently extended opposite an A and is not subject to proofreading. Proofreading mutations have been shown to result in tumor formation in both mice and humans; the results presented here can help explain the properties exhibited by those proofreading mutants. PMID:25742645

Mutational Analysis of Influenza A Virus Nucleoprotein: Identification of Mutations That Affect RNA Replication

PubMed Central

Mena, Ignacio; Jambrina, Enrique; Albo, Carmen; Perales, Beatriz; Ortín, Juan; Arrese, Marta; Vallejo, Dolores; Portela, Agustín

1999-01-01

The influenza A virus nucleoprotein (NP) is a multifunctional polypeptide which plays a pivotal role in virus replication. To get information on the domains and specific residues involved in the different NP activities, we describe here the preparation and characterization of 20 influenza A virus mutant NPs. The mutations, mostly single-amino-acid substitutions, were introduced in a cDNA copy of the A/Victoria/3/75 NP gene and, in most cases, affected residues located in regions that were highly conserved across the NPs of influenza A, B, and C viruses. The mutant NPs were characterized (i) in vivo (cell culture) by analyzing their intracellular localization and their functionality in replication, transcription, and expression of model RNA templates; and (ii) in vitro by analyzing their RNA-binding and sedimentation properties. The results obtained allowed us to identify both a mutant protein that accumulated in the cytoplasm and mutations that altered the functionality and/or the oligomerization state of the NP polypeptide. Among the mutations that reduced the NP capability to express chloramphenicol acetyltransferase protein from a model viral RNA (vRNA) template, some displayed a temperature-sensitive phenotype. Interestingly, four mutant NPs, which showed a reduced functionality in synthesizing cRNA molecules from a vRNA template, were fully competent to reconstitute complementary ribonucleoproteins (cRNPs) capable of synthesizing vRNAs, which in turn yielded mRNA molecules. Based on the phenotype of these mutants and on previously published observations, it is proposed that these mutant NPs have a reduced capability to interact with the polymerase complex and that this NP-polymerase interaction is responsible for making vRNPs switch from mRNA to cRNA synthesis. PMID:9882320

A Study of Toca 511, a Retroviral Replicating Vector, Combined With Toca FC in Patients With Solid Tumors or Lymphoma (Toca 6)

ClinicalTrials.gov

2018-01-05

Colorectal Cancer; Triple Negative Breast Cancer; Pancreatic Cancer; Non-Small Cell Lung Cancer; Head and Neck Cancer; Ovarian Cancer; Lymphoma; Sarcoma; Bladder Cancer; Melanoma; IDH1 Mutated Solid Tumors; IDH1 Mutated or MGMT Methylated Recurrent HGG (Not Recruiting)

Extraordinary genome stability in the ciliate Paramecium tetraurelia

PubMed Central

Sung, Way; Tucker, Abraham E.; Doak, Thomas G.; Choi, Eunjin; Thomas, W. Kelley; Lynch, Michael

2012-01-01

Mutation plays a central role in all evolutionary processes and is also the basis of genetic disorders. Established base-substitution mutation rates in eukaryotes range between ∼5 × 10−10 and 5 × 10−8 per site per generation, but here we report a genome-wide estimate for Paramecium tetraurelia that is more than an order of magnitude lower than any previous eukaryotic estimate. Nevertheless, when the mutation rate per cell division is extrapolated to the length of the sexual cycle for this protist, the measure obtained is comparable to that for multicellular species with similar genome sizes. Because Paramecium has a transcriptionally silent germ-line nucleus, these results are consistent with the hypothesis that natural selection operates on the cumulative germ-line replication fidelity per episode of somatic gene expression, with the germ-line mutation rate per cell division evolving downward to the lower barrier imposed by random genetic drift. We observe ciliate-specific modifications of widely conserved amino acid sites in DNA polymerases as one potential explanation for unusually high levels of replication fidelity. PMID:23129619

Mutations in ORC1, encoding the largest subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome.

PubMed

Bicknell, Louise S; Walker, Sarah; Klingseisen, Anna; Stiff, Tom; Leitch, Andrea; Kerzendorfer, Claudia; Martin, Carol-Anne; Yeyati, Patricia; Al Sanna, Nouriya; Bober, Michael; Johnson, Diana; Wise, Carol; Jackson, Andrew P; O'Driscoll, Mark; Jeggo, Penny A

2011-02-27

Studies into disorders of extreme growth failure (for example, Seckel syndrome and Majewski osteodysplastic primordial dwarfism type II) have implicated fundamental cellular processes of DNA damage response signaling and centrosome function in the regulation of human growth. Here we report that mutations in ORC1, encoding a subunit of the origin recognition complex, cause microcephalic primordial dwarfism resembling Meier-Gorlin syndrome. We establish that these mutations disrupt known ORC1 functions including pre-replicative complex formation and origin activation. ORC1 deficiency perturbs S-phase entry and S-phase progression. Additionally, we show that Orc1 depletion in zebrafish is sufficient to markedly reduce body size during rapid embryonic growth. Our data suggest a model in which ORC1 mutations impair replication licensing, slowing cell cycle progression and consequently impeding growth during development, particularly at times of rapid proliferation. These findings establish a novel mechanism for the pathogenesis of microcephalic dwarfism and show a surprising but important developmental impact of impaired origin licensing.

Mutations in Nonconserved Domains of Ty3 Integrase Affect Multiple Stages of the Ty3 Life Cycle

PubMed Central

Nymark-McMahon, M. Henrietta; Sandmeyer, Suzanne B.

1999-01-01

Ty3, a retroviruslike element of Saccharomyces cerevisiae, transposes into positions immediately upstream of RNA polymerase III-transcribed genes. The Ty3 integrase (IN) protein is required for integration of the replicated, extrachromosomal Ty3 DNA. In retroviral IN, a conserved core region is sufficient for strand transfer activity. In this study, charged-to-alanine scanning mutagenesis was used to investigate the roles of the nonconserved amino- and carboxyl-terminal regions of Ty3 IN. Each of the 20 IN mutants was defective for transposition, but no mutant was grossly defective for capsid maturation. All mutations affecting steady-state levels of mature IN protein resulted in reduced levels of replicated DNA, even when polymerase activity was not grossly defective as measured by exogenous reverse transcriptase activity assay. Thus, IN could contribute to nonpolymerase functions required for DNA production in vivo or to the stability of the DNA product. Several mutations in the carboxyl-terminal domain resulted in relatively low levels of processed 3′ ends of the replicated DNA, suggesting that this domain may be important for binding of IN to the long terminal repeat. Another class of mutants produced wild-type amounts of DNA with correctly processed 3′ ends. This class could include mutants affected in nuclear entry and target association. Collectively, these mutations demonstrate that in vivo, within the preintegration complex, IN performs a central role in coordinating multiple late stages of the retrotransposition life cycle. PMID:9847351

A Library of Infectious Hepatitis C Viruses with Engineered Mutations in the E2 Gene Reveals Growth-Adaptive Mutations That Modulate Interactions with Scavenger Receptor Class B Type I.

PubMed

Zuiani, Adam; Chen, Kevin; Schwarz, Megan C; White, James P; Luca, Vincent C; Fremont, Daved H; Wang, David; Evans, Matthew J; Diamond, Michael S

2016-12-01

While natural hepatitis C virus (HCV) infection results in highly diverse quasispecies of related viruses over time, mutations accumulate more slowly in tissue culture, in part because of the inefficiency of replication in cells. To create a highly diverse population of HCV particles in cell culture and identify novel growth-enhancing mutations, we engineered a library of infectious HCV with all codons represented at most positions in the ectodomain of the E2 gene. We identified many putative growth-adaptive mutations and selected nine highly represented E2 mutants for further study: Q412R, T416R, S449P, T563V, A579R, L619T, V626S, K632T, and L644I. We evaluated these mutants for changes in particle-to-infectious-unit ratio, sensitivity to neutralizing antibody or CD81 large extracellular loop (CD81-LEL) inhibition, entry factor usage, and buoyant density profiles. Q412R, T416R, S449P, T563V, and L619T were neutralized more efficiently by anti-E2 antibodies and T416R, T563V, and L619T by CD81-LEL. Remarkably, all nine variants showed reduced dependence on scavenger receptor class B type I (SR-BI) for infection. This shift from SR-BI usage did not correlate with a change in the buoyant density profiles of the variants, suggesting an altered E2-SR-BI interaction rather than changes in the virus-associated lipoprotein-E2 interaction. Our results demonstrate that residues influencing SR-BI usage are distributed across E2 and support the development of large-scale mutagenesis studies to identify viral variants with unique functional properties. Characterizing variant viruses can reveal new information about the life cycle of HCV and the roles played by different viral genes. However, it is difficult to recapitulate high levels of diversity in the laboratory because of limitations in the HCV culture system. To overcome this limitation, we engineered a library of mutations into the E2 gene in the context of an infectious clone of the virus. We used this library of viruses to identify nine mutations that enhance the growth rate of HCV. These growth-enhancing mutations reduced the dependence on a key entry receptor, SR-BI. By generating a highly diverse library of infectious HCV, we mapped regions of the E2 protein that influence a key virus-host interaction and provide proof of principle for the generation of large-scale mutant libraries for the study of pathogens with great sequence variability. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Susceptibility of chickens, quail, and pigeons to an H7N9 human influenza virus and subsequent egg-passaged strains.

PubMed

Uchida, Yuko; Kanehira, Katsushi; Takemae, Nobuhiro; Hikono, Hirokazu; Saito, Takehiko

2017-01-01

H7N9 human influenza virus A/Anhui/1/2013 (Anhui2013) showed low pathogenicity in chickens, quail, and pigeons, with quail being the most susceptible among the species tested. IVPIE1-1, which was recovered from a dead chicken after intravenous inoculation of Anhui 2013, had broader tissue tropism in chickens than did the original inoculum, as well as amino acid substitutions in the polymerase acidic gene and neuraminidase gene segments, but its pathogenicity was not enhanced. Viruses obtained after passage of Anhui 2013 in 10- and 14-day-old embryonated eggs showed rapid accumulation of amino acid substitutions at the receptor-binding site of the hemagglutinin protein. Two strains obtained through egg passage, 10E4/14E17 and 10E4/10E13, replicated better in intranasally infected chickens than did the original Anhui 2013 strain, yet the new isolates showed low pathogenicity in chickens despite their amino acid substitutions. The increased virus replication in chickens of 10E4/14E17 and 10E4/10E13 was not correlated with temperature-sensitive replication, given that virus replication was suppressed at increased temperatures. The existence of highly susceptible hosts, such as quail, which permit asymptomatic infection, facilitates increased mutation of the virus through amino acid substitution at the receptor-binding site, and this might be one of the mechanisms underlying the prolonged circulation of H7N9 influenza virus.

Human mitochondrial DNA replication machinery and disease

PubMed Central

Young, Matthew J.; Copeland, William C.

2016-01-01

The human mitochondrial genome is replicated by DNA polymerase γ in concert with key components of the mitochondrial DNA (mtDNA) replication machinery. Defects in mtDNA replication or nucleotide metabolism cause deletions, point mutations, or depletion of mtDNA. The resulting loss of cellular respiration ultimately induces mitochondrial genetic diseases, including mtDNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mtDNA deletion disorders such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. Here we review the current literature regarding human mtDNA replication and heritable disorders caused by genetic changes of the POLG, POLG2, Twinkle, RNASEH1, DNA2 and MGME1 genes. PMID:27065468

The effect of DNA replication on mutation of the Saccharomyces cerevisiae CDC8 gene.

PubMed

Zaborowska, D; Zuk, J

1990-04-01

Incubation in YPD medium under permissive conditions when DNA replication is going on, strongly stimulates the induction of cdc+ colonies of UV-irradiated cells of yeast strains HB23 (cdc8-1/cdc8-3), HB26 (cdc8-3/cdc8-3) and HB7 (cdc8-1/cdc8-1). Inhibition of DNA replication by hydroxyurea, araCMP, cycloheximide or caffeine or else by incubation in phosphate buffer pH 7.0, abolishes this stimulation. Thus the replication of DNA is strongly correlated with the high induction of cdc+ colonies by UV irradiation. It is postulated that these UV-induced cdc+ colonies arise as the result infidelity in DNA replication.

Yeast Cells Expressing the Human Mitochondrial DNA Polymerase Reveal Correlations between Polymerase Fidelity and Human Disease Progression*

PubMed Central

Qian, Yufeng; Kachroo, Aashiq H.; Yellman, Christopher M.; Marcotte, Edward M.; Johnson, Kenneth A.

2014-01-01

Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-γ with their physiological consequences, we created “humanized” yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-γ. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-γ suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-γ mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans. PMID:24398692

Scanning mutagenesis studies reveal a potential intramolecular interaction within the C-terminal half of dengue virus NS2A involved in viral RNA replication and virus assembly and secretion.

PubMed

Wu, Ren-Huang; Tsai, Ming-Han; Chao, Day-Yu; Yueh, Andrew

2015-04-01

The NS2A protein of dengue virus (DENV) has eight predicted transmembrane segments (pTMSs; pTMS1 to pTMS8). NS2A has been shown to participate in RNA replication, virion assembly, and the host antiviral response. However, the role of the amino acid residues within the pTMS regions of NS2A during the virus life cycle is poorly understood. In the study described here, we explored the function of DENV NS2A by introducing a series of double or triple alanine substitutions into the C-terminal half (pTMS4 to pTMS8) of NS2A in the context of a DENV infectious clone or subgenomic replicon. Fourteen (8 within pTMS8) of 35 NS2A mutants displayed a lethal phenotype due to impairment of RNA replication by a replicon assay. Three NS2A mutants with mutations within pTMS7, the CM20, CM25, and CM27 mutants, displayed similar phenotypes, low virus yields (>100-fold reduction), wild-type-like replicon activity, and low infectious virus-like particle yields by transient trans-packaging experiments, suggesting a defect in virus assembly and secretion. The sequencing of revertant viruses derived from CM20, CM25, and CM27 mutant viruses revealed a consensus reversion mutation, leucine (L) to phenylalanine (F), at codon 181 within pTMS7. The introduction of an L181F mutation into a full-length NS2A mutant, i.e., the CM20, CM25, and CM27 constructs, completely restored wild-type infectivity. Notably, L181F also substantially rescued the other severely RNA replication-defective mutants with mutations within pTMS4, pTMS6, and pTMS8, i.e., the CM2, CM3, CM13, CM31, and CM32 mutants. In conclusion, the results revealed the essential roles of pTMS4 to pTMS8 of NS2A in RNA replication and/or virus assembly and secretion. The intramolecular interaction between pTMS7 and pTMS4, pTMS6, or pTMS8 of the NS2A protein was also implicated. The reported characterization of the C-terminal half of dengue virus NS2A is the first comprehensive mutagenesis study to investigate the function of flavivirus NS2A involved in the steps of the virus life cycle. In particular, detailed mapping of the amino acid residues within the predicted transmembrane segments (pTMSs) of NS2A involved in RNA replication and/or virus assembly and secretion was performed. A revertant genetics study also revealed that L181F within pTMS7 is a consensus reversion mutation that rescues both RNA replication-defective and virus assembly- and secretion-defective mutants with mutations within the other three pTMSs of NS2A. Collectively, these findings elucidate the role played by NS2A during the virus life cycle, possibly through the intricate intramolecular interaction between pTMS7 and other pTMSs within the NS2A protein. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Human embryonic stem cells fail to activate CHK1 and commit to apoptosis in response to DNA replication stress.

PubMed

Desmarais, Joëlle A; Hoffmann, Michele J; Bingham, Gregg; Gagou, Mary E; Meuth, Mark; Andrews, Peter W

2012-07-01

Pluripotent cells of the early embryo, to which embryonic stem cells (ESCs) correspond, give rise to all the somatic cells of the developing fetus. Any defects that occur in their genome or epigenome would have devastating consequences. Genetic and epigenetic change in human ESCs appear to be an inevitable consequence of long-term culture, driven by selection of variant cells that have a higher propensity for self-renewal rather than either differentiation or death. Mechanisms underlying the potentially separate events of mutation and subsequent selection of variants are poorly understood. Here, we show that human ESCs and their malignant counterpart, embryonal carcinoma (EC) cells, both fail to activate critical S-phase checkpoints when exposed to DNA replication inhibitors and commit to apoptosis instead. Human ESCs and EC cells also fail to form replication protein A, γH2AX, or RAD51 foci or load topoisomerase (DNA) II binding protein 1 onto chromatin in response to replication inhibitors. Furthermore, direct measurements of single-stranded DNA (ssDNA) show that these cells fail to generate the ssDNA regions in response to replication stress that are necessary for the activation of checkpoints and the initiation of homologous recombination repair to protect replication fork integrity and restart DNA replication. Taken together, our data suggest that pluripotent cells control genome integrity by the elimination of damaged cells through apoptosis rather than DNA repair, and therefore, mutations or epigenetic modifications resulting in an imbalance in cell death control could lead to genetic instability. Copyright © 2012 AlphaMed Press.

The leukemia-associated Rho guanine nucleotide exchange factor LARG is required for efficient replication stress signaling

PubMed Central

Beveridge, Ryan D; Staples, Christopher J; Patil, Abhijit A; Myers, Katie N; Maslen, Sarah; Skehel, J Mark; Boulton, Simon J; Collis, Spencer J

2014-01-01

We previously identified and characterized TELO2 as a human protein that facilitates efficient DNA damage response (DDR) signaling. A subsequent yeast 2-hybrid screen identified LARG; Leukemia-Associated Rho Guanine Nucleotide Exchange Factor (also known as Arhgef12), as a potential novel TELO2 interactor. LARG was previously shown to interact with Pericentrin (PCNT), which, like TELO2, is required for efficient replication stress signaling. Here we confirm interactions between LARG, TELO2 and PCNT and show that a sub-set of LARG co-localizes with PCNT at the centrosome. LARG-deficient cells exhibit replication stress signaling defects as evidenced by; supernumerary centrosomes, reduced replication stress-induced γH2AX and RPA nuclear foci formation, and reduced activation of the replication stress signaling effector kinase Chk1 in response to hydroxyurea. As such, LARG-deficient cells are sensitive to replication stress-inducing agents such as hydroxyurea and mitomycin C. Conversely we also show that depletion of TELO2 and the replication stress signaling kinase ATR leads to RhoA signaling defects. These data therefore reveal a level of crosstalk between the RhoA and DDR signaling pathways. Given that mutations in both ATR and PCNT can give rise to the related primordial dwarfism disorders of Seckel Syndrome and Microcephalic osteodysplastic primordial dwarfism type II (MOPDII) respectively, which both exhibit defects in ATR-dependent checkpoint signaling, these data also raise the possibility that mutations in LARG or disruption to RhoA signaling may be contributory factors to the etiology of a sub-set of primordial dwarfism disorders. PMID:25485589

Characterization of conserved arginine residues on Cdt1 that affect licensing activity and interaction with Geminin or Mcm complex.

PubMed

You, Zhiying; Ode, Koji L; Shindo, Mayumi; Takisawa, Haruhiko; Masai, Hisao

2016-05-02

All organisms ensure once and only once replication during S phase through a process called replication licensing. Cdt1 is a key component and crucial loading factor of Mcm complex, which is a central component for the eukaryotic replicative helicase. In higher eukaryotes, timely inhibition of Cdt1 by Geminin is essential to prevent rereplication. Here, we address the mechanism of DNA licensing using purified Cdt1, Mcm and Geminin proteins in combination with replication in Xenopus egg extracts. We mutagenized the 223th arginine of mouse Cdt1 (mCdt1) to cysteine or serine (R-S or R-C, respectively) and 342nd and 346th arginines constituting an arginine finger-like structure to alanine (RR-AA). The RR-AA mutant of Cdt1 could not only rescue the DNA replication activity in Cdt1-depleted extracts but also its specific activity for DNA replication and licensing was significantly increased compared to the wild-type protein. In contrast, the R223 mutants were partially defective in rescue of DNA replication and licensing. Biochemical analyses of these mutant Cdt1 proteins indicated that the RR-AA mutation disabled its functional interaction with Geminin, while R223 mutations resulted in ablation in interaction with the Mcm2∼7 complex. Intriguingly, the R223 mutants are more susceptible to the phosphorylation-induced inactivation or chromatin dissociation. Our results show that conserved arginine residues play critical roles in interaction with Geminin and Mcm that are crucial for proper conformation of the complexes and its licensing activity.

Internal Disequilibria and Phenotypic Diversification during Replication of Hepatitis C Virus in a Noncoevolving Cellular Environment.

PubMed

Moreno, Elena; Gallego, Isabel; Gregori, Josep; Lucía-Sanz, Adriana; Soria, María Eugenia; Castro, Victoria; Beach, Nathan M; Manrubia, Susanna; Quer, Josep; Esteban, Juan Ignacio; Rice, Charles M; Gómez, Jordi; Gastaminza, Pablo; Domingo, Esteban; Perales, Celia

2017-05-15

Viral quasispecies evolution upon long-term virus replication in a noncoevolving cellular environment raises relevant general issues, such as the attainment of population equilibrium, compliance with the molecular-clock hypothesis, or stability of the phenotypic profile. Here, we evaluate the adaptation, mutant spectrum dynamics, and phenotypic diversification of hepatitis C virus (HCV) in the course of 200 passages in human hepatoma cells in an experimental design that precluded coevolution of the cells with the virus. Adaptation to the cells was evidenced by increase in progeny production. The rate of accumulation of mutations in the genomic consensus sequence deviated slightly from linearity, and mutant spectrum analyses revealed a complex dynamic of mutational waves, which was sustained beyond passage 100. The virus underwent several phenotypic changes, some of which impacted the virus-host relationship, such as enhanced cell killing, a shift toward higher virion density, and increased shutoff of host cell protein synthesis. Fluctuations in progeny production and failure to reach population equilibrium at the genomic level suggest internal instabilities that anticipate an unpredictable HCV evolution in the complex liver environment. IMPORTANCE Long-term virus evolution in an unperturbed cellular environment can reveal features of virus evolution that cannot be explained by comparing natural viral isolates. In the present study, we investigate genetic and phenotypic changes that occur upon prolonged passage of hepatitis C virus (HCV) in human hepatoma cells in an experimental design in which host cell evolutionary change is prevented. Despite replication in a noncoevolving cellular environment, the virus exhibited internal population disequilibria that did not decline with increased adaptation to the host cells. The diversification of phenotypic traits suggests that disequilibria inherent to viral populations may provide a selective advantage to viruses that can be fully exploited in changing environments. Copyright © 2017 American Society for Microbiology.

Mutation in West Nile Virus Structural Protein prM during Human Infection.

PubMed

Lustig, Yaniv; Lanciotti, Robert S; Hindiyeh, Musa; Keller, Nathan; Milo, Ron; Mayan, Shlomo; Mendelson, Ella

2016-09-01

A mutation leading to substitution of a key amino acid in the prM protein of West Nile virus (WNV) occurred during persistent infection of an immunocompetent patient. WNV RNA persisted in the patient's urine and serum in the presence of low-level neutralizing antibodies. This case demonstrates active replication of WNV during persistent infection.

Mutations in CypA Binding Region of HIV-1 Capsid Affect Capsid Stability and Viral Replication in Primary Macrophages.

PubMed

Setiawan, Laurentia C; van Dort, Karel A; Rits, Maarten A N; Kootstra, Neeltje A

2016-04-01

Mutations in the cyclophilin A (CypA) binding region in the HIV-1 capsid affect their dependency on the known HIV-1 cofactor CypA and allow escape from the HIV-1 restriction factor Trim5α in human and simian cells. Here we study the effect of these mutations in the CypA binding region of capsid on cofactor binding, capsid destabilization, and viral replication in primary cells. We showed that the viral capsid with mutations in the CypA binding region (CypA-BR) interacted efficiently with CypA, but had an increased stability upon infection as compared to the wild-type capsid. Interestingly, the wild-type virus was able to infect monocyte-derived macrophages (MDM) more efficiently as compared to the CypA-BR mutant variant. The lower infectivity of the CypA-BR mutant virus in MDM was associated with lower levels of reverse transcription products. Similar to the wild-type virus, the CypA-BR mutant variant was unable to induce a strong innate response in primary macrophages. These data demonstrate that mutations in the CypA binding site of the capsid resulted in higher capsid stability and hampered infectivity in macrophages.

In cell mutational interference mapping experiment (in cell MIME) identifies the 5' polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging.

PubMed

Smyth, Redmond P; Smith, Maureen R; Jousset, Anne-Caroline; Despons, Laurence; Laumond, Géraldine; Decoville, Thomas; Cattenoz, Pierre; Moog, Christiane; Jossinet, Fabrice; Mougel, Marylène; Paillart, Jean-Christophe; von Kleist, Max; Marquet, Roland

2018-05-18

Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5' region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5' PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production.

In cell mutational interference mapping experiment (in cell MIME) identifies the 5′ polyadenylation signal as a dual regulator of HIV-1 genomic RNA production and packaging

PubMed Central

Smith, Maureen R; Jousset, Anne-Caroline; Despons, Laurence; Laumond, Géraldine; Decoville, Thomas; Cattenoz, Pierre; Moog, Christiane; Jossinet, Fabrice; Mougel, Marylène; Paillart, Jean-Christophe

2018-01-01

Abstract Non-coding RNA regulatory elements are important for viral replication, making them promising targets for therapeutic intervention. However, regulatory RNA is challenging to detect and characterise using classical structure-function assays. Here, we present in cell Mutational Interference Mapping Experiment (in cell MIME) as a way to define RNA regulatory landscapes at single nucleotide resolution under native conditions. In cell MIME is based on (i) random mutation of an RNA target, (ii) expression of mutated RNA in cells, (iii) physical separation of RNA into functional and non-functional populations, and (iv) high-throughput sequencing to identify mutations affecting function. We used in cell MIME to define RNA elements within the 5′ region of the HIV-1 genomic RNA (gRNA) that are important for viral replication in cells. We identified three distinct RNA motifs controlling intracellular gRNA production, and two distinct motifs required for gRNA packaging into virions. Our analysis reveals the 73AAUAAA78 polyadenylation motif within the 5′ PolyA domain as a dual regulator of gRNA production and gRNA packaging, and demonstrates that a functional polyadenylation signal is required for viral packaging even though it negatively affects gRNA production. PMID:29514260

The resistance mutation R155K in the NS3/4A protease of hepatitis C virus also leads the virus to escape from HLA-A*68-restricted CD8 T cells.

PubMed

Salloum, Shadi; Kluge, Silvia F; Kim, Arthur Y; Roggendorf, Michael; Timm, Joerg

2010-08-01

The NS3/4A serine protease of the hepatitis C virus (HCV) is one of the most attractive targets for specific antiviral agents. However, mutations conferring resistance may decrease the efficacy of these drugs. Although the level of resistance associated with specific mutations differs between different compounds, substitutions R155K and A156T reduce susceptibility to all protease inhibitors published so far. Interestingly, variants harboring the resistant mutation R155K were also detected as the predominant quasispecies in some treatment-naïve patients. Of note, key positions for resistance overlap with the HLA-A*68-restricted epitope HAVGIFRAAV(1175-1184). The aim of our study was to analyze the impact of protease inhibitor resistance mutations on the replication level and the antiviral CD8 T cell response against this HCV epitope. Our findings suggest that the R155K variant is associated with a relatively high replication level and with a substantial loss of cross-recognition by specific CD8 T cells targeting the epitope HAVGIFRAAV(1175-1184), providing a possible explanation for its existence in the absence of drug selection pressure. Copyright 2010 Elsevier B.V. All rights reserved.

Identification of conserved amino acids in the herpes simplex virus type 1 UL8 protein required for DNA synthesis and UL52 primase interaction in the virus replisome.

PubMed

Muylaert, Isabella; Zhao, Zhiyuan; Andersson, Torbjörn; Elias, Per

2012-09-28

We have used oriS-dependent transient replication assays to search for species-specific interactions within the herpes simplex virus replisome. Hybrid replisomes derived from herpes simplex virus type 1 (HSV-1) and equine herpesvirus type 1 (EHV-1) failed to support DNA replication in cells. Moreover, the replisomes showed a preference for their cognate origin of replication. The results demonstrate that the herpesvirus replisome behaves as a molecular machine relying on functionally important interactions. We then searched for functional interactions in the replisome context by subjecting HSV-1 UL8 protein to extensive mutagenesis. 52 mutants were made by replacing single or clustered charged amino acids with alanines. Four mutants showed severe replication defects. Mutant A23 exhibited a lethal phenotype, and mutants A49, A52 and A53 had temperature-sensitive phenotypes. Mutants A49 and A53 did not interact with UL52 primase as determined by co-immunoprecipitation experiments. Using GFP-tagged UL8, we demonstrate that all mutants were unable to support formation of ICP8-containing nuclear replication foci. Extended mutagenesis suggested that a highly conserved motif corresponding to mutant A49 serves an important role for establishing a physical contact between UL8 and UL52. The replication-defective mutations affected conserved amino acids, and similar phenotypes were observed when the corresponding mutations were introduced into EHV-1 UL8.

A distinct first replication cycle of DNA introduced in mammalian cells

PubMed Central

Chandok, Gurangad S.; Kapoor, Kalvin K.; Brick, Rachel M.; Sidorova, Julia M.; Krasilnikova, Maria M.

2011-01-01

Many mutation events in microsatellite DNA sequences were traced to the first embryonic divisions. It was not known what makes the first replication cycles of embryonic DNA different from subsequent replication cycles. Here we demonstrate that an unusual replication mode is involved in the first cycle of replication of DNA introduced in mammalian cells. This alternative replication starts at random positions, and occurs before the chromatin is fully assembled. It is detected in various cell lines and primary cells. The presence of single-stranded regions increases the efficiency of this alternative replication mode. The alternative replication cannot progress through the A/T-rich FRA16B fragile site, while the regular replication mode is not affected by it. A/T-rich microsatellites are associated with the majority of chromosomal breakpoints in cancer. We suggest that the alternative replication mode may be initiated at the regions with immature chromatin structure in embryonic and cancer cells resulting in increased genomic instability. This work demonstrates, for the first time, differences in the replication progression during the first and subsequent replication cycles in mammalian cells. PMID:21062817

Interest of proviral HIV-1 DNA genotypic resistance testing in virologically suppressed patients candidate for maintenance therapy.

PubMed

Allavena, C; Rodallec, A; Leplat, A; Hall, N; Luco, C; Le Guen, L; Bernaud, C; Bouchez, S; André-Garnier, E; Boutoille, D; Ferré, V; Raffi, F

2018-01-01

Switch of antiretroviral therapy in virologically suppressed HIV-infected patients is frequent, to prevent toxicities, for simplification or convenience reasons. Pretherapeutic genotypic resistance testing on RNA can be lacking in some patients, which could enhance the risk of virologic failure, if resistance-associated mutations of the new regimen are not taken into account. Proviral DNA resistance testing in 69 virologically suppressed patients on antiretroviral treatment with no history of virological failure were pair-wised compared with pre-ART plasma RNA resistance testing. The median time between plasma (RNA testing) and whole blood (proviral DNA testing) was 47 months (IQR 29-63). A stop codon was evidenced in 23% (16/69) of proviral DNA sequences; these strains were considered as defective, non-replicative, and not taken into consideration. Within the non defective strains, concordance rate between plasma RNA and non-defective proviral DNA was high both on protease (194/220 concordant resistance-associated mutations=88%) and reverse transcriptase (28/37 concordant resistance-associated mutations=76%) genes. This study supports that proviral DNA testing might be an informative tool before switching antiretrovirals in virologically suppressed patients with no history of virological failure, but the interpretation should be restricted to non-defective viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

RPA and Rad51 constitute a cell intrinsic mechanism to protect the cytosol from self DNA.

PubMed

Wolf, Christine; Rapp, Alexander; Berndt, Nicole; Staroske, Wolfgang; Schuster, Max; Dobrick-Mattheuer, Manuela; Kretschmer, Stefanie; König, Nadja; Kurth, Thomas; Wieczorek, Dagmar; Kast, Karin; Cardoso, M Cristina; Günther, Claudia; Lee-Kirsch, Min Ae

2016-05-27

Immune recognition of cytosolic DNA represents a central antiviral defence mechanism. Within the host, short single-stranded DNA (ssDNA) continuously arises during the repair of DNA damage induced by endogenous and environmental genotoxic stress. Here we show that short ssDNA traverses the nuclear membrane, but is drawn into the nucleus by binding to the DNA replication and repair factors RPA and Rad51. Knockdown of RPA and Rad51 enhances cytosolic leakage of ssDNA resulting in cGAS-dependent type I IFN activation. Mutations in the exonuclease TREX1 cause type I IFN-dependent autoinflammation and autoimmunity. We demonstrate that TREX1 is anchored within the outer nuclear membrane to ensure immediate degradation of ssDNA leaking into the cytosol. In TREX1-deficient fibroblasts, accumulating ssDNA causes exhaustion of RPA and Rad51 resulting in replication stress and activation of p53 and type I IFN. Thus, the ssDNA-binding capacity of RPA and Rad51 constitutes a cell intrinsic mechanism to protect the cytosol from self DNA.

Transposon Mutagenesis of the Zika Virus Genome Highlights Regions Essential for RNA Replication and Restricted for Immune Evasion.

PubMed

Fulton, Benjamin O; Sachs, David; Schwarz, Megan C; Palese, Peter; Evans, Matthew J

2017-08-01

The molecular constraints affecting Zika virus (ZIKV) evolution are not well understood. To investigate ZIKV genetic flexibility, we used transposon mutagenesis to add 15-nucleotide insertions throughout the ZIKV MR766 genome and subsequently deep sequenced the viable mutants. Few ZIKV insertion mutants replicated, which likely reflects a high degree of functional constraints on the genome. The NS1 gene exhibited distinct mutational tolerances at different stages of the screen. This result may define regions of the NS1 protein that are required for the different stages of the viral life cycle. The ZIKV structural genes showed the highest degree of insertional tolerance. Although the envelope (E) protein exhibited particular flexibility, the highly conserved envelope domain II (EDII) fusion loop of the E protein was intolerant of transposon insertions. The fusion loop is also a target of pan-flavivirus antibodies that are generated against other flaviviruses and neutralize a broad range of dengue virus and ZIKV isolates. The genetic restrictions identified within the epitopes in the EDII fusion loop likely explain the sequence and antigenic conservation of these regions in ZIKV and among multiple flaviviruses. Thus, our results provide insights into the genetic restrictions on ZIKV that may affect the evolution of this virus. IMPORTANCE Zika virus recently emerged as a significant human pathogen. Determining the genetic constraints on Zika virus is important for understanding the factors affecting viral evolution. We used a genome-wide transposon mutagenesis screen to identify where mutations were tolerated in replicating viruses. We found that the genetic regions involved in RNA replication were mostly intolerant of mutations. The genes coding for structural proteins were more permissive to mutations. Despite the flexibility observed in these regions, we found that epitopes bound by broadly reactive antibodies were genetically constrained. This finding may explain the genetic conservation of these epitopes among flaviviruses. Copyright © 2017 American Society for Microbiology.

Requirement of the cyclic adenosine monophosphate response element-binding protein for hepatitis B virus replication.

PubMed

Kim, Bo Kyung; Lim, Seoung Ok; Park, Yun Gyu

2008-08-01

The cyclic adenosine monophosphate-response element (CRE)-transcription factor complex participates in the regulation of viral gene expression and pathologic processes caused by various viruses. The hepatitis B virus (HBV) enhancer I directs liver-specific transcription of viral genes and contains a CRE sequence (HBV-CRE); however, whether the HBV-CRE and CRE-binding protein (CREB) are required for the HBV life cycle remains to be determined. This study was designed to investigate the role of CREB in HBV replication and gene expression. Sequence-comparison analysis of 984 HBVs reported worldwide showed that the HBV-CRE sequence is highly conserved, indicating the possibility that it plays an important role in the HBV life cycle. The binding of CREB to the HBV-CRE site was markedly inhibited by oligonucleotides containing HBV-CRE and consensus CRE sequences in vitro and in vivo. The HBV promoter activity was demonstrated to be dependent upon the transactivation activity of CREB. Treatment with CRE decoy oligonucleotides reduced HBV promoter activity, and this was reversed by CREB overexpression. The levels of viral transcripts, DNA, and antigens were remarkably decreased in response to the overexpression of CREB mutants or treatment with the CRE decoy oligonucleotides, whereas enhancing CREB activity increased the levels of viral transcripts. In addition, introduction of a three-base mutation into the HBV-CRE led to a marked reduction in HBV messenger RNA synthesis. Taken together, our results demonstrate that both replication and gene expression of HBV require a functional CREB and HBV-CRE. We have also demonstrated that CRE decoy oligonucleotides and the overexpression of CREB mutants can effectively block the HBV life cycle, suggesting that interventions against CREB activity could provide a new avenue to treat HBV infection.

Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites.

PubMed

Tamura, Tomokazu; Nagashima, Naofumi; Ruggli, Nicolas; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro

2014-04-17

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein Npro of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE-, Npro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE- vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE- vaccine virus in pigs, the IRF3-degrading function of Npro was not recovered. Therefore, we examined whether restoring the ability of Npro to block IFN-α/β induction of both the avirulent and moderately virulent GPE--derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in Npro regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional Npro significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional Npro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.

A priA Mutant Expressed in Two Pieces Has Almost Full Activity in Escherichia coli K-12

PubMed Central

Leroux, Maxime; Jani, Niketa

2017-01-01

ABSTRACT The ability to restart broken DNA replication forks is essential across all domains of life. In Escherichia coli, the priA, priB, priC, and dnaT genes encode the replication restart proteins (RRPs) to accomplish this task. PriA plays a critical role in replication restart such that its absence reveals a dramatic phenotype: poor growth, high basal levels of SOS expression, poorly partitioned nucleoids (Par−), UV sensitivity, and recombination deficiency (Rec−). PriA has 733 amino acids, and its structure is composed of six domains that enable it to bind to DNA replication fork-like structures, remodel the strands of DNA, interact with SSB (single-stranded DNA binding protein), PriB, and DnaT, and display ATPase, helicase, and translocase activities. We have characterized a new priA mutation called priA316::cat. It is a composite mutation involving an insertion that truncates the protein within the winged-helix domain (at the 154th codon) and an ACG (Thr)-to-ATG (Met) mutation that allows reinitiation of translation at the 157th codon such that PriA is expressed in two pieces. priA316::cat phenotypes are like those of the wild type for growth, recombination, and UV resistance, revealing only a slightly increased level of SOS expression and defects in nucleoid partitioning in the mutant. Both parts of PriA are required for activity, and the N-terminal fragment can be optimized to yield wild-type activity. A deletion of the lon protease suppresses priA316::cat phenotypes. We hypothesize the two parts of PriA form a complex that supplies most of the PriA activity needed in the cell. IMPORTANCE PriA is a highly conserved multifunctional protein that plays a crucial role in the essential process of replication restart. Here we characterize an insertion mutation of priA with an intragenic suppressor such that it is now made in two parts. These two pieces split the winged-helix domain to separate the N-terminal 3′ DNA-binding domain from the C-terminal domain of PriA. It is hypothesized that the two pieces form a complex that is capable of almost wild type priA function. The composite mutation leads to a moderate level of SOS expression and defects in partitioning of the chromosomes. Full function is restored by deletion of lon, suggesting that stability of this complex may be a reason for the partial phenotypes seen. PMID:28607160

The (not so) immortal strand hypothesis.

PubMed

Tomasetti, Cristian; Bozic, Ivana

2015-03-01

Non-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an "immortal" DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented. Using a novel methodology that utilizes cancer sequencing data we are able to estimate the rate of accumulation of mutations in healthy stem cells of the colon, blood and head and neck tissues. We find that in these tissues mutations in stem cells accumulate at rates strikingly similar to those expected without the protection from the immortal strand mechanism. Utilizing an approach that is fundamentally different from previous efforts to confirm or refute the immortal strand hypothesis, we provide evidence against non-random segregation of DNA during stem cell replication. Our results strongly suggest that parental DNA is passed randomly to stem cell daughters and provides new insight into the mechanism of DNA replication in stem cells. Copyright © 2015. Published by Elsevier B.V.

The (not so) Immortal Strand Hypothesis

PubMed Central

Tomasetti, Cristian; Bozic, Ivana

2015-01-01

Background Non-random segregation of DNA strands during stem cell replication has been proposed as a mechanism to minimize accumulated genetic errors in stem cells of rapidly dividing tissues. According to this hypothesis, an “immortal” DNA strand is passed to the stem cell daughter and not the more differentiated cell, keeping the stem cell lineage replication error-free. After it was introduced, experimental evidence both in favor and against the hypothesis has been presented. Principal Findings Using a novel methodology that utilizes cancer sequencing data we are able to estimate the rate of accumulation of mutations in healthy stem cells of the colon, blood and head and neck tissues. We to find that in these tissues mutations in stem cells accumulate at rates strikingly similar to those expected without the protection from the immortal strand mechanism. Significance Utilizing an approach that is fundamentally different from previous efforts to confirm or refute the immortal strand hypothesis, we provide strong evidence against non-random segregation of DNA during stem cell replication. Our results strongly suggest that parental DNA is passed randomly to stem cell daughters and provides new insight into the mechanism of DNA replication in stem cells. PMID:25700960

Genomic instability--an evolving hallmark of cancer.

PubMed

Negrini, Simona; Gorgoulis, Vassilis G; Halazonetis, Thanos D

2010-03-01

Genomic instability is a characteristic of most cancers. In hereditary cancers, genomic instability results from mutations in DNA repair genes and drives cancer development, as predicted by the mutator hypothesis. In sporadic (non-hereditary) cancers the molecular basis of genomic instability remains unclear, but recent high-throughput sequencing studies suggest that mutations in DNA repair genes are infrequent before therapy, arguing against the mutator hypothesis for these cancers. Instead, the mutation patterns of the tumour suppressor TP53 (which encodes p53), ataxia telangiectasia mutated (ATM) and cyclin-dependent kinase inhibitor 2A (CDKN2A; which encodes p16INK4A and p14ARF) support the oncogene-induced DNA replication stress model, which attributes genomic instability and TP53 and ATM mutations to oncogene-induced DNA damage.

Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

PubMed

Loutre, Romuald; Heckel, Anne-Marie; Jeandard, Damien; Tarassov, Ivan; Entelis, Nina

2018-01-01

Mutations in mitochondrial DNA are an important source of severe and incurable human diseases. The vast majority of these mutations are heteroplasmic, meaning that mutant and wild-type genomes are present simultaneously in the same cell. Only a very high proportion of mutant mitochondrial DNA (heteroplasmy level) leads to pathological consequences. We previously demonstrated that mitochondrial targeting of small RNAs designed to anneal with mutant mtDNA can decrease the heteroplasmy level by specific inhibition of mutant mtDNA replication, thus representing a potential therapy. We have also shown that 5S ribosomal RNA, partially imported into human mitochondria, can be used as a vector to deliver anti-replicative oligoribonucleotides into human mitochondria. So far, the efficiency of cellular expression of recombinant 5S rRNA molecules bearing therapeutic insertions remained very low. In the present study, we designed new versions of anti-replicative recombinant 5S rRNA targeting a large deletion in mitochondrial DNA which causes the KSS syndrome, analyzed their specific annealing to KSS mitochondrial DNA and demonstrated their import into mitochondria of cultured human cells. To obtain an increased level of the recombinant 5S rRNA stable expression, we created transmitochondrial cybrid cell line bearing a site for Flp-recombinase and used this system for the recombinase-mediated integration of genes coding for the anti-replicative recombinant 5S rRNAs into nuclear genome. We demonstrated that stable expression of anti-replicative 5S rRNA versions in human transmitochondrial cybrid cells can induce a shift in heteroplasmy level of KSS mutation in mtDNA. This shift was directly dependent on the level of the recombinant 5S rRNA expression and the sequence of the anti-replicative insertion. Quantification of mtDNA copy number in transfected cells revealed the absence of a non-specific effect on wild type mtDNA replication, indicating that the decreased proportion between mutant and wild type mtDNA molecules is not a consequence of a random repopulation of depleted pool of mtDNA genomes. The heteroplasmy change could be also modulated by cell growth conditions, namely increased by cells culturing in a carbohydrate-free medium, thus forcing them to use oxidative phosphorylation and providing a selective advantage for cells with improved respiration capacities. We discuss the advantages and limitations of this approach and propose further development of the anti-replicative strategy based on the RNA import into human mitochondria.

Shifts in Host Range of a Promiscuous Plasmid through Parallel Evolution of its Replication Initiation Protein

PubMed Central

Sota, Masahiro; Yano, Hirokazu; Hughes, Julie; Daughdrill, Gary W.; Abdo, Zaid; Forney, Larry J.; Top, Eva M.

2011-01-01

The ability of bacterial plasmids to adapt to novel hosts and thereby shift their host range is key to their long-term persistence in bacterial communities. Promiscuous plasmids of the IncP-1 group can colonize a wide range of hosts, but it is not known if and how they can contract, shift or further expand their host range. To understand the evolutionary mechanisms of host range shifts of IncP-1 plasmids, an IncP-1β mini-replicon was experimentally evolved in four hosts wherein it was initially unstable. After 1000 generations in serial batch cultures under antibiotic selection for plasmid maintenance (kanamycin resistance), the stability of the mini-plasmid had dramatically improved in all coevolved hosts. However, only plasmids evolved in Shewanella oneidensis showed improved stability in the ancestor, indicating that adaptive mutations had occurred in the plasmid itself. Complete genome sequence analysis of nine independently evolved plasmids showed seven unique plasmid genotypes that had various kinds of single mutations at one locus, namely the N-terminal region of the replication initiation protein TrfA. Such parallel evolution indicates that this region was under strong selection. In five of the seven evolved plasmids these trfA mutations resulted in a significantly higher plasmid copy number. Evolved plasmids were found to be stable in four other naïve hosts, but could no longer replicate in Pseudomonas aeruginosa. This study demonstrates that plasmids can specialize to a novel host through trade-offs between improved stability in the new host and the ability to replicate in a previously permissive host. PMID:20520653

Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B.

PubMed

Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

2017-02-01

Feline coronavirus (FCoV) causes the fatal disease feline infectious peritonitis, which is currently incurable by drug treatment, and no effective vaccines are available. Cyclosporin A (CsA), a cyclophilin (Cyp) inhibitor, inhibits the replication of FCoV in vitro and in vivo as well as the replication of human and animal coronaviruses. However, the mechanism underlying the regulation of coronavirus replication by CsA is unknown. In this study, we analysed the role of Cyps in FCoV replication using knockdown and knockout cells specific to Cyps. Inhibition of CypA and CypB reduced FCoV replication, with replication in knockout cells being much less than that in knockdown cells. Furthermore, the proteins expressed by CypA and CypB harbouring mutations in their respective predicted peptidyl-prolyl cis-transisomerase active sites, which also alter the affinities between Cyps and CsA, inhibited FCoV replication. These findings indicate that the peptidyl-prolyl cis-transisomerase active sites of Cyps might be required for FCoV replication.

Role of Fanconi Anemia FANCG in Preventing Double-Strand Breakage and Chromosomal Rearrangement during DNA Replication

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tebbs, R S; Hinz, J M; Yamada, N A

The Fanconi anemia (FA) proteins overlap with those of homologous recombination through FANCD1/BRCA2, but the biochemical functions of other FA proteins are unknown. By constructing and characterizing a null fancg mutant of hamster CHO cells, we present several new insights for FA. The fancg cells show a broad sensitivity to genotoxic agents, not supporting the conventional concept of sensitivity to only DNA crosslinking agents. The aprt mutation rate is normal, but hprt mutations are reduced, which we ascribe to the lethality of large deletions. CAD and dhfr gene amplification rates are increased, implying excess chromosomal breakage during DNA replication, andmore » suggesting amplification as a contributing factor to cancer-proneness in FA patients. In S-phase cells, both spontaneous and mutagen-induced Rad51 nuclear foci are elevated. These results support a model in which FancG protein helps to prevent collapse of replication forks by allowing translesion synthesis or lesion bypass through homologous recombination.« less

Re-wiring of energy metabolism promotes viability during hyperreplication stress in E. coli

PubMed Central

Campion, Christopher; Weimann, Allan

2017-01-01

Chromosome replication in Escherichia coli is initiated by DnaA. DnaA binds ATP which is essential for formation of a DnaA-oriC nucleoprotein complex that promotes strand opening, helicase loading and replisome assembly. Following initiation, DnaAATP is converted to DnaAADP primarily by the Regulatory Inactivation of DnaA process (RIDA). In RIDA deficient cells, DnaAATP accumulates leading to uncontrolled initiation of replication and cell death by accumulation of DNA strand breaks. Mutations that suppress RIDA deficiency either dampen overinitiation or permit growth despite overinitiation. We characterize mutations of the last group that have in common that distinct metabolic routes are rewired resulting in the redirection of electron flow towards the cytochrome bd-1. We propose a model where cytochrome bd-1 lowers the formation of reactive oxygen species and hence oxidative damage to the DNA in general. This increases the processivity of replication forks generated by overinitiation to a level that sustains viability. PMID:28129339

In Vitro Analysis of the Role of Replication Protein A (RPA) and RPA Phosphorylation in ATR-mediated Checkpoint Signaling*

PubMed Central

Lindsey-Boltz, Laura A.; Reardon, Joyce T.; Wold, Marc S.; Sancar, Aziz

2012-01-01

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair. PMID:22948311

In vitro analysis of the role of replication protein A (RPA) and RPA phosphorylation in ATR-mediated checkpoint signaling.

PubMed

Lindsey-Boltz, Laura A; Reardon, Joyce T; Wold, Marc S; Sancar, Aziz

2012-10-19

Replication protein A (RPA) plays essential roles in DNA metabolism, including replication, checkpoint, and repair. Recently, we described an in vitro system in which the phosphorylation of human Chk1 kinase by ATR (ataxia telangiectasia mutated and Rad3-related) is dependent on RPA bound to single-stranded DNA. Here, we report that phosphorylation of other ATR targets, p53 and Rad17, has the same requirements and that RPA is also phosphorylated in this system. At high p53 or Rad17 concentrations, RPA phosphorylation is inhibited and, in this system, RPA with phosphomimetic mutations cannot support ATR kinase function, whereas a non-phosphorylatable RPA mutant exhibits full activity. Phosphorylation of these ATR substrates depends on the recruitment of ATR and the substrates by RPA to the RPA-ssDNA complex. Finally, mutant RPAs lacking checkpoint function exhibit essentially normal activity in nucleotide excision repair, revealing RPA separation of function for checkpoint and excision repair.

Genome instabilities arising from ribonucleotides in DNA.

PubMed

Klein, Hannah L

2017-08-01

Genomic DNA is transiently contaminated with ribonucleotide residues during the process of DNA replication through misincorporation by the replicative DNA polymerases α, δ and ε, and by the normal replication process on the lagging strand, which uses RNA primers. These ribonucleotides are efficiently removed during replication by RNase H enzymes and the lagging strand synthesis machinery. However, when ribonucleotides remain in DNA they can distort the DNA helix, affect machineries for DNA replication, transcription and repair, and can stimulate genomic instabilities which are manifest as increased mutation, recombination and chromosome alterations. The genomic instabilities associated with embedded ribonucleotides are considered here, along with a discussion of the origin of the lesions that stimulate particular classes of instabilities. Copyright © 2017 Elsevier B.V. All rights reserved.

Inhibition of polyomavirus ori-dependent DNA replication by mSin3B.

PubMed

Xie, An-Yong; Folk, William R

2002-12-01

When tethered in cis to DNA, the transcriptional corepressor mSin3B inhibits polyomavirus (Py) ori-dependent DNA replication in vivo. Histone deacetylases (HDACs) appear not to be involved, since tethering class I and class II HDACs in cis does not inhibit replication and treating the cells with trichostatin A does not specifically relieve inhibition by mSin3B. However, the mSin3B L59P mutation that impairs mSin3B interaction with N-CoR/SMRT abrogates inhibition of replication, suggesting the involvement of N-CoR/SMRT. Py large T antigen interacts with mSin3B, suggesting an HDAC-independent mechanism by which mSin3B inhibits DNA replication.

Environmental change drives accelerated adaptation through stimulated copy number variation

PubMed Central

Hull, Ryan M.; Cruz, Cristina; Jack, Carmen V.

2017-01-01

Copy number variation (CNV) is rife in eukaryotic genomes and has been implicated in many human disorders, particularly cancer, in which CNV promotes both tumorigenesis and chemotherapy resistance. CNVs are considered random mutations but often arise through replication defects; transcription can interfere with replication fork progression and stability, leading to increased mutation rates at highly transcribed loci. Here we investigate whether inducible promoters can stimulate CNV to yield reproducible, environment-specific genetic changes. We propose a general mechanism for environmentally-stimulated CNV and validate this mechanism for the emergence of copper resistance in budding yeast. By analysing a large cohort of individual cells, we directly demonstrate that CNV of the copper-resistance gene CUP1 is stimulated by environmental copper. CNV stimulation accelerates the formation of novel alleles conferring enhanced copper resistance, such that copper exposure actively drives adaptation to copper-rich environments. Furthermore, quantification of CNV in individual cells reveals remarkable allele selectivity in the rate at which specific environments stimulate CNV. We define the key mechanistic elements underlying this selectivity, demonstrating that CNV is regulated by both promoter activity and acetylation of histone H3 lysine 56 (H3K56ac) and that H3K56ac is required for CUP1 CNV and efficient copper adaptation. Stimulated CNV is not limited to high-copy CUP1 repeat arrays, as we find that H3K56ac also regulates CNV in 3 copy arrays of CUP1 or SFA1 genes. The impact of transcription on DNA damage is well understood, but our research reveals that this apparently problematic association forms a pathway by which mutations can be directed to particular loci in particular environments and furthermore that this mutagenic process can be regulated through histone acetylation. Stimulated CNV therefore represents an unanticipated and remarkably controllable pathway facilitating organismal adaptation to new environments. PMID:28654659

A conserved mechanism for replication origin recognition and binding in archaea.

PubMed

Majerník, Alan I; Chong, James P J

2008-01-15

To date, methanogens are the only group within the archaea where firing DNA replication origins have not been demonstrated in vivo. In the present study we show that a previously identified cluster of ORB (origin recognition box) sequences do indeed function as an origin of replication in vivo in the archaeon Methanothermobacter thermautotrophicus. Although the consensus sequence of ORBs in M. thermautotrophicus is somewhat conserved when compared with ORB sequences in other archaea, the Cdc6-1 protein from M. thermautotrophicus (termed MthCdc6-1) displays sequence-specific binding that is selective for the MthORB sequence and does not recognize ORBs from other archaeal species. Stabilization of in vitro MthORB DNA binding by MthCdc6-1 requires additional conserved sequences 3' to those originally described for M. thermautotrophicus. By testing synthetic sequences bearing mutations in the MthORB consensus sequence, we show that Cdc6/ORB binding is critically dependent on the presence of an invariant guanine found in all archaeal ORB sequences. Mutation of a universally conserved arginine residue in the recognition helix of the winged helix domain of archaeal Cdc6-1 shows that specific origin sequence recognition is dependent on the interaction of this arginine residue with the invariant guanine. Recognition of a mutated origin sequence can be achieved by mutation of the conserved arginine residue to a lysine or glutamine residue. Thus despite a number of differences in protein and DNA sequences between species, the mechanism of origin recognition and binding appears to be conserved throughout the archaea.

Overexpressed DNA polymerase iota regulated by JNK/c-Jun contributes to hypermutagenesis in bladder cancer.

PubMed

Yuan, Fang; Xu, Zhigang; Yang, Mingzhen; Wei, Quanfang; Zhang, Yi; Yu, Jin; Zhi, Yi; Liu, Yang; Chen, Zhiwen; Yang, Jin

2013-01-01

Human DNA polymerase iota (pol ι) possesses high error-prone DNA replication features and performs translesion DNA synthesis. It may be specialized and strictly regulated in normal mammalian cells. Dysregulation of pol ι may contribute to the acquisition of a mutator phenotype. However, there are few reports describing the transcription regulatory mechanism of pol ι, and there is controversy regarding its role in carcinogenesis. In this study, we performed the deletion and point-mutation experiment, EMSA, ChIP, RNA interference and western blot assay to prove that c-Jun activated by c-Jun N-terminal kinase (JNK) regulates the transcription of pol ι in normal and cancer cells. Xeroderma pigmentosum group C protein (XPC) and ataxia-telangiectasia mutated related protein (ATR) promote early JNK activation in response to DNA damage and consequently enhance the expression of pol ι, indicating that the novel role of JNK signal pathway is involved in DNA damage response. Furthermore, associated with elevated c-Jun activity, the overexpression of pol ι is positively correlated with the clinical tumor grade in 97 bladder cancer samples and may contribute to the hypermutagenesis. The overexpressed pol ι-involved mutagenesis is dependent on JNK/c-Jun pathway in bladder cancer cells identifying by the special mutation spectra. Our results support the conclusion that dysregulation of pol ι by JNK/c-Jun is involved in carcinogenesis and offer a novel understanding of the role of pol ι or c-Jun in mutagenesis.

Overexpressed DNA Polymerase Iota Regulated by JNK/c-Jun Contributes to Hypermutagenesis in Bladder Cancer

PubMed Central

Yuan, Fang; Xu, Zhigang; Yang, Mingzhen; Wei, Quanfang; Zhang, Yi; Yu, Jin; Zhi, Yi; Liu, Yang; Chen, Zhiwen; Yang, Jin

2013-01-01

Human DNA polymerase iota (pol ι) possesses high error-prone DNA replication features and performs translesion DNA synthesis. It may be specialized and strictly regulated in normal mammalian cells. Dysregulation of pol ι may contribute to the acquisition of a mutator phenotype. However, there are few reports describing the transcription regulatory mechanism of pol ι, and there is controversy regarding its role in carcinogenesis. In this study, we performed the deletion and point-mutation experiment, EMSA, ChIP, RNA interference and western blot assay to prove that c-Jun activated by c-Jun N-terminal kinase (JNK) regulates the transcription of pol ι in normal and cancer cells. Xeroderma pigmentosum group C protein (XPC) and ataxia-telangiectasia mutated related protein (ATR) promote early JNK activation in response to DNA damage and consequently enhance the expression of pol ι, indicating that the novel role of JNK signal pathway is involved in DNA damage response. Furthermore, associated with elevated c-Jun activity, the overexpression of pol ι is positively correlated with the clinical tumor grade in 97 bladder cancer samples and may contribute to the hypermutagenesis. The overexpressed pol ι-involved mutagenesis is dependent on JNK/c-Jun pathway in bladder cancer cells identifying by the special mutation spectra. Our results support the conclusion that dysregulation of pol ι by JNK/c-Jun is involved in carcinogenesis and offer a novel understanding of the role of pol ι or c-Jun in mutagenesis. PMID:23922701

The role of BRCA1 in homologous recombination repair in response to replication stress: significance in tumorigenesis and cancer therapy

PubMed Central

2013-01-01

Germ line mutations in breast cancer gene 1 (BRCA1) predispose women to breast and ovarian cancers. Although BRCA1 is involved in many important biological processes, the function of BRCA1 in homologous recombination (HR) mediated repair is considered one of the major mechanisms contributing to its tumor suppression activity, and the cause of hypersensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors when BRCA1 is defective. Mounting evidence suggests that the mechanism of repairing DNA double strand breaks (DSBs) by HR is different than the mechanism operating when DNA replication is blocked. Although BRCA1 has been recognized as a central component in HR, the precise role of BRCA1 in HR, particularly under replication stress, has remained largely unknown. Given the fact that DNA lesions caused by replication blockages are the primary substrates for HR in mitotic cells, functional analysis of BRCA1 in HR repair in the context of replication stress should benefit our understanding of the molecular mechanisms underlying tumorigenesis associated with BRCA1 deficiencies, as well as the development of therapeutic approaches for cancer patients carrying BRCA1 mutations or reduced BRCA1 expression. This review focuses on the current advances in this setting and also discusses the significance in tumorigenesis and cancer therapy. PMID:23388117

Identification of cellular and viral factors related to anti-hepatitis C virus activity of cyclophilin inhibitor.

PubMed

Goto, Kaku; Watashi, Koichi; Inoue, Daisuke; Hijikata, Makoto; Shimotohno, Kunitada

2009-10-01

We have so far reported that an immunosuppressant cyclosporin A (CsA), a well-known cyclophilin (CyP) inhibitor (CPI), strongly suppressed hepatitis C virus (HCV) replication in cell culture, and that CyPB was a cellular cofactor for viral replication. To further investigate antiviral mechanisms of CPI, we here developed cells carrying CsA-resistant HCV replicons, by culturing the HCV subgenomic replicon cells for 4 weeks in the presence of CsA with G418. Transfection of total RNA from the isolated CsA-resistant cells to naïve Huh7 cells conferred CsA resistance, suggesting that the replicon RNA itself was responsible for the resistant phenotype. Of the identified amino acid mutations, D320E in NS5A conferred the CsA resistance. The replicon carrying the D320E mutation was sensitive to interferon-alpha, but was resistant to CsA and other CPIs including NIM811 and sanglifehrin A. Knockdown of individual CyP subtypes revealed CyP40, in addition to CyPA and CyPB, contributed to viral replication, and CsA-resistant replicons acquired independence from CyPA for efficient replication. These data provide important evidence on the mechanisms underlying the regulation of HCV replication by CyP and for designing novel and specific anti-HCV strategies with CPIs.

Temperature-sensitive mutations for live-attenuated Rift Valley fever vaccines: implications from other RNA viruses

PubMed Central

Nishiyama, Shoko; Ikegami, Tetsuro

2015-01-01

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to the African continent. RVF is characterized by high rate of abortions in ruminants and hemorrhagic fever, encephalitis, or blindness in humans. RVF is caused by the Rift Valley fever virus (RVFV: genus Phlebovirus, family Bunyaviridae). Vaccination is the only known effective strategy to prevent the disease, but there are no licensed RVF vaccines available for humans. A live-attenuated vaccine candidate derived from the wild-type pathogenic Egyptian ZH548 strain, MP-12, has been conditionally licensed for veterinary use in the U.S. MP-12 displays a temperature-sensitive (ts) phenotype and does not replicate at 41°C. The ts mutation limits viral replication at a specific body temperature and may lead to an attenuation of the virus. Here we will review well-characterized ts mutations for RNA viruses, and further discuss the potential in designing novel live-attenuated vaccines for RVF. PMID:26322023

Mutations in Alström protein impair terminal differentiation of cardiomyocytes.

PubMed

Shenje, Lincoln T; Andersen, Peter; Halushka, Marc K; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A S; Chen, Yan; Chelko, Stephen; Crosson, Jane E; Scheel, Janet; Vricella, Luca; Craig, Brian D; Marosy, Beth A; Mohr, David W; Hetrick, Kurt N; Romm, Jane M; Scott, Alan F; Valle, David; Naggert, Jürgen K; Kwon, Chulan; Doheny, Kimberly F; Judge, Daniel P

2014-03-04

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

Mutations in Alström Protein Impair Terminal Differentiation of Cardiomyocytes

PubMed Central

Shenje, Lincoln T.; Andersen, Peter; Halushka, Marc K.; Lui, Cecillia; Fernandez, Laviel; Collin, Gayle B.; Amat-Alarcon, Nuria; Meschino, Wendy; Cutz, Ernest; Chang, Kenneth; Yonescu, Raluca; Batista, Denise A. S.; Chen, Yan; Chelko, Stephen; Crosson, Jane E.; Scheel, Janet; Vricella, Luca; Craig, Brian D.; Marosy, Beth A.; Mohr, David W.; Hetrick, Kurt N.; Romm, Jane M.; Scott, Alan F.; Valle, David; Naggert, Jürgen K.; Kwon, Chulan; Doheny, Kimberly F.; Judge, Daniel P.

2014-01-01

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognise homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at two weeks postnatal compared to wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest. PMID:24595103

Seven Activities for Enhancing the Replicability of Evidence-Based Practices. Research-to-Results Brief. Publication #2007-30

ERIC Educational Resources Information Center

Metz, Allison J. R.; Bowie, Lillian; Blase, Karen

2007-01-01

This brief will define program replication, describe the critical role of "core components" in program replication, and outline seven activities that program developers and researchers can conduct to enhance the replicability of effective program models and facilitate their adoption by other organizations and programs. Outlined is seven specific…

The Roles of Hemagglutinin Phe-95 in Receptor Binding and Pathogenicity of Influenza B Virus

PubMed Central

Ni, Fengyun; Mbawuike, Innocent Nnadi; Kondrashkina, Elena; Wang, Qinghua

2014-01-01

Diverged ~4,000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and highly restricted host range. Based on our prior structural studies, we hypothesized that a single-residue difference in the receptor-binding site of hemagglutinin (HA), Phe-95 in influenza B virus versus Tyr-98 in influenza A/H1~H15, is possibly a key determinant for the low receptor-binding affinity. Here we demonstrate that the mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA and has the potential to enhance receptor binding. However, the full realization of this potential is influenced by the local environment into which the mutation is introduced. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of HA. These results are discussed in relation to the roles of Phe-95 in receptor binding and pathogenicity of influenza B virus. PMID:24503069

Germline and somatic polymerase ε and δ mutations define a new class of hypermutated colorectal and endometrial cancers

PubMed Central

Briggs, Sarah; Tomlinson, Ian

2013-01-01

Polymerases ϵ and δ are the main enzymes that replicate eukaryotic DNA. Accurate replication occurs through Watson–Crick base pairing and also through the action of the polymerases' exonuclease (proofreading) domains. We have recently shown that germline exonuclease domain mutations (EDMs) of POLE and POLD1 confer a high risk of multiple colorectal adenomas and carcinoma (CRC). POLD1 mutations also predispose to endometrial cancer (EC). These mutations are associated with high penetrance and dominant inheritance, although the phenotype can be variable. We have named the condition polymerase proofreading-associated polyposis (PPAP). Somatic POLE EDMs have also been found in sporadic CRCs and ECs, although very few somatic POLD1 EDMs have been detected. Both the germline and the somatic DNA polymerase EDMs cause an ‘ultramutated’, apparently microsatellite-stable, type of cancer, sometimes leading to over a million base substitutions per tumour. Here, we present the evidence for POLE and POLD1 as important contributors to the pathogenesis of CRC and EC, and highlight some of the key questions in this emerging field. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd PMID:23447401

Hypomorphic PCNA mutation underlies a human DNA repair disorder

PubMed Central

Baple, Emma L.; Chambers, Helen; Cross, Harold E.; Fawcett, Heather; Nakazawa, Yuka; Chioza, Barry A.; Harlalka, Gaurav V.; Mansour, Sahar; Sreekantan-Nair, Ajith; Patton, Michael A.; Muggenthaler, Martina; Rich, Phillip; Wagner, Karin; Coblentz, Roselyn; Stein, Constance K.; Last, James I.; Taylor, A. Malcolm R.; Jackson, Andrew P.; Ogi, Tomoo; Lehmann, Alan R.; Green, Catherine M.; Crosby, Andrew H.

2014-01-01

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA’s interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration. PMID:24911150

The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

PubMed Central

Prasanth, K. Reddisiva; Barajas, Daniel

2014-01-01

ABSTRACT RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a “matchmaker” that brings the viral p92pol replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. IMPORTANCE RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination. PMID:25540361

Checkpoint independence of most DNA replication origins in fission yeast

PubMed Central

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-01-01

Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells. Conclusion The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks. PMID:18093330

Potential genetic modifiers of disease risk and age at onset in patients with frontotemporal lobar degeneration and GRN mutations: a genome-wide association study.

PubMed

Pottier, Cyril; Zhou, Xiaolai; Perkerson, Ralph B; Baker, Matt; Jenkins, Gregory D; Serie, Daniel J; Ghidoni, Roberta; Benussi, Luisa; Binetti, Giuliano; López de Munain, Adolfo; Zulaica, Miren; Moreno, Fermin; Le Ber, Isabelle; Pasquier, Florence; Hannequin, Didier; Sánchez-Valle, Raquel; Antonell, Anna; Lladó, Albert; Parsons, Tammee M; Finch, NiCole A; Finger, Elizabeth C; Lippa, Carol F; Huey, Edward D; Neumann, Manuela; Heutink, Peter; Synofzik, Matthis; Wilke, Carlo; Rissman, Robert A; Slawek, Jaroslaw; Sitek, Emilia; Johannsen, Peter; Nielsen, Jørgen E; Ren, Yingxue; van Blitterswijk, Marka; DeJesus-Hernandez, Mariely; Christopher, Elizabeth; Murray, Melissa E; Bieniek, Kevin F; Evers, Bret M; Ferrari, Camilla; Rollinson, Sara; Richardson, Anna; Scarpini, Elio; Fumagalli, Giorgio G; Padovani, Alessandro; Hardy, John; Momeni, Parastoo; Ferrari, Raffaele; Frangipane, Francesca; Maletta, Raffaele; Anfossi, Maria; Gallo, Maura; Petrucelli, Leonard; Suh, EunRan; Lopez, Oscar L; Wong, Tsz H; van Rooij, Jeroen G J; Seelaar, Harro; Mead, Simon; Caselli, Richard J; Reiman, Eric M; Noel Sabbagh, Marwan; Kjolby, Mads; Nykjaer, Anders; Karydas, Anna M; Boxer, Adam L; Grinberg, Lea T; Grafman, Jordan; Spina, Salvatore; Oblak, Adrian; Mesulam, M-Marsel; Weintraub, Sandra; Geula, Changiz; Hodges, John R; Piguet, Olivier; Brooks, William S; Irwin, David J; Trojanowski, John Q; Lee, Edward B; Josephs, Keith A; Parisi, Joseph E; Ertekin-Taner, Nilüfer; Knopman, David S; Nacmias, Benedetta; Piaceri, Irene; Bagnoli, Silvia; Sorbi, Sandro; Gearing, Marla; Glass, Jonathan; Beach, Thomas G; Black, Sandra E; Masellis, Mario; Rogaeva, Ekaterina; Vonsattel, Jean-Paul; Honig, Lawrence S; Kofler, Julia; Bruni, Amalia C; Snowden, Julie; Mann, David; Pickering-Brown, Stuart; Diehl-Schmid, Janine; Winkelmann, Juliane; Galimberti, Daniela; Graff, Caroline; Öijerstedt, Linn; Troakes, Claire; Al-Sarraj, Safa; Cruchaga, Carlos; Cairns, Nigel J; Rohrer, Jonathan D; Halliday, Glenda M; Kwok, John B; van Swieten, John C; White, Charles L; Ghetti, Bernardino; Murell, Jill R; Mackenzie, Ian R A; Hsiung, Ging-Yuek R; Borroni, Barbara; Rossi, Giacomina; Tagliavini, Fabrizio; Wszolek, Zbigniew K; Petersen, Ronald C; Bigio, Eileen H; Grossman, Murray; Van Deerlin, Vivianna M; Seeley, William W; Miller, Bruce L; Graff-Radford, Neill R; Boeve, Bradley F; Dickson, Dennis W; Biernacka, Joanna M; Rademakers, Rosa

2018-06-01

Loss-of-function mutations in GRN cause frontotemporal lobar degeneration (FTLD). Patients with GRN mutations present with a uniform subtype of TAR DNA-binding protein 43 (TDP-43) pathology at autopsy (FTLD-TDP type A); however, age at onset and clinical presentation are variable, even within families. We aimed to identify potential genetic modifiers of disease onset and disease risk in GRN mutation carriers. The study was done in three stages: a discovery stage, a replication stage, and a meta-analysis of the discovery and replication data. In the discovery stage, genome-wide logistic and linear regression analyses were done to test the association of genetic variants with disease risk (case or control status) and age at onset in patients with a GRN mutation and controls free of neurodegenerative disorders. Suggestive loci (p<1 × 10 -5 ) were genotyped in a replication cohort of patients and controls, followed by a meta-analysis. The effect of genome-wide significant variants at the GFRA2 locus on expression of GFRA2 was assessed using mRNA expression studies in cerebellar tissue samples from the Mayo Clinic brain bank. The effect of the GFRA2 locus on progranulin concentrations was studied using previously generated ELISA-based expression data. Co-immunoprecipitation experiments in HEK293T cells were done to test for a direct interaction between GFRA2 and progranulin. Individuals were enrolled in the current study between Sept 16, 2014, and Oct 5, 2017. After quality control measures, statistical analyses in the discovery stage included 382 unrelated symptomatic GRN mutation carriers and 1146 controls free of neurodegenerative disorders collected from 34 research centres located in the USA, Canada, Australia, and Europe. In the replication stage, 210 patients (67 symptomatic GRN mutation carriers and 143 patients with FTLD without GRN mutations pathologically confirmed as FTLD-TDP type A) and 1798 controls free of neurodegenerative diseases were recruited from 26 sites, 20 of which overlapped with the discovery stage. No genome-wide significant association with age at onset was identified in the discovery or replication stages, or in the meta-analysis. However, in the case-control analysis, we replicated the previously reported TMEM106B association (rs1990622 meta-analysis odds ratio [OR] 0·54, 95% CI 0·46-0·63; p=3·54 × 10 -16 ), and identified a novel genome-wide significant locus at GFRA2 on chromosome 8p21.3 associated with disease risk (rs36196656 meta-analysis OR 1·49, 95% CI 1·30-1·71; p=1·58 × 10 -8 ). Expression analyses showed that the risk-associated allele at rs36196656 decreased GFRA2 mRNA concentrations in cerebellar tissue (p=0·04). No effect of rs36196656 on plasma and CSF progranulin concentrations was detected by ELISA; however, co-immunoprecipitation experiments in HEK293T cells did suggest a direct binding of progranulin and GFRA2. TMEM106B-related and GFRA2-related pathways might be future targets for treatments for FTLD, but the biological interaction between progranulin and these potential disease modifiers requires further study. TMEM106B and GFRA2 might also provide opportunities to select and stratify patients for future clinical trials and, when more is known about their potential effects, to inform genetic counselling, especially for asymptomatic individuals. National Institute on Aging, National Institute of Neurological Disorders and Stroke, Canadian Institutes of Health Research, Italian Ministry of Health, UK National Institute for Health Research, National Health and Medical Research Council of Australia, and the French National Research Agency. Copyright © 2018 Elsevier Ltd. All rights reserved.

Reduced repair capacity of a DNA clustered damage site comprised of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 2-deoxyribonolactone results in an increased mutagenic potential of these lesions

DOE PAGES

Cunniffe, Siobhan; O’Neill, Peter; Greenberg, Marc M.; ...

2014-04-01

A signature of ionizing radiation is the induction of DNA clustered damaged sites. Non-double strand break (DSB) clustered damage has been shown to compromise the base excision repair pathway, extending the lifetimes of the lesions within the cluster, compared to isolated lesions. This increases the likelihood the lesions persist to replication and thus increasing the mutagenic potential of the lesions within the cluster. Lesions formed by ionizing radiation include 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 2-deoxyribonolactone (dL). dL poses an additional challenge to the cell as it is not repaired by the short-patch base excision repair pathway. Here we show recalcitrant dL repairmore » is reflected in mutations observed when DNA containing it and a proximal 8-oxodGuo is replicated in Escherichia coli. 8-oxodGuo in close proximity to dL on the opposing DNA strand results in an enhanced frequency of mutation of the lesions within the cluster and a 20 base sequence flanking the clustered damage site in an E. coli based plasmid assay. In vitro repair of a dL lesion is reduced when compared to the repair of an abasic (AP) site and a tetrahydrofuran (THF), and this is due mainly to a reduction in the activity of polymerase β, leading to retarded FEN1 and ligase 1 activities. This study has given insights in to the biological effects of clusters containing dL.« less

Multiple Legionella pneumophila effector virulence phenotypes revealed through high-throughput analysis of targeted mutant libraries

PubMed Central

Shames, Stephanie R.; Liu, Luying; Havey, James C.; Schofield, Whitman B.; Goodman, Andrew L.; Roy, Craig R.

2017-01-01

Legionella pneumophila is the causative agent of a severe pneumonia called Legionnaires’ disease. A single strain of L. pneumophila encodes a repertoire of over 300 different effector proteins that are delivered into host cells by the Dot/Icm type IV secretion system during infection. The large number of L. pneumophila effectors has been a limiting factor in assessing the importance of individual effectors for virulence. Here, a transposon insertion sequencing technology called INSeq was used to analyze replication of a pool of effector mutants in parallel both in a mouse model of infection and in cultured host cells. Loss-of-function mutations in genes encoding effector proteins resulted in host-specific or broad virulence phenotypes. Screen results were validated for several effector mutants displaying different virulence phenotypes using genetic complementation studies and infection assays. Specifically, loss-of-function mutations in the gene encoding LegC4 resulted in enhanced L. pneumophila in the lungs of infected mice but not within cultured host cells, which indicates LegC4 augments bacterial clearance by the host immune system. The effector proteins RavY and Lpg2505 were important for efficient replication within both mammalian and protozoan hosts. Further analysis of Lpg2505 revealed that this protein functions as a metaeffector that counteracts host cytotoxicity displayed by the effector protein SidI. Thus, this study identified a large cohort of effectors that contribute to L. pneumophila virulence positively or negatively and has demonstrated regulation of effector protein activities by cognate metaeffectors as being critical for host pathogenesis. PMID:29133401

Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1

PubMed Central

Clark, Iain C.; Melnyk, Ryan A.; Youngblut, Matthew D.; Carlson, Hans K.; Iavarone, Anthony T.

2015-01-01

ABSTRACT Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. PMID:25991681

Investigation of Influenza Virus Polymerase Activity in Pig Cells

PubMed Central

Moncorgé, Olivier; Long, Jason S.; Cauldwell, Anna V.; Zhou, Hongbo; Lycett, Samantha J.

2013-01-01

Reassortant influenza viruses with combinations of avian, human, and/or swine genomic segments have been detected frequently in pigs. As a consequence, pigs have been accused of being a “mixing vessel” for influenza viruses. This implies that pig cells support transcription and replication of avian influenza viruses, in contrast to human cells, in which most avian influenza virus polymerases display limited activity. Although influenza virus polymerase activity has been studied in human and avian cells for many years by use of a minigenome assay, similar investigations in pig cells have not been reported. We developed the first minigenome assay for pig cells and compared the activities of polymerases of avian or human influenza virus origin in pig, human, and avian cells. We also investigated in pig cells the consequences of some known mammalian host range determinants that enhance influenza virus polymerase activity in human cells, such as PB2 mutations E627K, D701N, G590S/Q591R, and T271A. The two typical avian influenza virus polymerases used in this study were poorly active in pig cells, similar to what is seen in human cells, and mutations that adapt the avian influenza virus polymerase for human cells also increased activity in pig cells. In contrast, a different pattern was observed in avian cells. Finally, highly pathogenic avian influenza virus H5N1 polymerase activity was tested because this subtype has been reported to replicate only poorly in pigs. H5N1 polymerase was active in swine cells, suggesting that other barriers restrict these viruses from becoming endemic in pigs. PMID:23077313

DNA Replication Origin Function Is Promoted by H3K4 Di-methylation in Saccharomyces cerevisiae

PubMed Central

Rizzardi, Lindsay F.; Dorn, Elizabeth S.; Strahl, Brian D.; Cook, Jeanette Gowen

2012-01-01

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication. PMID:22851644

DNA replication origin function is promoted by H3K4 di-methylation in Saccharomyces cerevisiae.

PubMed

Rizzardi, Lindsay F; Dorn, Elizabeth S; Strahl, Brian D; Cook, Jeanette Gowen

2012-10-01

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.

Rapid DNA Synthesis During Early Drosophila Embryogenesis Is Sensitive to Maternal Humpty Dumpty Protein Function.

PubMed

Lesly, Shera; Bandura, Jennifer L; Calvi, Brian R

2017-11-01

Problems with DNA replication cause cancer and developmental malformations. It is not fully understood how DNA replication is coordinated with development and perturbed in disease. We had previously identified the Drosophila gene humpty dumpty ( hd ), and showed that null alleles cause incomplete DNA replication, tissue undergrowth, and lethality. Animals homozygous for the missense allele, hd 272-9 , were viable, but adult females had impaired amplification of eggshell protein genes in the ovary, resulting in the maternal effects of thin eggshells and embryonic lethality. Here, we show that expression of an hd transgene in somatic cells of the ovary rescues amplification and eggshell synthesis but not embryo viability. The germline of these mothers remain mutant for the hd 272-9 allele, resulting in reduced maternal Hd protein and embryonic arrest during mitosis of the first few S/M nuclear cleavage cycles with chromosome instability and chromosome bridges. Epistasis analysis of hd with the rereplication mutation plutonium indicates that the chromosome bridges of hd embryos are the result of a failed attempt to segregate incompletely replicated sister chromatids. This study reveals that maternally encoded Humpty dumpty protein is essential for DNA replication and genome integrity during the little-understood embryonic S/M cycles. Moreover, the two hd 272-9 maternal-effect phenotypes suggest that ovarian gene amplification and embryonic cleavage are two time periods in development that are particularly sensitive to mild deficits in DNA replication function. This last observation has broader relevance for interpreting why mild mutations in the human ortholog of humpty dumpty and other DNA replication genes cause tissue-specific malformations of microcephalic dwarfisms. Copyright © 2017 by the Genetics Society of America.

"Bad Luck Mutations": DNA Mutations Are not the Whole Answer to Understanding Cancer Risk.

PubMed

Trosko, James E; Carruba, Giuseppe

2017-01-01

It has been proposed that many human cancers are generated by intrinsic mechanisms that produce "Bad Luck" mutations by the proliferation of organ-specific adult stem cells. There have been serious challenges to this interpretation, including multiple extrinsic factors thought to be correlated with mutations found in cancers associated with these exposures. While support for both interpretations provides some validity, both interpretations ignore several concepts of the multistage, multimechanism process of carcinogenesis, namely, (1) mutations can be generated by both "errors of DNA repair" and "errors of DNA replication," during the "initiation" process of carcinogenesis; (2) "initiated" stem cells must be clonally amplified by nonmutagenic, intrinsic or extrinsic epigenetic mechanisms; (3) organ-specific stem cell numbers can be modified during in utero development, thereby altering the risk to cancer later in life; and (4) epigenetic tumor promoters are characterized by species, individual genetic-, gender-, developmental state-specificities, and threshold levels to be active; sustained and long-term exposures; and exposures in the absence of antioxidant "antipromoters." Because of the inevitability of some of the stem cells generating "initiating" mutations by either "errors of DNA repair" or "errors of DNA replication," a tumor is formed depending on the promotion phase of carcinogenesis. While it is possible to reduce our frequencies of mutagenic "initiated" cells, one can never reduce it to zero. Because of the extended period of the promotion phase of carcinogenesis, strategies to reduce the appearance of cancers must involve the interruption of the promotion of these initiated cells.

Avoidance of APOBEC3B-induced mutation by error-free lesion bypass

PubMed Central

Hoopes, James I.; Hughes, Amber L.; Hobson, Lauren A.; Cortez, Luis M.; Brown, Alexander J.

2017-01-01

Abstract APOBEC cytidine deaminases mutate cancer genomes by converting cytidines into uridines within ssDNA during replication. Although uracil DNA glycosylases limit APOBEC-induced mutation, it is unknown if subsequent base excision repair (BER) steps function on replication-associated ssDNA. Hence, we measured APOBEC3B-induced CAN1 mutation frequencies in yeast deficient in BER endonucleases or DNA damage tolerance proteins. Strains lacking Apn1, Apn2, Ntg1, Ntg2 or Rev3 displayed wild-type frequencies of APOBEC3B-induced canavanine resistance (CanR). However, strains without error-free lesion bypass proteins Ubc13, Mms2 and Mph1 displayed respective 4.9-, 2.8- and 7.8-fold higher frequency of APOBEC3B-induced CanR. These results indicate that mutations resulting from APOBEC activity are avoided by deoxyuridine conversion to abasic sites ahead of nascent lagging strand DNA synthesis and subsequent bypass by error-free template switching. We found this mechanism also functions during telomere re-synthesis, but with a diminished requirement for Ubc13. Interestingly, reduction of G to C substitutions in Ubc13-deficient strains uncovered a previously unknown role of Ubc13 in controlling the activity of the translesion synthesis polymerase, Rev1. Our results highlight a novel mechanism for error-free bypass of deoxyuridines generated within ssDNA and suggest that the APOBEC mutation signature observed in cancer genomes may under-represent the genomic damage these enzymes induce. PMID:28334887

Enhancement of the RAD51 Recombinase Activity by the Tumor Suppressor PALB2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dray, Eloise; Etchin, Julia; Wiese, Claudia

2010-08-24

Homologous recombination mediated by the RAD51 recombinase helps eliminate chromosomal lesions, such as DNA double-stranded breaks induced by radiation or arising from injured DNA replication forks. The tumor suppressors BRCA2 and PALB2 act together to deliver RAD51 to chromosomal lesions to initiate repair. Here we document a new function of PALB2 in the enhancement of RAD51's ability to form the D-loop. We show that PALB2 binds DNA and physically interacts with RAD51. Importantly, while PALB2 alone stimulates D-loop formation, a cooperative effect is seen with RAD51AP1, an enhancer of RAD51. This stimulation stems from PALB2's ability to function with RAD51more » and RAD51AP1 to assemble the synaptic complex. Our results help unveil a multi-faceted role of PALB2 in chromosome damage repair. Since PALB2 mutations can cause breast and other tumors or lead to Fanconi anemia, our findings are important for understanding the mechanism of tumor suppression in humans.« less

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

PubMed Central

Bastia, Deepak; Srivastava, Pankaj; Zaman, Shamsu; Choudhury, Malay; Mohanty, Bidyut K.; Bacal, Julien; Langston, Lance D.; Pasero, Philippe; O’Donnell, Michael E.

2016-01-01

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex. PMID:27298353

G4 motifs affect origin positioning and efficiency in two vertebrate replicators

PubMed Central

Valton, Anne-Laure; Hassan-Zadeh, Vahideh; Lema, Ingrid; Boggetto, Nicole; Alberti, Patrizia; Saintomé, Carole; Riou, Jean-François; Prioleau, Marie-Noëlle

2014-01-01

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome-wide studies in vertebrates have recently identified a consensus G-rich motif potentially able to form G-quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation. PMID:24521668

Inhibition of coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand.

PubMed

Yuan, Ji; Cheung, Paul K M; Zhang, Huifang M; Chau, David; Yang, Decheng

2005-02-01

Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic potential. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4 was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence match in the central region of the target, but mismatches were more tolerated near the 3' end than the 5' end of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new possibility for antiviral intervention.

Multiple mutant clones in blood rarely coexist

NASA Astrophysics Data System (ADS)

Dingli, David; Pacheco, Jorge M.; Traulsen, Arne

2008-02-01

Leukemias arise due to mutations in the genome of hematopoietic (blood) cells. Hematopoiesis has a multicompartment architecture, with cells exhibiting different rates of replication and differentiation. At the root of this process, one finds a small number of stem cells, and hence the description of the mutation-selection dynamics of blood cells calls for a stochastic approach. We use stochastic dynamics to investigate to which extent acquired hematopoietic disorders are associated with mutations of single or multiple genes within developing blood cells. Our analysis considers the appearance of mutations both in the stem cell compartment as well as in more committed compartments. We conclude that in the absence of genomic instability, acquired hematopoietic disorders due to mutations in multiple genes are most likely very rare events, as multiple mutations typically require much longer development times compared to those associated with a single mutation.

Synergistic interactions between the NS3(hel) and E proteins contribute to the virulence of dengue virus type 1.

PubMed

de Borba, Luana; Strottmann, Daisy M; de Noronha, Lucia; Mason, Peter W; Dos Santos, Claudia N Duarte

2012-01-01

Dengue includes a broad range of symptoms, ranging from fever to hemorrhagic fever and may occasionally have alternative clinical presentations. Many possible viral genetic determinants of the intrinsic virulence of dengue virus (DENV) in the host have been identified, but no conclusive evidence of a correlation between viral genotype and virus transmissibility and pathogenicity has been obtained. We used reverse genetics techniques to engineer DENV-1 viruses with subsets of mutations found in two different neuroadapted derivatives. The mutations were inserted into an infectious clone of DENV-1 not adapted to mice. The replication and viral production capacity of the recombinant viruses were assessed in vitro and in vivo. The results demonstrated that paired mutations in the envelope protein (E) and in the helicase domain of the NS3 (NS3(hel)) protein had a synergistic effect enhancing viral fitness in human and mosquito derived cell lines. E mutations alone generated no detectable virulence in the mouse model; however, the combination of these mutations with NS3(hel) mutations, which were mildly virulent on their own, resulted in a highly neurovirulent phenotype. The generation of recombinant viruses carrying specific E and NS3(hel) proteins mutations increased viral fitness both in vitro and in vivo by increasing RNA synthesis and viral load (these changes being positively correlated with central nervous system damage), the strength of the immune response and animal mortality. The introduction of only pairs of amino acid substitutions into the genome of a non-mouse adapted DENV-1 strain was sufficient to alter viral fitness substantially. Given current limitations to our understanding of the molecular basis of dengue neuropathogenesis, these results could contribute to the development of attenuated strains for use in vaccinations and provide insights into virus/host interactions and new information about the mechanisms of basic dengue biology.

Comparative analysis of seven viral nuclear export signals (NESs) reveals the crucial role of nuclear export mediated by the third NES consensus sequence of nucleoprotein (NP) in influenza A virus replication.

PubMed

Chutiwitoonchai, Nopporn; Kakisaka, Michinori; Yamada, Kazunori; Aida, Yoko

2014-01-01

The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES) consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ) residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4) was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues) within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function during viral replication.

Adaptive Mutations That Occurred during Circulation in Humans of H1N1 Influenza Virus in the 2009 Pandemic Enhance Virulence in Mice.

PubMed

Otte, A; Sauter, M; Daxer, M A; McHardy, A C; Klingel, K; Gabriel, G

2015-07-01

During the 2009 H1N1 influenza pandemic, infection attack rates were particularly high among young individuals who suffered from pneumonia with occasional death. Moreover, previously reported determinants of mammalian adaptation and pathogenicity were not present in 2009 pandemic H1N1 influenza A viruses. Thus, it was proposed that unknown viral factors might have contributed to disease severity in humans. In this study, we performed a comparative analysis of two clinical 2009 pandemic H1N1 strains that belong to the very early and later phases of the pandemic. We identified mutations in the viral hemagglutinin (HA) and the nucleoprotein (NP) that occurred during pandemic progression and mediate increased virulence in mice. Lethal disease outcome correlated with elevated viral replication in the alveolar epithelium, increased proinflammatory cytokine and chemokine responses, pneumonia, and lymphopenia in mice. These findings show that viral mutations that have occurred during pandemic circulation among humans are associated with severe disease in mice. In this study, novel determinants of 2009 pandemic H1N1 influenza pathogenicity were identified in the viral hemagglutinin (HA) and the nucleoprotein (NP) genes. In contrast to highly pathogenic avian influenza viruses, increased virulence in mice did not correlate with enhanced polymerase activity but with reduced activity. Lethal 2009 pandemic H1N1 infection in mice correlated with lymphopenia and severe pneumonia. These studies suggest that molecular mechanisms that mediate 2009 pandemic H1N1 influenza pathogenicity are distinct from those that mediate avian influenza virus pathogenicity in mice. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Replication-Competent Controlled Herpes Simplex Virus

PubMed Central

Bloom, David C.; Feller, Joyce; McAnany, Peterjon; Vilaboa, Nuria

2015-01-01

ABSTRACT We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCE The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without sacrificing safety. PMID:26269179

Cardiac-Targeted Transgenic Mutant Mitochondrial Enzymes

PubMed Central

Kohler, James J.; Hosseini, Seyed H.; Green, Elgin; Hoying-Brandt, Amy; Cucoranu, Ioan; Haase, Chad P.; Russ, Rodney; Srivastava, Jaya; Ivey, Kristopher; Ludaway, Tomika; Kapoor, Victor; Abuin, Allison; Shapoval, Alexsey; Santoianni, Robert; Saada, Ann; Elpeleg, Orly; Lewis, William

2009-01-01

Mitochondrial (mt) DNA biogenesis is critical to cardiac contractility. DNA polymerase gamma (pol γ) replicates mtDNA, whereas thymidine kinase 2 (TK2) monophosphorylates pyrimidines intramitochondrially. Point mutations in POLG and TK2 result in clinical diseases associated with mtDNA depletion and organ dysfunction. Pyrimidine analogs (NRTIs) inhibit Pol γ and mtDNA replication. Cardiac “dominant negative” murine transgenes (TGs; Pol γ Y955G, and TK2 H121N or I212N) defined the role of each in the heart. mtDNA abundance, histopathological features, histochemistry, mitochondrial protein abundance, morphometry, and echocardiography were determined for TGs in “2 × 2” studies with or without pyrimidine analogs. Cardiac mtDNA abundance decreased in Y955C TGs (∼50%) but increased in H121N and I212N TGs (20-70%). Succinate dehydrogenase (SDH) increased in hearts of all mutants. Ultrastructural changes occurred in Y955C and H121N TGs. Histopathology demonstrated hypertrophy in H121N, LV dilation in I212N, and both hypertrophy and dilation in Y955C TGs. Antiretrovirals increased LV mass (≈50%) for all three TGs which combined with dilation indicates cardiomyopathy. Taken together, these studies demonstrate three manifestations of cardiac dysfunction that depend on the nature of the specific mutation and antiretroviral treatment. Mutations in genes for mtDNA biogenesis increase risk for defective mtDNA replication, leading to LV hypertrophy. PMID:18446447

New Small-Molecule Inhibitors Effectively Blocking Picornavirus Replication

PubMed Central

Ford Siltz, Lauren A.; Viktorova, Ekaterina G.; Zhang, Ben; Kouiavskaia, Diana; Dragunsky, Eugenia; Chumakov, Konstantin; Isaacs, Lyle

2014-01-01

ABSTRACT Few drugs targeting picornaviruses are available, making the discovery of antivirals a high priority. Here, we identified and characterized three compounds from a library of kinase inhibitors that block replication of poliovirus, coxsackievirus B3, and encephalomyocarditis virus. Using an in vitro translation-replication system, we showed that these drugs inhibit different stages of the poliovirus life cycle. A4(1) inhibited both the formation and functioning of the replication complexes, while E5(1) and E7(2) were most effective during the formation but not the functioning step. Neither of the compounds significantly inhibited VPg uridylylation. Poliovirus resistant to E7(2) had a G5318A mutation in the 3A protein. This mutation was previously found to confer resistance to enviroxime-like compounds, which target a phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ)-dependent step in viral replication. Analysis of host protein recruitment showed that E7(2) reduced the amount of GBF1 on the replication complexes; however, the level of PI4KIIIβ remained intact. E7(2) as well as another enviroxime-like compound, GW5074, interfered with viral polyprotein processing affecting both 3C- and 2A-dependent cleavages, and the resistant G5318A mutation partially rescued this defect. Moreover, E7(2) induced abnormal recruitment to membranes of the viral proteins; thus, enviroxime-like compounds likely severely compromise the interaction of the viral polyprotein with membranes. A4(1) demonstrated partial protection from paralysis in a murine model of poliomyelitis. Multiple attempts to isolate resistant mutants in the presence of A4(1) or E5(1) were unsuccessful, showing that effective broad-spectrum antivirals could be developed on the basis of these compounds. IMPORTANCE Diverse picornaviruses can trigger multiple human maladies, yet currently, only hepatitis A virus and poliovirus can be controlled with vaccination. The development of antipicornavirus therapeutics is also facing significant difficulties because these viruses readily generate resistance to compounds targeting either viral or cellular factors. Here, we describe three novel compounds that effectively block replication of distantly related picornaviruses with minimal toxicity to cells. The compounds prevent viral RNA replication after the synthesis of the uridylylated VPg primer. Importantly, two of the inhibitors are strongly refractory to the emergence of resistant mutants, making them promising candidates for further broad-spectrum therapeutic development. Evaluation of one of the compounds in an in vivo model of poliomyelitis demonstrated partial protection from the onset of paralysis. PMID:25008939

microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure.

PubMed

Gerresheim, Gesche K; Dünnes, Nadia; Nieder-Röhrmann, Anika; Shalamova, Lyudmila A; Fricke, Markus; Hofacker, Ivo; Höner Zu Siederdissen, Christian; Marz, Manja; Niepmann, Michael

2017-02-01

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

Alteration of the carbohydrate for deoxyguanosine analogs markedly changes DNA replication fidelity, cell cycle progression and cytotoxicity

PubMed Central

O’Konek, Jessica J.; Ladd, Brendon; Flanagan, Sheryl A.; Im, Mike M.; Boucher, Paul D.; Thepsourinthone, Tico S.; Secrist, John A.; Shewach, Donna S.

2011-01-01

Nucleoside analogs are efficacious cancer chemotherapeutics due to their incorporation into tumor cell DNA. However, they exhibit vastly different antitumor efficacies, suggesting that incorporation produces divergent effects on DNA replication. Here we have evaluated the consequences of incorporation on DNA replication and its fidelity for three structurally related deoxyguanosine analogs: ganciclovir (GCV), currently in clinical trials in a suicide gene therapy approach for cancer, D-carbocyclic 2′-deoxyguanosine (CdG) and penciclovir (PCV). GCV and CdG elicited similar cytotoxicity at low concentrations, whereas PCV was 10–100-fold less cytotoxic in human tumor cells. DNA replication fidelity was evaluated using a supF plasmid-based mutation assay. Only GCV induced a dose-dependent increase in mutation frequency, predominantly GC→TA transversions, which contributed to cytotoxicity and implicated the ether oxygen in mutagenicity. Activation of mismatch repair with hydroxyurea decreased mutations but failed to repair the GC→TA transversions. GCV slowed S-phase progression and CdG also induced a G2/M block, but both drugs allowed completion of one cell cycle after drug treatment followed by cell death in the second cell cycle. In contrast, PCV induced a lengthy early S-phase block due to profound suppression of DNA synthesis, with cell death in the first cell cycle after drug treatment. These data suggest that GCV and CdG elicit superior cytotoxicity due to their effects in template DNA, whereas strong inhibition of nascent strand synthesis by PCV may protect against cytotoxicity. Nucleoside analogs based on the carbohydrate structures of GCV and CdG is a promising area for antitumor drug development. PMID:20004674

A magic bullet to specifically eliminate mutated mitochondrial genomes from patients' cells

PubMed Central

Moraes, Carlos T

2014-01-01

When mitochondrial diseases result from mutations found in the mitochondrial DNA, engineered mitochondrial-targeted nucleases such as mitochondrial-targeted zinc finger nucleases are shown to specifically eliminate the mutated molecules, leaving the wild-type mitochondrial DNA intact to replicate and restore normal copy number. In this issue, Gammage and colleagues successfully apply this improved technology on patients' cells with two types of genetic alterations responsible for neuropathy ataxia and retinitis pigmentosa (NARP) syndrome and Kearns Sayre syndrome and progressive external ophthalmoplegia (PEO). PMID:24623377

DNA--How Sweet It Is!

ERIC Educational Resources Information Center

Banta, Linda

1997-01-01

Describes how to use various types of licorice ropes to illustrate the similarities between RNA and DNA. Specifically addresses illustrations of replication, transcription, protein synthesis, mitosis, meiosis, mutations, and anomalies. (DDR)

DNA polymerase-α regulates type I interferon activation through cytosolic RNA:DNA synthesis

PubMed Central

Starokadomskyy, Petro; Gemelli, Terry; Rios, Jonathan J.; Xing, Chao; Wang, Richard C.; Li, Haiying; Pokatayev, Vladislav; Dozmorov, Igor; Khan, Shaheen; Miyata, Naoteru; Fraile, Guadalupe; Raj, Prithvi; Xu, Zhe; Xu, Zigang; Ma, Lin; Lin, Zhimiao; Wang, Huijun; Yang, Yong; Ben-Amitai, Dan; Orenstein, Naama; Mussaffi, Huda; Baselga, Eulalia; Tadini, Gianluca; Grunebaum, Eyal; Sarajlija, Adrijan; Krzewski, Konrad; Wakeland, Edward K.; Yan, Nan; de la Morena, Maria Teresa; Zinn, Andrew R.; Burstein, Ezra

2016-01-01

Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations disrupting nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts expression of POLA1, the gene encoding the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency results in increased type I interferon production. This enzyme is necessary for RNA:DNA primer synthesis during DNA replication and strikingly, POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Altogether, this work identified POLA1 as a critical regulator of the type I interferon response. PMID:27019227

Inactivation of Hepatitis B Virus Replication in Cultured Cells and In Vivo with Engineered Transcription Activator-Like Effector Nucleases

PubMed Central

Bloom, Kristie; Ely, Abdullah; Mussolino, Claudio; Cathomen, Toni; Arbuthnot, Patrick

2013-01-01

Chronic hepatitis B virus (HBV) infection remains an important global health problem. Stability of the episomal covalently closed circular HBV DNA (cccDNA) is largely responsible for the modest curative efficacy of available therapy. Since licensed anti-HBV drugs have a post-transcriptional mechanism of action, disabling cccDNA is potentially of therapeutic benefit. To develop this approach, we engineered mutagenic transcription activator-like effector nucleases (TALENs) that target four HBV-specific sites within the viral genome. TALENs with cognate sequences in the S or C open-reading frames (ORFs) efficiently disrupted sequences at the intended sites and suppressed markers of viral replication. Following triple transfection of cultured HepG2.2.15 cells under mildly hypothermic conditions, the S TALEN caused targeted mutation in ~35% of cccDNA molecules. Markers of viral replication were also inhibited in vivo in a murine hydrodynamic injection model of HBV replication. HBV target sites within S and C ORFs of the injected HBV DNA were mutated without evidence of toxicity. These findings are the first to demonstrate a targeted nuclease-mediated disruption of HBV cccDNA. Efficacy in vivo also indicates that these engineered nucleases have potential for use in treatment of chronic HBV infection. PMID:23883864

Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing.

PubMed

Laun, Peter; Bruschi, Carlo V; Dickinson, J Richard; Rinnerthaler, Mark; Heeren, Gino; Schwimbersky, Richard; Rid, Raphaela; Breitenbach, Michael

2007-01-01

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast.

Yeast mother cell-specific ageing, genetic (in)stability, and the somatic mutation theory of ageing

PubMed Central

Laun, Peter; Bruschi, Carlo V.; Dickinson, J. Richard; Rinnerthaler, Mark; Heeren, Gino; Schwimbersky, Richard; Rid, Raphaela; Breitenbach, Michael

2007-01-01

Yeast mother cell-specific ageing is characterized by a limited capacity to produce daughter cells. The replicative lifespan is determined by the number of cell cycles a mother cell has undergone, not by calendar time, and in a population of cells its distribution follows the Gompertz law. Daughter cells reset their clock to zero and enjoy the full lifespan characteristic for the strain. This kind of replicative ageing of a cell population based on asymmetric cell divisions is investigated as a model for the ageing of a stem cell population in higher organisms. The simple fact that the daughter cells can reset their clock to zero precludes the accumulation of chromosomal mutations as the cause of ageing, because semiconservative replication would lead to the same mutations in the daughters. However, nature is more complicated than that because, (i) the very last daughters of old mothers do not reset the clock; and (ii) mutations in mitochondrial DNA could play a role in ageing due to the large copy number in the cell and a possible asymmetric distribution of damaged mitochondrial DNA between mother and daughter cell. Investigation of the loss of heterozygosity in diploid cells at the end of their mother cell-specific lifespan has shown that genomic rearrangements do occur in old mother cells. However, it is not clear if this kind of genomic instability is causative for the ageing process. Damaged material other than DNA, for instance misfolded, oxidized or otherwise damaged proteins, seem to play a major role in ageing, depending on the balance between production and removal through various repair processes, for instance several kinds of proteolysis and autophagy. We are reviewing here the evidence for genetic change and its causality in the mother cell-specific ageing process of yeast. PMID:17986449

Mutation rates among RNA viruses

PubMed Central

Drake, John W.; Holland, John J.

1999-01-01

The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viruses (specifically in riboviruses rather than retroviruses) was constrained by the quality and quantity of available measurements and by the lack of a specific theoretical framework for converting mutation frequencies into mutation rates in this group of organisms. Here, we describe a simple relation between ribovirus mutation frequencies and mutation rates, apply it to the best (albeit far from satisfactory) available data, and observe a central value for the mutation rate per genome per replication of μg ≈ 0.76. (The rate per round of cell infection is twice this value or about 1.5.) This value is so large, and ribovirus genomes are so informationally dense, that even a modest increase extinguishes the population. PMID:10570172

The Role of Viral, Host, and Secondary Bacterial Factors in Influenza Pathogenesis

PubMed Central

Kash, John C.; Taubenberger, Jeffery K.

2016-01-01

Influenza A virus infections in humans generally cause self-limited infections, but can result in severe disease, secondary bacterial pneumonias, and death. Influenza viruses can replicate in epithelial cells throughout the respiratory tree and can cause tracheitis, bronchitis, bronchiolitis, diffuse alveolar damage with pulmonary edema and hemorrhage, and interstitial and airspace inflammation. The mechanisms by which influenza infections result in enhanced disease, including development of pneumonia and acute respiratory distress, are multifactorial, involving host, viral, and bacterial factors. Host factors that enhance risk of severe influenza disease include underlying comorbidities, such as cardiac and respiratory disease, immunosuppression, and pregnancy. Viral parameters enhancing disease risk include polymerase mutations associated with host switch and adaptation, viral proteins that modulate immune and antiviral responses, and virulence factors that increase disease severity, which can be especially prominent in pandemic viruses and some zoonotic influenza viruses causing human infections. Influenza viral infections result in damage to the respiratory epithelium that facilitates secondary infection with common bacterial pneumopathogens and can lead to secondary bacterial pneumonias that greatly contribute to respiratory distress, enhanced morbidity, and death. Understanding the molecular mechanisms by which influenza and secondary bacterial infections, coupled with the role of host risk factors, contribute to enhanced morbidity and mortality is essential to develop better therapeutic strategies to treat severe influenza. PMID:25747532

Abnormal, Error-Prone Bypass of Photoproducts by Xeroderma Pigmentosum Variant Cell Extracts Results in Extreme Strand Bias for the Kinds of Mutations Induced by UV Light

PubMed Central

McGregor, W. Glenn; Wei, Dong; Maher, Veronica M.; McCormick, J. Justin

1999-01-01

Xeroderma pigmentosum (XP) is a rare genetic disease characterized by a greatly increased susceptibility to sunlight-induced skin cancer. Cells from the majority of patients are defective in nucleotide excision repair. However, cells from one set of patients, XP variants, exhibit normal repair but are abnormally slow in replicating DNA containing UV photoproducts. The frequency of UV radiation-induced mutations in the XP variant cells is significantly higher than that in normal human cells. Furthermore, the kinds of UV-induced mutations differ very significantly from normal. Instead of transitions, mainly C→T, 30% of the base substitutions consist of C→A transversions, all arising from photoproducts located in one strand. Mutations involving cytosine in the other strand are almost all C→T transitions. Forty-five percent of the substitutions involve thymine, and the majority are transversions. To test the hypothesis that the UV hypermutability and the abnormal spectrum of mutations result from abnormal bypass of photoproducts in DNA, we compared extracts from XP variant cells with those from HeLa cells and a fibroblast cell strain, MSU-1.2, for the ability to replicate a UV-irradiated form I M13 phage. The M13 template contains a simian virus 40 origin of replication located directly to the left or to the right of the target gene, lacZα, so that the template for the leading and lagging strands of DNA replication is defined. Reduction of replication to ∼37% of the control value required only 1 photoproduct per template for XP variant cell extracts, but ∼2.2 photoproducts for HeLa or MSU-1.2 cell extracts. The frequency of mutants induced was four times higher with XP variant cell extracts than with HeLa or MSU-1.2 cell extracts. With XP variant cell extracts, the proportion of C→A transversions reached as high as 43% with either M13 template and arose from photoproducts located in the template for leading-strand synthesis; with HeLa or MSU-1.2 cell extracts, this value was only 5%, and these arose from photoproducts in either strand. With the XP variant extracts, 26% of the substitutions involved thymine, and virtually all were T→A transversions. Sequence analysis of the coding region of the catalytic subunit of DNA polymerase delta in XP variant cell lines revealed two polymorphisms, but these do not account for the reduced bypass fidelity. Our data indicate that the UV hypermutability of XP variant cells results from reduced bypass fidelity and that unlike for normal cells, bypass of photoproducts involving cytosine in the template for the leading strand differs significantly from that of photoproducts in the lagging strand. PMID:9858539

Rapid evolution of the human mutation spectrum

PubMed Central

Harris, Kelley; Pritchard, Jonathan K

2017-01-01

DNA is a remarkably precise medium for copying and storing biological information. This high fidelity results from the action of hundreds of genes involved in replication, proofreading, and damage repair. Evolutionary theory suggests that in such a system, selection has limited ability to remove genetic variants that change mutation rates by small amounts or in specific sequence contexts. Consistent with this, using SNV variation as a proxy for mutational input, we report here that mutational spectra differ substantially among species, human continental groups and even some closely related populations. Close examination of one signal, an increased TCC→TTC mutation rate in Europeans, indicates a burst of mutations from about 15,000 to 2000 years ago, perhaps due to the appearance, drift, and ultimate elimination of a genetic modifier of mutation rate. Our results suggest that mutation rates can evolve markedly over short evolutionary timescales and suggest the possibility of mapping mutational modifiers. DOI: http://dx.doi.org/10.7554/eLife.24284.001 PMID:28440220

Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases

PubMed Central

Lada, Artem G.; Stepchenkova, Elena I.; Zhuk, Anna S.; Kliver, Sergei F.; Rogozin, Igor B.; Polev, Dmitrii E.; Dhar, Alok; Pavlov, Youri I.

2017-01-01

DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters (kataegis). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by APOBECs in cell’s history and to acquisition of new mutations near original break. PMID:29312434

Recombination Is Responsible for the Increased Recovery of Drug-Resistant Mutants with Hypermutated Genomes in Resting Yeast Diploids Expressing APOBEC Deaminases.

PubMed

Lada, Artem G; Stepchenkova, Elena I; Zhuk, Anna S; Kliver, Sergei F; Rogozin, Igor B; Polev, Dmitrii E; Dhar, Alok; Pavlov, Youri I

2017-01-01

DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters ( kataegis ). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by APOBECs in cell's history and to acquisition of new mutations near original break.

Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

PubMed

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

Requirement of RecBC enzyme and an elevated level of activated RecA for induced stable DNA replication in Escherichia coli.

PubMed Central

Magee, T R; Kogoma, T

1990-01-01

During SOS induction, Escherichia coli cells acquire the ability to replicate DNA in the absence of protein synthesis, i.e., induced stable DNA replication (iSDR). Initiation of iSDR can occur in the absence of transcription and DnaA protein activity, which are both required for initiation of normal DNA replication at the origin of replication, oriC. In this study we examined the requirement of recB, recC, and recA for the induction and maintenance of iSDR. We found that recB and recC mutations blocked the induction of iSDR by UV irradiation and nalidixic acid treatment. In recB(Ts) strains, iSDR activity induced at 30 degrees C was inhibited by subsequent incubation at 42 degrees C. In addition, iSDR that was induced after heat activation of the RecA441 protein was abolished by the recB21 mutation. These results indicated that the RecBC enzyme was essential not only for SOS signal generation but also for the reinitiation of DNA synthesis following DNA damage. recAo(Con) lexA3(Ind-) strains were found to be capable of iSDR after nalidixic acid treatment, indicating that the derepression of the recA gene and the activation of the elevated level of RecA protein were the necessary and sufficient conditions for the induction of iSDR. PMID:2180906

Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

PubMed Central

Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Suárez, Cristina; Salas, José

2013-01-01

The function of the African swine fever virus (ASFV) reparative DNA polymerase, Pol X, was investigated in the context of virus infection. Pol X is a late structural protein that localizes at cytoplasmic viral factories during DNA replication. Using an ASFV deletion mutant lacking the Pol X gene, we have shown that Pol X is not required for virus growth in Vero cells or swine macrophages under one-step growth conditions. However, at a low multiplicity of infection, when multiple rounds of replication occur, the growth of the mutant virus is impaired in swine macrophages but not in Vero cells, suggesting that Pol X is needed to repair the accumulated DNA damage. The replication of the mutant virus in Vero cells presents sensitivity to oxidative damage, and mutational analysis of viral DNA shows that deletion of Pol X results in an increase in the mutation frequency in macrophages. Therefore, our data reveal a biological role for ASFV Pol X in the context of the infected cell in the preservation of viral genetic information. PMID:23824796

Human-Specific Adaptations in Vpu Conferring Anti-tetherin Activity Are Critical for Efficient Early HIV-1 Replication In Vivo.

PubMed

Yamada, Eri; Nakaoka, Shinji; Klein, Lukas; Reith, Elisabeth; Langer, Simon; Hopfensperger, Kristina; Iwami, Shingo; Schreiber, Gideon; Kirchhoff, Frank; Koyanagi, Yoshio; Sauter, Daniel; Sato, Kei

2018-01-10

The HIV-1-encoded accessory protein Vpu exerts several immunomodulatory functions, including counteraction of the host restriction factor tetherin, downmodulation of CD4, and inhibition of NF-κB activity to facilitate HIV-1 infection. However, the relative contribution of individual Vpu functions to HIV-1 infection in vivo remained unclear. Here, we used a humanized mouse model and HIV-1 strains with selective mutations in vpu to demonstrate that the anti-tetherin activity of Vpu is a prerequisite for efficient viral spread during the early phase of infection. Mathematical modeling and gain-of-function mutations in SIVcpz, the simian precursor of pandemic HIV-1, corroborate this finding. Blockage of interferon signaling combined with transcriptome analyses revealed that basal tetherin levels are sufficient to control viral replication. These results establish tetherin as a key effector of the intrinsic immune defense against HIV-1, and they demonstrate that Vpu-mediated tetherin antagonism is critical for efficient viral spread during the initial phase of HIV-1 replication. Copyright © 2017 Elsevier Inc. All rights reserved.

RPA-Mediated Recruitment of the E3 Ligase RFWD3 Is Vital for Interstrand Crosslink Repair and Human Health.

PubMed

Feeney, Laura; Muñoz, Ivan M; Lachaud, Christophe; Toth, Rachel; Appleton, Paul L; Schindler, Detlev; Rouse, John

2017-06-01

Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Analysis of recombinant H7N9 wild-type and mutant viruses in pigs shows that the Q226L mutation in HA is important for transmission.

PubMed

Liu, Qinfang; Zhou, Bin; Ma, Wenjun; Bawa, Bhupinder; Ma, Jingjiao; Wang, Wei; Lang, Yuekun; Lyoo, Young; Halpin, Rebecca A; Lin, Xudong; Stockwell, Timothy B; Webby, Richard; Wentworth, David E; Richt, Juergen A

2014-07-01

The fact that there have been more than 300 human infections with a novel avian H7N9 virus in China indicates that this emerging strain has pandemic potential. Furthermore, many of the H7N9 viruses circulating in animal reservoirs contain putative mammalian signatures in the HA and PB2 genes that are believed to be important in the adaptation of other avian strains to humans. To date, the definitive roles of these mammalian-signature substitutions in transmission and pathogenesis of H7N9 viruses remain unclear. To address this we analyzed the biological characteristics, pathogenicity, and transmissibility of A/Anhui/1/2013 (H7N9) virus and variants in vitro and in vivo using a synthetically created wild-type virus (rAnhui-WT) and two mutants (rAnhui-HA-226Q and rAnhui-PB2-627E). All three viruses replicated in lungs of intratracheally inoculated pigs, yet nasal shedding was limited. The rAnhui-WT and rAnhui-PB2-627E viruses were transmitted to contact animals. In contrast, the rAnhui-HA-226Q virus was not transmitted to sentinel pigs. Deep sequencing of viruses from the lungs of infected pigs identified substitutions arising in the viral population (e.g., PB2-T271A, PB2-D701N, HA-V195I, and PB2-E627K reversion) that may enhance viral replication in pigs. Collectively, the results demonstrate that critical mutations (i.e., HA-Q226L) enable the H7N9 viruses to be transmitted in a mammalian host and suggest that the myriad H7N9 genotypes circulating in avian species in China and closely related strains (e.g., H7N7) have the potential for further adaptation to human or other mammalian hosts (e.g., pigs), leading to strains capable of sustained human-to-human transmission. Importance: The genomes of the zoonotic avian H7N9 viruses emerging in China have mutations in critical genes (PB2-E627K and HA-Q226L) that may be important in their pandemic potential. This study shows that (i) HA-226L of zoonotic H7N9 strains is critical for binding the α-2,6-linked receptor and enables transmission in pigs; (ii) wild-type A/Anhui/1/2013 (H7N9) shows modest replication, virulence, and transmissibility in pigs, suggesting that it is not well adapted to the mammalian host; and (iii) both wild-type and variant H7N9 viruses rapidly develop additional mammalian-signature mutations in pigs, indicating that they represent an important potential intermediate host. This is the first study analyzing the phenotypic effects of specific mutations within the HA and PB2 genes of the novel H7N9 viruses created by reverse genetics in an important mammalian host model. Finally, this study illustrates that loss-of-function mutations can be used to effectively identify residues critical to zoonosis/transmission. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Integrative Genomics and Transcriptomics Analysis Reveals Potential Mechanisms for Favorable Prognosis of Patients with HPV-Positive Head and Neck Carcinomas

PubMed Central

Zhang, Wensheng; Edwards, Andrea; Fang, Zhide; Flemington, Erik K.; Zhang, Kun

2016-01-01

Patients with HPV-positive head neck squamous cell carcinomas (HNSCC) usually have a better prognosis than the HPV-negative cases while the underlying mechanism remains far from being well understood. We investigated this issue by an integrative analysis of clinically-annotated multi-omics HNSCC data released by the Cancer Genome Atlas. As confirmatory results, we found: (1) Co-occurrence of mutant TP53 and HPV infection was rare; (2) Regardless of HPV status, HNSCCs of wild-type TP53 implied a good survival chance for patients and had fewer genome-wide somatic mutations than those with a mutation burden on the gene. Our analysis further led to some novel observations. They included: (1) The genes involved in “DNA mismatch repair” pathway were up-regulated in HPV-positive tumors compared to normal tissue samples and HPV-negative cases, and thus constituted a strong predictive signature for the identification of HPV infection; (2) HPV infection could disrupt some regulatory miRNA-mRNA correlations operational in the HPV-negative tumors. In light of these results, we proposed a hypothesis for the favorable clinical outcomes of HPV-positive HNSCC patients. That is, the replication of HPV genome and/or its invasion into the genomes of cancer cells may enhance DNA repair mechanisms, which in turn limit the accumulation of lethal somatic mutations. PMID:27108969

Genetic evolution of Human Enterovirus A71 subgenotype C4 in Shenzhen, China, 1998-2013.

PubMed

He, Yaqing; Zou, Linjie; Chong, Marc Ka Chun; Men, Ruoting; Xu, Wenbo; Yang, Hong; Yao, Xiangjie; Chen, Long; Xian, Huixia; Zhang, Hailong; Luo, Min; Cheng, Jinquan; Ma, Hanwu; Feng, Qianjin; Huang, Yun; Wang, Yujie; Yeoh, Eng-Kiong; Zee, Benny Chung-Ying; Zhou, Yuanping; He, Ming-Liang; Wang, Maggie Haitian

2016-06-01

Human Enterovirus A71 (EV-A71) is one of the severest enteroviruses that causes hand, foot, and mouth disease (HFMD) among children. This study identified the mutations of EV-A71 VP1 amino acid residues over a number of years and explored the possible association of identified mutations and HFMD epidemic outbreaks in Shenzhen, China. A total of 3760 stool specimens were collected from HFMD patients by Shenzhen Centers for Disease Control and Prevention (CDC) between 1998 and 2013. In total 289 VP1 strains were sequenced in this study, and amino acids mutation frequency was calculated. There were 2040 China nationwide sequences downloaded from Genebank as replication data. In our samples, 1036 subjects (27.6%) were EV-A71 infected. Three amino acid positions on VP1 protein were found to have high mutation prevalence. These are Q22H, S283T, and A289H. Site 22 showed a fast mutation fixation in the year 2008, at the time of the large scale epidemic outbreak in Shenzhen. Analysis of the nationwide data replicated the same trend of mutation prevalence of the three sites. The switching from Q to H on site 22 of the EV-A71 VP1 strain might be associated with the HFMD outbreak in Shenzhen in 2008. The identified amino acid sites 22, 283 and 289 provided information for developing anti-viral drugs against EV-A71 in the future. Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Use of mutation spectra analysis software.

PubMed

Rogozin, I; Kondrashov, F; Glazko, G

2001-02-01

The study and comparison of mutation(al) spectra is an important problem in molecular biology, because these spectra often reflect on important features of mutations and their fixation. Such features include the interaction of DNA with various mutagens, the function of repair/replication enzymes, and properties of target proteins. It is known that mutability varies significantly along nucleotide sequences, such that mutations often concentrate at certain positions, called "hotspots," in a sequence. In this paper, we discuss in detail two approaches for mutation spectra analysis: the comparison of mutation spectra with a HG-PUBL program, (FTP: sunsite.unc.edu/pub/academic/biology/dna-mutations/hyperg) and hotspot prediction with the CLUSTERM program (www.itba.mi.cnr.it/webmutation; ftp.bionet.nsc.ru/pub/biology/dbms/clusterm.zip). Several other approaches for mutational spectra analysis, such as the analysis of a target protein structure, hotspot context revealing, multiple spectra comparisons, as well as a number of mutation databases are briefly described. Mutation spectra in the lacI gene of E. coli and the human p53 gene are used for illustration of various difficulties of such analysis. Copyright 2001 Wiley-Liss, Inc.

Characterizing in vivo stability and potential interactions of a UL5 helicase-primase mutation previously shown to reduce virulence and in vivo replication of Marek's disease virus

USDA-ARS?s Scientific Manuscript database

The unpredictable yet recurrent emergence of more virulent field strains of Marek’s disease virus (MDV) in Marek’s disease (MD) vaccinated flocks of chickens has prompted concerns regarding the sustainability of MD vaccines. A single non-synonymous point mutation (I682R) within the UL5 helicase-prim...

Alteration of Mature Nucleocapsid and Enhancement of Covalently Closed Circular DNA Formation by Hepatitis B Virus Core Mutants Defective in Complete-Virion Formation.

PubMed

Cui, Xiuji; Luckenbaugh, Laurie; Bruss, Volker; Hu, Jianming

2015-10-01

Assembly of hepatitis B virus (HBV) begins with packaging of the pregenomic RNA (pgRNA) into immature nucleocapsids (NC), which are converted to mature NCs containing the genomic relaxed circular (RC) DNA as a result of reverse transcription. Mature NCs have two alternative fates: (i) envelopment by viral envelope proteins, leading to secretion extracellularly as virions, or (ii) disassembly (uncoating) to deliver their RC DNA content into the host cell nucleus for conversion to the covalently closed circular (CCC) DNA, the template for viral transcription. How these two alternative fates are regulated remains to be better understood. The NC shell is composed of multiple copies of a single viral protein, the HBV core (HBc) protein. HBc mutations located on the surface of NC have been identified that allow NC maturation but block its envelopment. The potential effects of some of these mutations on NC uncoating and CCC DNA formation have been analyzed by transfecting HBV replication constructs into hepatoma cells. All envelopment-defective HBc mutations tested were competent for CCC DNA formation, indicating that core functions in envelopment and uncoating/nuclear delivery of RC DNA were genetically separable. Some of the envelopment-defective HBc mutations were found to alter specifically the integrity of mature, but not immature, NCs such that RC DNA became susceptible to nuclease digestion. Furthermore, CCC DNA formation could be enhanced by NC surface mutations that did or did not significantly affect mature NC integrity, indicating that the NC surface residues may be closely involved in NC uncoating and/or nuclear delivery of RC DNA. Hepatitis B virus (HBV) infection is a major health issue worldwide. HBV assembly begins with the packaging into immature nucleocapsids (NCs) of a viral RNA pregenome, which is converted to the DNA genome in mature NCs. Mature NCs are then selected for envelopment and secretion as complete-virion particles or, alternatively, can deliver their DNA to the host cell nucleus to maintain the viral genome as nuclear episomes, which are the basis for virus persistence. Previous studies have identified mutations on the capsid surface that selectively block NC envelopment without affecting NC maturation. We have now discovered that some of the same mutations result in preferential alteration of mature NCs and increased viral nuclear episomes. These findings provide important new insights into the regulation of the two alternative fates of mature NCs and suggest new ways to perturb viral persistence by manipulating levels of viral nuclear episomes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak.

PubMed

Dietzel, Erik; Schudt, Gordian; Krähling, Verena; Matrosovich, Mikhail; Becker, Stephan

2017-01-15

The Ebola virus (EBOV) outbreak in West Africa started in December 2013, claimed more than 11,000 lives, threatened to destabilize a whole region, and showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and disease remains obscure. In this study, we investigated the functional significance of three nonsynonymous mutations that emerged early during the West African EBOV outbreak. Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three mutations: a D759G substitution in the active center of the L polymerase, an A82V substitution in the receptor binding domain of surface glycoprotein GP, and an R111C substitution in the self-assembly domain of RNA-encapsidating nucleoprotein NP. Using a newly developed virus-like particle system and reverse genetics, we found that the mutations have an impact on the functions of the respective viral proteins and on the growth of recombinant EBOVs. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth advantage over the prototype isolate Makona C7 lacking the mutations. This study showed that virus variants with improved fitness emerged early during the West African EBOV outbreak. The dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral L polymerase, surface glycoprotein GP, and nucleocapsid protein NP emerged, were fixed early in the outbreak, and were found in almost 90% of the sequences. Here we showed that these mutations affected the functional activity of viral proteins and improved viral growth in cell culture. Our results demonstrate emergence of adaptive changes in the Ebola virus genome during virus circulation in humans and prompt further studies on the potential role of these changes in virus transmissibility and pathogenicity. Copyright © 2017 American Society for Microbiology.

The proteasomal Rpn11 metalloprotease suppresses tombusvirus RNA recombination and promotes viral replication via facilitating assembly of the viral replicase complex.

PubMed

Prasanth, K Reddisiva; Barajas, Daniel; Nagy, Peter D

2015-03-01

RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92(pol) replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. RNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cyclin-dependent kinase regulates the length of S phase through TICRR/TRESLIN phosphorylation.

PubMed

Sansam, Courtney G; Goins, Duane; Siefert, Joseph C; Clowdus, Emily A; Sansam, Christopher L

2015-03-01

S-phase cyclin-dependent kinases (CDKs) stimulate replication initiation and accelerate progression through the replication timing program, but it is unknown which CDK substrates are responsible for these effects. CDK phosphorylation of the replication factor TICRR (TopBP1-interacting checkpoint and replication regulator)/TRESLIN is required for DNA replication. We show here that phosphorylated TICRR is limiting for S-phase progression. Overexpression of a TICRR mutant with phosphomimetic mutations at two key CDK-phosphorylated residues (TICRR(TESE)) stimulates DNA synthesis and shortens S phase by increasing replication initiation. This effect requires the TICRR region that is necessary for its interaction with MDM two-binding protein. Expression of TICRR(TESE) does not grossly alter the spatial organization of replication forks in the nucleus but does increase replication clusters and the number of replication forks within each cluster. In contrast to CDK hyperactivation, the acceleration of S-phase progression by TICRR(TESE) does not induce DNA damage. These results show that CDK can stimulate initiation and compress the replication timing program by phosphorylating a single protein, suggesting a simple mechanism by which S-phase length is controlled. © 2015 Sansam et al.; Published by Cold Spring Harbor Laboratory Press.

Increased Functional Stability and Homogeneity of Viral Envelope Spikes through Directed Evolution

PubMed Central

Leaman, Daniel P.; Zwick, Michael B.

2013-01-01

The functional HIV-1 envelope glycoprotein (Env) trimer, the target of anti-HIV-1 neutralizing antibodies (Abs), is innately labile and coexists with non-native forms of Env. This lability and heterogeneity in Env has been associated with its tendency to elicit non-neutralizing Abs. Here, we use directed evolution to overcome instability and heterogeneity of a primary Env spike. HIV-1 virions were subjected to iterative cycles of destabilization followed by replication to select for Envs with enhanced stability. Two separate pools of stable Env variants with distinct sequence changes were selected using this method. Clones isolated from these viral pools could withstand heat, denaturants and other destabilizing conditions. Seven mutations in Env were associated with increased trimer stability, primarily in the heptad repeat regions of gp41, but also in V1 of gp120. Combining the seven mutations generated a variant Env with superior homogeneity and stability. This variant spike moreover showed resistance to proteolysis and to dissociation by detergent. Heterogeneity within the functional population of hyper-stable Envs was also reduced, as evidenced by a relative decrease in a proportion of virus that is resistant to the neutralizing Ab, PG9. The latter result may reflect a change in glycans on the stabilized Envs. The stabilizing mutations also increased the proportion of secreted gp140 existing in a trimeric conformation. Finally, several Env-stabilizing substitutions could stabilize Env spikes from HIV-1 clades A, B and C. Spike stabilizing mutations may be useful in the development of Env immunogens that stably retain native, trimeric structure. PMID:23468626

Compartmentalized self-replication under fast PCR cycling conditions yields Taq DNA polymerase mutants with increased DNA-binding affinity and blood resistance.

PubMed

Arezi, Bahram; McKinney, Nancy; Hansen, Connie; Cayouette, Michelle; Fox, Jeffrey; Chen, Keith; Lapira, Jennifer; Hamilton, Sarah; Hogrefe, Holly

2014-01-01

Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd ) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.

A single R36Q mutation in the matrix protein of pigeon paramyxovirus type 1 reduces virus replication and shedding in pigeons.

PubMed

Xu, Haixu; Song, Qingqing; Zhu, Jie; Liu, Jiajia; Cheng, Xin; Hu, Shunlin; Wu, Shuang; Wang, Xiaoquan; Liu, Xiaowen; Liu, Xiufan

2016-07-01

Pigeon paramyxovirus type 1 (PPMV-1) is considered an antigenic and variant of avian paramyxovirus type 1 (APMV-1) that has adapted to pigeons as hosts. However, how this host-specific adaption of PPMV-1 is related to its biological characteristics is unknown. In this study, seven unique amino acids in PPMV-1 that are not present in other APMV-1 strains (n = 39 versus n = 106) were identified. R36 of the M protein was found to be not only a unique amino acid but also a positive-selection site. To investigate the role of R36 in host adaptation, a recombinant PPMV-1 with R36Q mutation was constructed. Our results indicated that the an R36Q mutation significantly attenuates pathogenicity in chickens, viral growth in both chicken embryo fibroblasts (CEFs) and pigeon embryo fibroblasts (PEFs), and virus replication and shedding in pigeons in comparison with the wild-type virus, suggesting that R36 is a key residue that evolved during the adaptation of PPMV-1 in pigeons.

Telomere length maintenance--an ALTernative mechanism.

PubMed

Royle, N J; Foxon, J; Jeyapalan, J N; Mendez-Bermudez, A; Novo, C L; Williams, J; Cotton, V E

2008-01-01

The Alternative Lengthening of Telomeres (ALT) mechanism is utilised by approximately 10% of human tumours and a higher proportion of some types of sarcomas. ALT+ cell lines and tumours show heterogeneous telomere length, extra-chromosomal circular and linear telomeric DNA, ALT associated promyelocytic bodies (APBs), a high frequency of post-replication exchanges in telomeres (designated as telomere-sister chromatid exchanges, T-SCE) and high instability at a GC-rich minisatellite, MS32 (D1S8). It is clear that there is a link between the minisatellite instability and the mechanism that underpins ALT, however currently the nature of this relationship is uncertain. Single molecule analysis of telomeric DNA from ALT+ cell lines and tumours has revealed complex telomere mutations that have not been seen in cell lines or tumours that express telomerase. These complex telomere mutations cannot be explained by T-SCE but must arise by another inter-molecular process. The break-induced replication (BIR) model that may explain the observed high frequency of T-SCE and the presence of complex telomere mutations is reviewed. Copyright 2008 S. Karger AG, Basel.

Hepatitis B "e" antigen-mediated inhibition of HBV replication fitness and transcription efficiency in vitro.

PubMed

Samal, Jasmine; Kandpal, Manish; Vivekanandan, Perumal

2015-10-01

A mutation at nucleotide 1896 (G1896A) is the most common cause for the loss of HBeAg. In contrast to clinical data, cell culture studies report a high-replicating phenotype for the G1896A mutant. Differences between the wild-type and the G1896A mutant in early steps of HBV replication including the synthesis of pre-genomic RNA and transcripts have not been investigated. The G1896A mutant is associated with higher replication fitness, transcription efficiency and higher levels of secreted HBsAg than the wild-type. Interestingly, trans-complementation of the G1896A mutant with HBeAg lowers the replication fitness and transcriptionefficiency to levels comparable to that of the wild-type. Our results highlight the role of HBeAg in modulating the early steps in HBV replication. In sum, our findings highlight the role of HBeAg in regulating hepatitis B virus replication fitness and transcription efficiency and new insights on the early steps of replication in the G1896A mutant. Copyright © 2015 Elsevier Inc. All rights reserved.

High throughput generation and characterization of replication-competent clade C transmitter-founder simian human immunodeficiency viruses

PubMed Central

Dutta, Debashis; Johnson, Samuel; Dalal, Alisha; Deymier, Martin J.; Hunter, Eric

2018-01-01

Traditional restriction endonuclease-based cloning has been routinely used to generate replication-competent simian-human immunodeficiency viruses (SHIV) and simian tropic HIV (stHIV). This approach requires the existence of suitable restriction sites or the introduction of nucleotide changes to create them. Here, using an In-Fusion cloning technique that involves homologous recombination, we generated SHIVs and stHIVs based on epidemiologically linked clade C transmitted/founder HIV molecular clones from Zambia. Replacing vif from these HIV molecular clones with vif of SIVmac239 resulted in chimeric genomes used to generate infectious stHIV viruses. Likewise, exchanging HIV env genes and introducing N375 mutations to enhance macaque CD4 binding site and cloned into a SHIVAD8-EO backbone. The generated SHIVs and stHIV were infectious in TZMbl and ZB5 cells, as well as macaque PBMCs. Therefore, this method can replace traditional methods and be a valuable tool for the rapid generation and testing of molecular clones of stHIV and SHIV based on primary clinical isolates will be valuable to generate rapid novel challenge viruses for HIV vaccine/cure studies. PMID:29758076

A Constant Rate of Spontaneous Mutation in DNA-Based Microbes

NASA Astrophysics Data System (ADS)

Drake, John W.

1991-08-01

In terms of evolution and fitness, the most significant spontaneous mutation rate is likely to be that for the entire genome (or its nonfrivolous fraction). Information is now available to calculate this rate for several DNA-based haploid microbes, including bacteriophages with single- or double-stranded DNA, a bacterium, a yeast, and a filamentous fungus. Their genome sizes vary by ≈6500-fold. Their average mutation rates per base pair vary by ≈16,000-fold, whereas their mutation rates per genome vary by only ≈2.5-fold, apparently randomly, around a mean value of 0.0033 per DNA replication. The average mutation rate per base pair is inversely proportional to genome size. Therefore, a nearly invariant microbial mutation rate appears to have evolved. Because this rate is uniform in such diverse organisms, it is likely to be determined by deep general forces, perhaps by a balance between the usually deleterious effects of mutation and the physiological costs of further reducing mutation rates.

Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus.

PubMed

Li, Xiao-Dan; Deng, Cheng-Lin; Ye, Han-Qing; Zhang, Hong-Lei; Zhang, Qiu-Yan; Chen, Dong-Dong; Zhang, Pan-Tao; Shi, Pei-Yong; Yuan, Zhi-Ming; Zhang, Bo

2016-06-15

Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly. Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Systematic analysis of viral genes responsible for differential virulence between American and Australian West Nile virus strains.

PubMed

Setoh, Yin Xiang; Prow, Natalie A; Rawle, Daniel J; Tan, Cindy Si En; Edmonds, Judith H; Hall, Roy A; Khromykh, Alexander A

2015-06-01

A variant Australian West Nile virus (WNV) strain, WNVNSW2011, emerged in 2011 causing an unprecedented outbreak of encephalitis in horses in south-eastern Australia. However, no human cases associated with this strain have yet been reported. Studies using mouse models for WNV pathogenesis showed that WNVNSW2011 was less virulent than the human-pathogenic American strain of WNV, New York 99 (WNVNY99). To identify viral genes and mutations responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, we constructed chimeric viruses with substitution of large genomic regions coding for the structural genes, non-structural genes and untranslated regions, as well as seven individual non-structural gene chimeras, using a modified circular polymerase extension cloning method. Our results showed that the complete non-structural region of WNVNSW2011, when substituted with that of WNVNY99, significantly enhanced viral replication and the ability to suppress type I IFN response in cells, resulting in higher virulence in mice. Analysis of the individual non-structural gene chimeras showed a predominant contribution of WNVNY99 NS3 to increased virus replication and evasion of IFN response in cells, and to virulence in mice. Other WNVNY99 non-structural proteins (NS2A, NS4B and NS5) were shown to contribute to the modulation of IFN response. Thus a combination of non-structural proteins, likely NS2A, NS3, NS4B and NS5, is primarily responsible for the difference in virulence between WNVNSW2011 and WNVNY99 strains, and accumulative mutations within these proteins would likely be required for the Australian WNVNSW2011 strain to become significantly more virulent. © 2015 The Authors.

Deubiquitinase function of arterivirus papain-like protease 2 suppresses the innate immune response in infected host cells.

PubMed

van Kasteren, Puck B; Bailey-Elkin, Ben A; James, Terrence W; Ninaber, Dennis K; Beugeling, Corrine; Khajehpour, Mazdak; Snijder, Eric J; Mark, Brian L; Kikkert, Marjolein

2013-02-26

Protein ubiquitination regulates important innate immune responses. The discovery of viruses encoding deubiquitinating enzymes (DUBs) suggests they remove ubiquitin to evade ubiquitin-dependent antiviral responses; however, this has never been conclusively demonstrated in virus-infected cells. Arteriviruses are economically important positive-stranded RNA viruses that encode an ovarian tumor (OTU) domain DUB known as papain-like protease 2 (PLP2). This enzyme is essential for arterivirus replication by cleaving a site within the viral replicase polyproteins and also removes ubiquitin from cellular proteins. To dissect this dual specificity, which relies on a single catalytic site, we determined the crystal structure of equine arteritis virus PLP2 in complex with ubiquitin (1.45 Å). PLP2 binds ubiquitin using a zinc finger that is uniquely integrated into an exceptionally compact OTU-domain fold that represents a new subclass of zinc-dependent OTU DUBs. Notably, the ubiquitin-binding surface is distant from the catalytic site, which allowed us to mutate this surface to significantly reduce DUB activity without affecting polyprotein cleavage. Viruses harboring such mutations exhibited WT replication kinetics, confirming that PLP2-mediated polyprotein cleavage was intact, but the loss of DUB activity strikingly enhanced innate immune signaling. Compared with WT virus infection, IFN-β mRNA levels in equine cells infected with PLP2 mutants were increased by nearly an order of magnitude. Our findings not only establish PLP2 DUB activity as a critical factor in arteriviral innate immune evasion, but the selective inactivation of DUB activity also opens unique possibilities for developing improved live attenuated vaccines against arteriviruses and other viruses encoding similar dual-specificity proteases.

Mutalisk: a web-based somatic MUTation AnaLyIS toolKit for genomic, transcriptional and epigenomic signatures.

PubMed

Lee, Jongkeun; Lee, Andy Jinseok; Lee, June-Koo; Park, Jongkeun; Kwon, Youngoh; Park, Seongyeol; Chun, Hyonho; Ju, Young Seok; Hong, Dongwan

2018-05-22

Somatic genome mutations occur due to combinations of various intrinsic/extrinsic mutational processes and DNA repair mechanisms. Different molecular processes frequently generate different signatures of somatic mutations in their own favored contexts. As a result, the regional somatic mutation rate is dependent on the local DNA sequence, the DNA replication/RNA transcription dynamics and epigenomic chromatin organization landscape in the genome. Here, we propose an online computational framework, termed Mutalisk, which correlates somatic mutations with various genomic, transcriptional and epigenomic features in order to understand mutational processes that contribute to the generation of the mutations. This user-friendly tool explores the presence of localized hypermutations (kataegis), dissects the spectrum of mutations into the maximum likelihood combination of known mutational signatures and associates the mutation density with numerous regulatory elements in the genome. As a result, global patterns of somatic mutations in any query sample can be efficiently screened, thus enabling a deeper understanding of various mutagenic factors. This tool will facilitate more effective downstream analyses of cancer genome sequences to elucidate the diversity of mutational processes underlying the development and clonal evolution of cancer cells. Mutalisk is freely available at http://mutalisk.org.

Fragile DNA Motifs Trigger Mutagenesis at Distant Chromosomal Loci in Saccharomyces cerevisiae

PubMed Central

Saini, Natalie; Zhang, Yu; Nishida, Yuri; Sheng, Ziwei; Choudhury, Shilpa; Mieczkowski, Piotr; Lobachev, Kirill S.

2013-01-01

DNA sequences capable of adopting non-canonical secondary structures have been associated with gross-chromosomal rearrangements in humans and model organisms. Previously, we have shown that long inverted repeats that form hairpin and cruciform structures and triplex-forming GAA/TTC repeats induce the formation of double-strand breaks which trigger genome instability in yeast. In this study, we demonstrate that breakage at both inverted repeats and GAA/TTC repeats is augmented by defects in DNA replication. Increased fragility is associated with increased mutation levels in the reporter genes located as far as 8 kb from both sides of the repeats. The increase in mutations was dependent on the presence of inverted or GAA/TTC repeats and activity of the translesion polymerase Polζ. Mutagenesis induced by inverted repeats also required Sae2 which opens hairpin-capped breaks and initiates end resection. The amount of breakage at the repeats is an important determinant of mutations as a perfect palindromic sequence with inherently increased fragility was also found to elevate mutation rates even in replication-proficient strains. We hypothesize that the underlying mechanism for mutagenesis induced by fragile motifs involves the formation of long single-stranded regions in the broken chromosome, invasion of the undamaged sister chromatid for repair, and faulty DNA synthesis employing Polζ. These data demonstrate that repeat-mediated breaks pose a dual threat to eukaryotic genome integrity by inducing chromosomal aberrations as well as mutations in flanking genes. PMID:23785298

Synthetic and Evolutionary Construction of a Chlorate-Reducing Shewanella oneidensis MR-1.

PubMed

Clark, Iain C; Melnyk, Ryan A; Youngblut, Matthew D; Carlson, Hans K; Iavarone, Anthony T; Coates, John D

2015-05-19

Despite evidence for the prevalence of horizontal gene transfer of respiratory genes, little is known about how pathways functionally integrate within new hosts. One example of a mobile respiratory metabolism is bacterial chlorate reduction, which is frequently encoded on composite transposons. This implies that the essential components of the metabolism are encoded on these mobile elements. To test this, we heterologously expressed genes for chlorate reduction from Shewanella algae ACDC in the non-chlorate-reducing Shewanella oneidensis MR-1. The construct that ultimately endowed robust growth on chlorate included cld, a cytochrome c gene, clrABDC, and two genes of unknown function. Although strain MR-1 was unable to grow on chlorate after initial insertion of these genes into the chromosome, 11 derived strains capable of chlorate respiration were obtained through adaptive evolution. Genome resequencing indicated that all of the evolved chlorate-reducing strains replicated a large genomic region containing chlorate reduction genes. Contraction in copy number and loss of the ability to reduce chlorate were also observed, indicating that this phenomenon was extremely dynamic. Although most strains contained more than six copies of the replicated region, a single strain with less duplication also grew rapidly. This strain contained three additional mutations that we hypothesized compensated for the low copy number. We remade the mutations combinatorially in the unevolved strain and determined that a single nucleotide polymorphism (SNP) upstream of cld enabled growth on chlorate and was epistatic to a second base pair change in the NarP binding sequence between narQP and nrfA that enhanced growth. The ability of chlorate reduction composite transposons to form functional metabolisms after transfer to a new host is an important part of their propagation. To study this phenomenon, we engineered Shewanella oneidensis MR-1 into a chlorate reducer. We defined a set of genes sufficient to endow growth on chlorate from a plasmid, but found that chromosomal insertion of these genes was nonfunctional. Evolution of this inoperative strain into a chlorate reducer showed that tandem duplication was a dominant mechanism of activation. While copy number changes are a relatively rapid way of increasing gene dosage, replicating almost 1 megabase of extra DNA is costly. Mutations that alleviate the need for high copy number are expected to arise and eventually predominate, and we identified a single nucleotide polymorphism (SNP) that relieved the copy number requirement. This study uses both rational and evolutionary approaches to gain insight into the evolution of a fascinating respiratory metabolism. Copyright © 2015 Clark et al.

A reverse genetics system for the Great Lakes strain of viral hemorrhagic septicemia virus: the NV gene is required for pathogenicity

USGS Publications Warehouse

Ammayappan, Arun; Kurath, Gael; Thompson, Tarin M.; Vakharia, Vikram N.

2011-01-01

Viral hemorrhagic septicemia virus (VHSV), belonging to the genus Novirhabdovirus in the family of Rhabdoviridae, causes a highly contagious disease of fresh and saltwater fish worldwide. Recently, a novel genotype of VHSV, designated IVb, has invaded the Great Lakes in North America, causing large-scale epidemics in wild fish. An efficient reverse genetics system was developed to generate a recombinant VHSV of genotype IVb from cloned cDNA. The recombinant VHSV (rVHSV) was comparable to the parental wild-type strain both in vitro and in vivo, causing high mortality in yellow perch (Perca flavescens). A modified recombinant VHSV was generated in which the NV gene was substituted with an enhanced green fluorescent protein gene (rVHSV-ΔNV-EGFP), and another recombinant was made by inserting the EGFP gene into the full-length viral clone between the P and M genes (rVHSV-EGFP). The in vitro replication kinetics of rVHSV-EGFP was similar to rVHSV; however, the rVHSV-ΔNV-EGFP grew 2 logs lower. In yellow perch challenges, wtVHSV and rVHSV induced 82-100% cumulative per cent mortality (CPM), respectively, whereas rVHSV-EGFP produced 62% CPM and rVHSV-ΔNV-EGFP caused only 15% CPM. No reversion of mutation was detected in the recovered viruses and the recombinant viruses stably maintained the foreign gene after several passages. These results indicate that the NV gene of VHSV is not essential for viral replication in vitro and in vivo, but it plays an important role in viral replication efficiency and pathogenicity. This system will facilitate studies of VHSV replication, virulence, and production of viral vectored vaccines.

The Aspergillus nidulans npkA gene encodes a Cdc2-related kinase that genetically interacts with the UvsBATR kinase.

PubMed Central

Fagundes, Marcia R V Z Kress; Lima, Joel Fernandes; Savoldi, Marcela; Malavazi, Iran; Larson, Roy E; Goldman, Maria H S; Goldman, Gustavo H

2004-01-01

The DNA damage response is a protective mechanism that ensures the maintenance of genomic integrity. We have used Aspergillus nidulans as a model system to characterize the DNA damage response caused by the antitopoisomerase I drug, camptothecin. We report the molecular characterization of a p34Cdc2-related gene, npkA, from A. nidulans. The npkA gene is transcriptionally induced by camptothecin and other DNA-damaging agents, and its induction in the presence of camptothecin is dependent on the uvsBATR gene. There were no growth defects, changes in developmental patterns, increased sensitivity to DNA-damaging agents, or effects on septation or growth rate in the A. nidulans npkA deletion strain. However, the DeltanpkA mutation can partially suppress HU sensitivity caused by the DeltauvsBATR and uvsD153ATRIP checkpoint mutations. We demonstrated that the A. nidulans uvsBATR gene is involved in DNA replication and the intra-S-phase checkpoints and that the DeltanpkA mutation can suppress its intra-S-phase checkpoint deficiency. There is a defect in both the intra-S-phase and DNA replication checkpoints due to the npkA inactivation when DNA replication is slowed at 6 mm HU. Our results suggest that the npkA gene plays a role in cell cycle progression during S-phase as well as in a DNA damage signal transduction pathway in A. nidulans. PMID:15342504

Trans-Lesion DNA Polymerases May Be Involved in Yeast Meiosis

PubMed Central

Arbel-Eden, Ayelet; Joseph-Strauss, Daphna; Masika, Hagit; Printzental, Oxana; Rachi, Eléanor; Simchen, Giora

2013-01-01

Trans-lesion DNA polymerases (TLSPs) enable bypass of DNA lesions during replication and are also induced under stress conditions. Being only weakly dependent on their template during replication, TLSPs introduce mutations into DNA. The low processivity of these enzymes ensures that they fall off their template after a few bases are synthesized and are then replaced by the more accurate replicative polymerase. We find that the three TLSPs of budding yeast Saccharomyces cerevisiae Rev1, PolZeta (Rev3 and Rev7), and Rad30 are induced during meiosis at a time when DNA double-strand breaks (DSBs) are formed and homologous chromosomes recombine. Strains deleted for one or any combination of the three TLSPs undergo normal meiosis. However, in the triple-deletion mutant, there is a reduction in both allelic and ectopic recombination. We suggest that trans-lesion polymerases are involved in the processing of meiotic double-strand breaks that lead to mutations. In support of this notion, we report significant yeast two-hybrid (Y2H) associations in meiosis-arrested cells between the TLSPs and DSB proteins Rev1-Spo11, Rev1-Mei4, and Rev7-Rec114, as well as between Rev1 and Rad30. We suggest that the involvement of TLSPs in processing of meiotic DSBs could be responsible for the considerably higher frequency of mutations reported during meiosis compared with that found in mitotically dividing cells, and therefore may contribute to faster evolutionary divergence than previously assumed. PMID:23550131

Translational animal models of autism and neurodevelopmental disorders.

PubMed

Crawley, Jacqueline N

2012-09-01

Autism is a neurodevelopmental disorder whose diagnosis is based on three behavioral criteria: unusual reciprocal social interactions, deficits in communication, and stereotyped repetitive behaviors with restricted interests. A large number of de novo single gene mutations and chromosomal deletions are associated with autism spectrum disorders. Based on the strong genetic evidence, mice with targeted mutations in homologous genes have been generated as translational research tools. Mouse models of autism have revealed behavioral and biological outcomes of mutations in risk genes. The field is now poised to employ the most robust phenotypes in the most replicable mouse models for preclinical screening of novel therapeutics.

Methadone enhances human influenza A virus replication.

PubMed

Chen, Yun-Hsiang; Wu, Kuang-Lun; Tsai, Ming-Ta; Chien, Wei-Hsien; Chen, Mao-Liang; Wang, Yun

2017-01-01

Growing evidence has indicated that opioids enhance replication of human immunodeficiency virus and hepatitis C virus in target cells. However, it is unknown whether opioids can enhance replication of other clinically important viral pathogens. In this study, the interaction of opioid agonists and human influenza A/WSN/33 (H1N1) virus was examined in human lung epithelial A549 cells. Cells were exposed to morphine, methadone or buprenorphine followed by human H1N1 viral infection. Exposure to methadone differentially enhanced viral propagation, consistent with an increase in virus adsorption, susceptibility to virus infection and viral protein synthesis. In contrast, morphine or buprenorphine did not alter H1N1 replication. Because A549 cells do not express opioid receptors, methadone-enhanced H1N1 replication in human lung cells may not be mediated through these receptors. The interaction of methadone and H1N1 virus was also examined in adult mice. Treatment with methadone significantly increased H1N1 viral replication in lungs. Our data suggest that use of methadone facilitates influenza A viral infection in lungs and might raise concerns regarding the possible consequence of an increased risk of serious influenza A virus infection in people who receive treatment in methadone maintenance programs. © 2015 Society for the Study of Addiction.

A novel dysfunctional germline P53 mutation identified in a family with Li-Fraumeni syndrome.

PubMed

Ji, Min; Wang, Lin; Shao, Yuguo; Cao, Wei; Xu, Ting; Chen, Shujie; Wang, Zhiwei; He, Qi; Yang, Kuo

2018-01-01

Li-Fraumeni Syndrome (LFS), which is a rare dominantly inherited cancer predisposition syndrome, is associated with germline P53 mutations. Mutations of the tumor suppressor protein P53 are associated with more than 50% of human cancers; however, almost 30% of P53 mutations occur rarely and this has raised questions about their significance. It therefore appeared of particular interest that we identified a novel mutation in a patient suffering from breast cancer and fulfilling the diagnostic criteria of LFS. In this study, a patient with remarkable family history developed breast cancer and was diagnosed with LFS. By performing next-generation sequencing on the patient and subsequent verification by Sanger sequencing among other family members, a new germ-line P53 replication error, a trinucleotide repeat mutation in the coding region, was identified in two generations of this Li-Fraumeni family.

A novel Werner Syndrome mutation: pharmacological treatment by read-through of nonsense mutations and epigenetic therapies

PubMed Central

Agrelo, Ruben; Sutz, Miguel Arocena; Setien, Fernando; Aldunate, Fabian; Esteller, Manel; Da Costa, Valeria; Achenbach, Ricardo

2015-01-01

Werner Syndrome (WS) is a rare inherited disease characterized by premature aging and increased propensity for cancer. Mutations in the WRN gene can be of several types, including nonsense mutations, leading to a truncated protein form. WRN is a RecQ family member with both helicase and exonuclease activities, and it participates in several cell metabolic pathways, including DNA replication, DNA repair, and telomere maintenance. Here, we reported a novel homozygous WS mutation (c.3767 C > G) in 2 Argentinian brothers, which resulted in a stop codon and a truncated protein (p.S1256X). We also observed increased WRN promoter methylation in the cells of patients and decreased messenger WRN RNA (WRN mRNA) expression. Finally, we showed that the read-through of nonsense mutation pharmacologic treatment with both aminoglycosides (AGs) and ataluren (PTC-124) in these cells restores full-length protein expression and WRN functionality. PMID:25830902

Elevated germline mutation rate in teenage fathers.

PubMed

Forster, Peter; Hohoff, Carsten; Dunkelmann, Bettina; Schürenkamp, Marianne; Pfeiffer, Heidi; Neuhuber, Franz; Brinkmann, Bernd

2015-03-22

Men age and die, while cells in their germline are programmed to be immortal. To elucidate how germ cells maintain viable DNA despite increasing parental age, we analysed DNA from 24 097 parents and their children, from Europe, the Middle East and Africa. We chose repetitive microsatellite DNA that mutates (unlike point mutations) only as a result of cellular replication, providing us with a natural 'cell-cycle counter'. We observe, as expected, that the overall mutation rate for fathers is seven times higher than for mothers. Also as expected, mothers have a low and lifelong constant DNA mutation rate. Surprisingly, however, we discover that (i) teenage fathers already set out from a much higher mutation rate than teenage mothers (potentially equivalent to 77-196 male germline cell divisions by puberty); and (ii) ageing men maintain sperm DNA quality similar to that of teenagers, presumably by using fresh batches of stem cells known as 'A-dark spermatogonia'.

MLH1 mutations differentially affect meiotic functions in Saccharomyces cerevisiae.

PubMed Central

Hoffmann, Eva R; Shcherbakova, Polina V; Kunkel, Thomas A; Borts, Rhona H

2003-01-01

To test whether missense mutations in the cancer susceptibility gene MLH1 adversely affect meiosis, we examined 14 yeast MLH1 mutations for effects on meiotic DNA transactions and gamete viability in the yeast Saccharomyces cerevisiae. Mutations analogous to those associated with hereditary nonpolyposis colorectal cancer (HNPCC) or those that reduce Mlh1p interactions with ATP or DNA all impair replicative mismatch repair as measured by increased mutation rates. However, their effects on meiotic heteroduplex repair, crossing over, chromosome segregation, and gametogenesis vary from complete loss of meiotic functions to no meiotic defect, and mutants defective in one meiotic process are not necessarily defective in others. DNA binding and ATP binding but not ATP hydrolysis are required for meiotic crossing over. The results reveal clear separation of different Mlh1p functions in mitosis and meiosis, and they suggest that some, but not all, MLH1 mutations may be a source of human infertility. PMID:12618391

Determinants of monovalent oral polio vaccine mutagenesis in vaccinated elderly people.

PubMed

Boot, Hein J; Sonsma, Jan; van Nunen, Femke; Abbink, Frithjofna; Kimman, Tjeerd G; Buisman, Anne-Marie

2007-06-11

Live oral poliovirus vaccine (OPV) strains can mutate and recombine during replication in the host. Trivalent OPV has long been used to restrain wild-type poliovirus in developing countries. However, recently WHO advocates using monovalent OPV (mOPV) to finally eradicate poliovirus world-wide. We analysed polioviruses recovered from the faeces of 101 elderly patients (divided into three groups by immune status) challenged with mOPV-1 or mOPV-3. A high number of nucleotide mutations was found in the viral capsid-protein-encoding regions. Some of these mutations caused amino acid changes in or near regions with neutralizing epitopes, especially in mOPV-1-derived strains. The quantities of mutations in recovered poliovirus strains correlated with prevaccination immune status (seronegatives have more mutations) and excretion duration. Duration of excretion appears to be the dominant factor for the accumulation of mutations in mOPV-derived strains in vaccinated elderly people.

Loss-of-heterozygosity facilitates passage through Haldane's sieve for Saccharomyces cerevisiae undergoing adaptation.

PubMed

Gerstein, A C; Kuzmin, A; Otto, S P

2014-05-07

Haldane's sieve posits that the majority of beneficial mutations that contribute to adaptation should be dominant, as these are the mutations most likely to establish and spread when rare. It has been argued, however, that if the dominance of mutations in their current and previous environments are correlated, Haldane's sieve could be eliminated. We constructed heterozygous lines of Saccharomyces cerevisiae containing single adaptive mutations obtained during exposure to the fungicide nystatin. Here we show that no clear dominance relationship exists across environments: mutations exhibited a range of dominance levels in a rich medium, yet were exclusively recessive under nystatin stress. Surprisingly, heterozygous replicates exhibited variable-onset rapid growth when exposed to nystatin. Targeted Sanger sequencing demonstrated that loss-of-heterozygosity (LOH) accounted for these growth patterns. Our experiments demonstrate that recessive beneficial mutations can avoid Haldane's sieve in clonal organisms through rapid LOH and thus contribute to rapid evolutionary adaptation.

The HARP domain dictates the annealing helicase activity of HARP/SMARCAL1.

PubMed

Ghosal, Gargi; Yuan, Jingsong; Chen, Junjie

2011-06-01

Mutations in HepA-related protein (HARP, or SMARCAL1) cause Schimke immunoosseous dysplasia (SIOD). HARP has ATP-dependent annealing helicase activity, which helps to stabilize stalled replication forks and facilitate DNA repair during replication. Here, we show that the conserved tandem HARP (2HP) domain dictates this annealing helicase activity. Furthermore, chimeric proteins generated by fusing the 2HP domain of HARP with the SNF2 domain of BRG1 or HELLS show annealing helicase activity in vitro and, when targeted to replication forks, mimic the functions of HARP in vivo. We propose that the HARP domain endows HARP with this ATP-driven annealing helicase activity.

Quasispecies in population of compositional assemblies.

PubMed

Gross, Renan; Fouxon, Itzhak; Lancet, Doron; Markovitch, Omer

2014-12-30

The quasispecies model refers to information carriers that undergo self-replication with errors. A quasispecies is a steady-state population of biopolymer sequence variants generated by mutations from a master sequence. A quasispecies error threshold is a minimal replication accuracy below which the population structure breaks down. Theory and experimentation of this model often refer to biopolymers, e.g. RNA molecules or viral genomes, while its prebiotic context is often associated with an RNA world scenario. Here, we study the possibility that compositional entities which code for compositional information, intrinsically different from biopolymers coding for sequential information, could show quasispecies dynamics. We employed a chemistry-based model, graded autocatalysis replication domain (GARD), which simulates the network dynamics within compositional molecular assemblies. In GARD, a compotype represents a population of similar assemblies that constitute a quasi-stationary state in compositional space. A compotype's center-of-mass is found to be analogous to a master sequence for a sequential quasispecies. Using single-cycle GARD dynamics, we measured the quasispecies transition matrix (Q) for the probabilities of transition from one center-of-mass Euclidean distance to another. Similarly, the quasispecies' growth rate vector (A) was obtained. This allowed computing a steady state distribution of distances to the center of mass, as derived from the quasispecies equation. In parallel, a steady state distribution was obtained via the GARD equation kinetics. Rewardingly, a significant correlation was observed between the distributions obtained by these two methods. This was only seen for distances to the compotype center-of-mass, and not to randomly selected compositions. A similar correspondence was found when comparing the quasispecies time dependent dynamics towards steady state. Further, changing the error rate by modifying basal assembly joining rate of GARD kinetics was found to display an error catastrophe, similar to the standard quasispecies model. Additional augmentation of compositional mutations leads to the complete disappearance of the master-like composition. Our results show that compositional assemblies, as simulated by the GARD formalism, portray significant attributes of quasispecies dynamics. This expands the applicability of the quasispecies model beyond sequence-based entities, and potentially enhances validity of GARD as a model for prebiotic evolution.

Frequency-dependent selection can lead to evolution of high mutation rates.

PubMed

Rosenbloom, Daniel I S; Allen, Benjamin

2014-05-01

Theoretical and experimental studies have shown that high mutation rates can be advantageous, especially in novel or fluctuating environments. Here we examine how frequency-dependent competition may lead to fluctuations in trait frequencies that exert upward selective pressure on mutation rates. We use a mathematical model to show that cyclical trait dynamics generated by "rock-paper-scissors" competition can cause the mutation rate in a population to converge to a high evolutionarily stable mutation rate, reflecting a trade-off between generating novelty and reproducing past success. Introducing recombination lowers the evolutionarily stable mutation rate but allows stable coexistence between mutation rates above and below the evolutionarily stable rate. Even considering strong mutational load and ignoring the costs of faithful replication, evolution favors positive mutation rates if the selective advantage of prevailing in competition exceeds the ratio of recombining to nonrecombining offspring. We discuss a number of genomic mechanisms that may meet our theoretical requirements for the adaptive evolution of mutation. Overall, our results suggest that local mutation rates may be higher on genes influencing cyclical competition and that global mutation rates in asexual species may be higher in populations subject to strong cyclical competition.

Steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis.

PubMed

Mandl, Christian W

2005-08-01

Tick-borne encephalitis virus (TBEV) is an important human pathogen that causes severe neurological illness in large areas of Europe and Asia. The neuropathogenesis of this disease agent is determined by its capacity to enter the central nervous system (CNS) after peripheral inoculation ("neuroinvasiveness") and its ability to replicate and cause damage within the CNS ("neurovirulence"). TBEV is a small, enveloped flavivirus with an unsegmented, positive-stranded RNA genome. Mutations affecting various steps of its natural replication cycle were shown to influence its neuropathogenic properties. This review describes experimental approaches and summarizes results on molecular determinants of neurovirulence and neuroinvasiveness that have been identified for this virus. It focuses on molecular mechanisms of three particular steps of the viral life cycle that have been studied in some detail for TBEV and two closely related tick-borne flaviviruses (Louping ill virus (LIV) and Langat virus (LGTV)), namely (i) the envelope protein E and its role in viral attachment to the cell surface, (ii) the 3'-noncoding region of the genome and its importance for viral RNA replication, and (iii) the capsid protein C and its role in the assembly process of infectious virus particles. Mutations affecting each of these three molecular targets significantly influence neuropathogenesis of TBEV, particularly its neuroinvasiveness. The understanding of molecular determinants of TBEV neuropathogenesis is relevant for vaccine development, also against other flaviviruses.

Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae.

PubMed

Lindstrom, Derek L; Leverich, Christina K; Henderson, Kiersten A; Gottschling, Daniel E

2011-03-01

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.

A minimal threshold of FANCJ helicase activity is required for its response to replication stress or double-strand break repair.

PubMed

Bharti, Sanjay Kumar; Sommers, Joshua A; Awate, Sanket; Bellani, Marina A; Khan, Irfan; Bradley, Lynda; King, Graeme A; Seol, Yeonee; Vidhyasagar, Venkatasubramanian; Wu, Yuliang; Abe, Takuye; Kobayashi, Koji; Shin-Ya, Kazuo; Kitao, Hiroyuki; Wold, Marc S; Branzei, Dana; Neuman, Keir C; Brosh, Robert M

2018-05-21

Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj-/- cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj-/- cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers

PubMed Central

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-01-01

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks. PMID:26563448

Mutagenic consequences of a single G-quadruplex demonstrate mitotic inheritance of DNA replication fork barriers.

PubMed

Lemmens, Bennie; van Schendel, Robin; Tijsterman, Marcel

2015-11-13

Faithful DNA replication is vital to prevent disease-causing mutations, chromosomal aberrations and malignant transformation. However, accuracy conflicts with pace and flexibility and cells rely on specialized polymerases and helicases to ensure effective and timely replication of genomes that contain DNA lesions or secondary structures. If and how cells can tolerate a permanent barrier to replication is, however, unknown. Here we show that a single unresolved G-quadruplexed DNA structure can persist through multiple mitotic divisions without changing conformation. Failed replication across a G-quadruplex causes single-strand DNA gaps that give rise to DNA double-strand breaks in subsequent cell divisions, which are processed by polymerase theta (POLQ)-mediated alternative end joining. Lineage tracing experiments further reveal that persistent G-quadruplexes cause genetic heterogeneity during organ development. Our data demonstrate that a single lesion can cause multiple unique genomic rearrangements, and that alternative end joining enables cells to proliferate in the presence of mitotically inherited replication blocks.

Nuclear Mitochondrial DNA Activates Replication in Saccharomyces cerevisiae

PubMed Central

Chatre, Laurent; Ricchetti, Miria

2011-01-01

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in. PMID:21408151

Nuclear mitochondrial DNA activates replication in Saccharomyces cerevisiae.

PubMed

Chatre, Laurent; Ricchetti, Miria

2011-03-08

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in.

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

PubMed

Elia, Andrew E H; Wang, David C; Willis, Nicholas A; Boardman, Alexander P; Hajdu, Ildiko; Adeyemi, Richard O; Lowry, Elizabeth; Gygi, Steven P; Scully, Ralph; Elledge, Stephen J

2015-10-15

We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Poly-(ADP-ribose)-polymerase inhibitors as radiosensitizers: a systematic review of pre-clinical and clinical human studies.

PubMed

Lesueur, Paul; Chevalier, François; Austry, Jean-Baptiste; Waissi, Waisse; Burckel, Hélène; Noël, Georges; Habrand, Jean-Louis; Saintigny, Yannick; Joly, Florence

2017-09-15

Poly-(ADP-Ribose)-Polymerase (PARP) inhibitors are becoming important actors of anti-neoplasic agents landscape, with recent but narrow FDA's approvals for ovarian BRCA mutated cancers and prostatic cancer. Nevertheless, PARP inhibitors are also promising drugs for combined treatments particularly with radiotherapy. More than seven PARP inhibitors have been currently developed. Central Role of PARP in DNA repair, makes consider PARP inhibitor as potential radiosensitizers, especially for tumors with DNA repair defects, such as BRCA mutation, because of synthetic lethality. Furthermore the replication-dependent activity of PARP inhibitor helps to maintain the differential effect between tumoral and healthy tissues. Inhibition of chromatin remodeling, G2/M arrest, vasodilatory effect induced by PARP inhibitor, also participate to their radio-sensitization effect. Here, after highlighting mechanisms of PARP inhibitors radiosensitization we methodically searched PubMed, Google Scholar, Cochrane Databases and meeting proceedings for human pre-clinical and clinical studies that evaluated PARP inhibitor radiosensitizing effect. Enhancement ratio, when available, was systematically reported. Sixty four studies finally met our selection criteria and were included in the analysis. Only three pre-clinical studies didn't find any radiosensitizing effect. Median enhancement ratio vary from 1,3 for prostate tumors to 1,5 for lung cancers. Nine phase I or II trials assessed safety data. PARP inhibitors are promising radiosensitizers, but need more clinical investigation. The next ten years will be determining for judging their real potential.

MMS Exposure Promotes Increased MtDNA Mutagenesis in the Presence of Replication-Defective Disease-Associated DNA Polymerase γ Variants

PubMed Central

Stumpf, Jeffrey D.; Copeland, William C.

2014-01-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities. PMID:25340760

MMS exposure promotes increased MtDNA mutagenesis in the presence of replication-defective disease-associated DNA polymerase γ variants.

PubMed

Stumpf, Jeffrey D; Copeland, William C

2014-10-01

Mitochondrial DNA (mtDNA) encodes proteins essential for ATP production. Mutant variants of the mtDNA polymerase cause mutagenesis that contributes to aging, genetic diseases, and sensitivity to environmental agents. We interrogated mtDNA replication in Saccharomyces cerevisiae strains with disease-associated mutations affecting conserved regions of the mtDNA polymerase, Mip1, in the presence of the wild type Mip1. Mutant frequency arising from mtDNA base substitutions that confer erythromycin resistance and deletions between 21-nucleotide direct repeats was determined. Previously, increased mutagenesis was observed in strains encoding mutant variants that were insufficient to maintain mtDNA and that were not expected to reduce polymerase fidelity or exonuclease proofreading. Increased mutagenesis could be explained by mutant variants stalling the replication fork, thereby predisposing the template DNA to irreparable damage that is bypassed with poor fidelity. This hypothesis suggests that the exogenous base-alkylating agent, methyl methanesulfonate (MMS), would further increase mtDNA mutagenesis. Mitochondrial mutagenesis associated with MMS exposure was increased up to 30-fold in mip1 mutants containing disease-associated alterations that affect polymerase activity. Disrupting exonuclease activity of mutant variants was not associated with increased spontaneous mutagenesis compared with exonuclease-proficient alleles, suggesting that most or all of the mtDNA was replicated by wild type Mip1. A novel subset of C to G transversions was responsible for about half of the mutants arising after MMS exposure implicating error-prone bypass of methylated cytosines as the predominant mutational mechanism. Exposure to MMS does not disrupt exonuclease activity that suppresses deletions between 21-nucleotide direct repeats, suggesting the MMS-induce mutagenesis is not explained by inactivated exonuclease activity. Further, trace amounts of CdCl2 inhibit mtDNA replication but suppresses MMS-induced mutagenesis. These results suggest a novel mechanism wherein mutations that lead to hypermutation by DNA base-damaging agents and associate with mitochondrial disease may contribute to previously unexplained phenomena, such as the wide variation of age of disease onset and acquired mitochondrial toxicities.

Toward a theory of multilevel evolution: long-term information integration shapes the mutational landscape and enhances evolvability.

PubMed

Hogeweg, Paulien

2012-01-01

Most of evolutionary theory has abstracted away from how information is coded in the genome and how this information is transformed into traits on which selection takes place. While in the earliest stages of biological evolution, in the RNA world, the mapping from the genotype into function was largely predefined by the physical-chemical properties of the evolving entities (RNA replicators, e.g. from sequence to folded structure and catalytic sites), in present-day organisms, the mapping itself is the result of evolution. I will review results of several in silico evolutionary studies which examine the consequences of evolving the genetic coding, and the ways this information is transformed, while adapting to prevailing environments. Such multilevel evolution leads to long-term information integration. Through genome, network, and dynamical structuring, the occurrence and/or effect of random mutations becomes nonrandom, and facilitates rapid adaptation. This is what does happen in the in silico experiments. Is it also what did happen in biological evolution? I will discuss some data that suggest that it did. In any case, these results provide us with novel search images to tackle the wealth of biological data.

Radiation-induced genomic instability

NASA Technical Reports Server (NTRS)

Kronenberg, A.

1994-01-01

Quantitative assessment of the heritable somatic effects of ionizing radiation exposures has relied upon the assumption that radiation-induced lesions were 'fixed' in the DNA prior to the first postirradiation mitosis. Lesion conversion was thought to occur during the initial round of DNA replication or as a consequence of error-prone enzymatic processing of lesions. The standard experimental protocols for the assessment of a variety of radiation-induced endpoints (cell death, specific locus mutations, neoplastic transformation and chromosome aberrations) evaluate these various endpoints at a single snapshot in time. In contrast with the aforementioned approaches, some studies have specifically assessed radiation effects as a function of time following exposure. Evidence has accumulated in support of the hypothesis that radiation exposure induces a persistent destabilization of the genome. This instability has been observed as a delayed expression of lethal mutations, as an enhanced rate of accumulation of non-lethal heritable alterations, and as a progressive intraclonal chromosomal heterogeneity. The genetic controls and biochemical mechanisms underlying radiation-induced genomic instability have not yet been delineated. The aim is to integrate the accumulated evidence that suggests that radiation exposure has a persistent effect on the stability of the mammalian genome.

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.

PubMed

Belmonte, Frances R; Martin, James L; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A; Kaufman, Brett A

2016-04-28

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.

Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions

PubMed Central

Belmonte, Frances R.; Martin, James L.; Frescura, Kristin; Damas, Joana; Pereira, Filipe; Tarnopolsky, Mark A.; Kaufman, Brett A.

2016-01-01

Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error. PMID:27122135

The full-length microRNA cluster in the intron of large latency transcript is associated with the virulence of pseudorabies virus.

PubMed

Wang, Xin; Zhang, Mei-Mei; Yan, Kai; Tang, Qi; Wu, Yi-Quan; He, Wen-Bo; Chen, Huan-Chun; Liu, Zheng-Fei

2018-07-01

Pseudorabies virus (PRV), the etiological pathogen of Aujeszky's disease, belongs to the Alphaherpesvirus subfamily. Large latency transcript (LLT), the most abundant PRV transcript, harbors a ~ 4.6 kb microRNA (miRNA) cluster-encoding intron. To investigate the function of the LLT miRNA cluster during the life cycle of PRV, we generated a miRNA cluster mutation virus (PRV-∆miR cluster) and revertant virus. Analysis of the growth kinetics of PRV-ΔmiR cluster-infected cells revealed significantly smaller plaques and lower titers than the wild-type and revertant viruses. The mutation virus exhibited increased IE180 and decreased EP0 expression. The clinical symptoms observed in mice infected with PRV-ΔmiR cluster revealed that the miRNA cluster is involved in the pathogenesis of PRV. Physical parameters, virus shedding assays, and the SN 50 titers revealed that the miRNA cluster enhances PRV virulence in pigs. Collectively, our findings suggest that the full-length miRNA cluster is involved in PRV replication and virulence. Copyright © 2018 Elsevier Inc. All rights reserved.

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication

PubMed Central

Gagnon, David; Lehoux, Michaël

2015-01-01

ABSTRACT The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. IMPORTANCE The E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the molecular nature of the E1-UAF1 interaction and, more generally, how UAF1-USP complexes recognize their substrate proteins. To address this question, we characterized the UAF1-binding site (UBS) of PHLPP1, a protein unrelated to E1. Using a PHLPP1-E1 chimeric helicase, we show that the PHLPP1 UBS (PUBS) can partially substitute for the E1 UBS in stimulating HPV DNA replication. This stimulation required conserved sequences in PUBS that meditate its interaction with UAF1, including a motif common to the E1 UBS. These results indicate that UAF1-binding sequences function as transferable protein interaction modules and provide further evidence that UAF1-USP complexes stimulate HPV DNA replication. PMID:25833051

Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication.

PubMed

Gagnon, David; Lehoux, Michaël; Archambault, Jacques

2015-06-01

The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication. The E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the molecular nature of the E1-UAF1 interaction and, more generally, how UAF1-USP complexes recognize their substrate proteins. To address this question, we characterized the UAF1-binding site (UBS) of PHLPP1, a protein unrelated to E1. Using a PHLPP1-E1 chimeric helicase, we show that the PHLPP1 UBS (PUBS) can partially substitute for the E1 UBS in stimulating HPV DNA replication. This stimulation required conserved sequences in PUBS that meditate its interaction with UAF1, including a motif common to the E1 UBS. These results indicate that UAF1-binding sequences function as transferable protein interaction modules and provide further evidence that UAF1-USP complexes stimulate HPV DNA replication. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Pancreatic Agenesis due to Compound Heterozygosity for a Novel Enhancer and Truncating Mutation in the PTF1A Gene.

PubMed

Gabbay, Monica; Ellard, Sian; De Franco, Elisa; Moisés, Regina S

2017-09-01

Neonatal diabetes, defined as the onset of diabetes within the first six months of life, is very rarely caused by pancreatic agenesis. Homozygous truncating mutations in the PTF1A gene, which encodes a transcriptional factor, have been reported in patients with pancreatic and cerebellar agenesis, whilst mutations located in a distal pancreatic-specific enhancer cause isolated pancreatic agenesis. We report an infant, born to healthy non-consanguineous parents, with neonatal diabetes due to pancreatic agenesis. Initial genetic investigation included sequencing of KCNJ11, ABCC8 and INS genes, but no mutations were found. Following this, 22 neonatal diabetes associated genes were analyzed by a next generation sequencing assay. We found compound heterozygous mutations in the PTF1A gene: A frameshift mutation in exon 1 (c.437_462 del, p.Ala146Glyfs*116) and a mutation affecting a highly conserved nucleotide within the distal pancreatic enhancer (g.23508442A>G). Both mutations were confirmed by Sanger sequencing. Isolated pancreatic agenesis resulting from compound heterozygosity for truncating and enhancer mutations in the PTF1A gene has not been previously reported. This report broadens the spectrum of mutations causing pancreatic agenesis.

Pancreatic Agenesis due to Compound Heterozygosity for a Novel Enhancer and Truncating Mutation in the PTF1A Gene

PubMed Central

Gabbay, Monica; Ellard, Sian; De Franco, Elisa; Moisés, Regina S.

2017-01-01

Neonatal diabetes, defined as the onset of diabetes within the first six months of life, is very rarely caused by pancreatic agenesis. Homozygous truncating mutations in the PTF1A gene, which encodes a transcriptional factor, have been reported in patients with pancreatic and cerebellar agenesis, whilst mutations located in a distal pancreatic-specific enhancer cause isolated pancreatic agenesis. We report an infant, born to healthy non-consanguineous parents, with neonatal diabetes due to pancreatic agenesis. Initial genetic investigation included sequencing of KCNJ11, ABCC8 and INS genes, but no mutations were found. Following this, 22 neonatal diabetes associated genes were analyzed by a next generation sequencing assay. We found compound heterozygous mutations in the PTF1A gene: A frameshift mutation in exon 1 (c.437_462 del, p.Ala146Glyfs*116) and a mutation affecting a highly conserved nucleotide within the distal pancreatic enhancer (g.23508442A>G). Both mutations were confirmed by Sanger sequencing. Isolated pancreatic agenesis resulting from compound heterozygosity for truncating and enhancer mutations in the PTF1A gene has not been previously reported. This report broadens the spectrum of mutations causing pancreatic agenesis. PMID:28663161

DNA replication stress: from molecular mechanisms to human disease.

PubMed

Muñoz, Sergio; Méndez, Juan

2017-02-01

The genome of proliferating cells must be precisely duplicated in each cell division cycle. Chromosomal replication entails risks such as the possibility of introducing breaks and/or mutations in the genome. Hence, DNA replication requires the coordinated action of multiple proteins and regulatory factors, whose deregulation causes severe developmental diseases and predisposes to cancer. In recent years, the concept of "replicative stress" (RS) has attracted much attention as it impinges directly on genomic stability and offers a promising new avenue to design anticancer therapies. In this review, we summarize recent progress in three areas: (1) endogenous and exogenous factors that contribute to RS, (2) molecular mechanisms that mediate the cellular responses to RS, and (3) the large list of diseases that are directly or indirectly linked to RS.

Challenge for One Health: Co-Circulation of Zoonotic H5N1 and H9N2 Avian Influenza Viruses in Egypt.

PubMed

Kim, Shin-Hee

2018-03-09

Highly pathogenic avian influenza (HPAI) H5N1 viruses are currently endemic in poultry in Egypt. Eradication of the viruses has been unsuccessful due to improper application of vaccine-based control strategies among other preventive measures. The viruses have evolved rapidly with increased bird-to-human transmission efficacy, thus affecting both animal and public health. Subsequent spread of potentially zoonotic low pathogenic avian influenza (LPAI) H9N2 in poultry has also hindered efficient control of avian influenza. The H5N1 viruses acquired enhanced bird-to-human transmissibility by (1) altering amino acids in hemagglutinin (HA) that enable binding affinity to human-type receptors, (2) loss of the glycosylation site and 130 loop in the HA protein and (3) mutation of E627K in the PB2 protein to enhance viral replication in mammalian hosts. The receptor binding site of HA of Egyptian H9N2 viruses has been shown to contain the Q234L substitution along with a H191 mutation, which can increase human-like receptor specificity. Therefore, co-circulation of H5N1 and H9N2 viruses in poultry farming and live bird markets has increased the risk of human exposure, resulting in complication of the epidemiological situation and raising a concern for potential emergence of a new influenza A virus pandemic. For efficient control of infection and transmission, the efficacy of vaccine and vaccination needs to be improved with a comprehensive control strategy, including enhanced biosecurity, education, surveillance, rapid diagnosis and culling of infected poultry.

Challenge for One Health: Co-Circulation of Zoonotic H5N1 and H9N2 Avian Influenza Viruses in Egypt

PubMed Central

2018-01-01

Highly pathogenic avian influenza (HPAI) H5N1 viruses are currently endemic in poultry in Egypt. Eradication of the viruses has been unsuccessful due to improper application of vaccine-based control strategies among other preventive measures. The viruses have evolved rapidly with increased bird-to-human transmission efficacy, thus affecting both animal and public health. Subsequent spread of potentially zoonotic low pathogenic avian influenza (LPAI) H9N2 in poultry has also hindered efficient control of avian influenza. The H5N1 viruses acquired enhanced bird-to-human transmissibility by (1) altering amino acids in hemagglutinin (HA) that enable binding affinity to human-type receptors, (2) loss of the glycosylation site and 130 loop in the HA protein and (3) mutation of E627K in the PB2 protein to enhance viral replication in mammalian hosts. The receptor binding site of HA of Egyptian H9N2 viruses has been shown to contain the Q234L substitution along with a H191 mutation, which can increase human-like receptor specificity. Therefore, co-circulation of H5N1 and H9N2 viruses in poultry farming and live bird markets has increased the risk of human exposure, resulting in complication of the epidemiological situation and raising a concern for potential emergence of a new influenza A virus pandemic. For efficient control of infection and transmission, the efficacy of vaccine and vaccination needs to be improved with a comprehensive control strategy, including enhanced biosecurity, education, surveillance, rapid diagnosis and culling of infected poultry. PMID:29522492

Productive replication of human papillomavirus 31 requires DNA repair factor Nbs1.

PubMed

Anacker, Daniel C; Gautam, Dipendra; Gillespie, Kenric A; Chappell, William H; Moody, Cary A

2014-08-01

Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

PubMed Central

Anacker, Daniel C.; Gautam, Dipendra; Gillespie, Kenric A.; Chappell, William H.

2014-01-01

ABSTRACT Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. IMPORTANCE The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication. PMID:24850735

The L, M, and S Segments of Rift Valley Fever Virus MP-12 Vaccine Independently Contribute to a Temperature-Sensitive Phenotype

PubMed Central

Nishiyama, Shoko; Lokugamage, Nandadeva

2016-01-01

ABSTRACT Rift Valley fever (RVF) is endemic to Africa, and the mosquito-borne disease is characterized by “abortion storms” in ruminants and by hemorrhagic fever, encephalitis, and blindness in humans. Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) has a tripartite negative-stranded RNA genome (L, M, and S segments). A live-attenuated vaccine for RVF, the MP-12 vaccine, is conditionally licensed for veterinary use in the United States. MP-12 is fully attenuated by the combination of the partially attenuated L, M, and S segments. Temperature sensitivity (ts) limits viral replication at a restrictive temperature and may be involved with viral attenuation. In this study, we aimed to characterize the ts mutations for MP-12. The MP-12 vaccine showed restricted replication at 38°C and replication shutoff (100-fold or greater reduction in virus titer compared to that at 37°C) at 39°C in Vero and MRC-5 cells. Using rZH501 reassortants with either the MP-12 L, M, or S segment, we found that all three segments encode a temperature-sensitive phenotype. However, the ts phenotype of the S segment was weaker than that of the M or L segment. We identified Gn-Y259H, Gc-R1182G, L-V172A, and L-M1244I as major ts mutations for MP-12. The ts mutations in the L segment decreased viral RNA synthesis, while those in the M segment delayed progeny production from infected cells. We also found that a lack of NSs and/or 78kD/NSm protein expression minimally affected the ts phenotype. Our study revealed that MP-12 is a unique vaccine carrying ts mutations in the L, M, and S segments. IMPORTANCE Rift Valley fever (RVF) is a mosquito-borne viral disease endemic to Africa, characterized by high rates of abortion in ruminants and severe diseases in humans. Vaccination is important to prevent the spread of disease, and a live-attenuated MP-12 vaccine is currently the only vaccine with a conditional license in the United States. This study determined the temperature sensitivity (ts) of MP-12 vaccine to understand virologic characteristics. Our study revealed that MP-12 vaccine contains ts mutations independently in the L, M, and S segments and that MP-12 displays a restrictive replication at 38°C. PMID:26819307

The L, M, and S Segments of Rift Valley Fever Virus MP-12 Vaccine Independently Contribute to a Temperature-Sensitive Phenotype.

PubMed

Nishiyama, Shoko; Lokugamage, Nandadeva; Ikegami, Tetsuro

2016-01-27

Rift Valley fever (RVF) is endemic to Africa, and the mosquito-borne disease is characterized by "abortion storms" in ruminants and by hemorrhagic fever, encephalitis, and blindness in humans. Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus) has a tripartite negative-stranded RNA genome (L, M, and S segments). A live-attenuated vaccine for RVF, the MP-12 vaccine, is conditionally licensed for veterinary use in the United States. MP-12 is fully attenuated by the combination of the partially attenuated L, M, and S segments. Temperature sensitivity (ts) limits viral replication at a restrictive temperature and may be involved with viral attenuation. In this study, we aimed to characterize the ts mutations for MP-12. The MP-12 vaccine showed restricted replication at 38°C and replication shutoff (100-fold or greater reduction in virus titer compared to that at 37°C) at 39°C in Vero and MRC-5 cells. Using rZH501 reassortants with either the MP-12 L, M, or S segment, we found that all three segments encode a temperature-sensitive phenotype. However, the ts phenotype of the S segment was weaker than that of the M or L segment. We identified Gn-Y259H, Gc-R1182G, L-V172A, and L-M1244I as major ts mutations for MP-12. The ts mutations in the L segment decreased viral RNA synthesis, while those in the M segment delayed progeny production from infected cells. We also found that a lack of NSs and/or 78kD/NSm protein expression minimally affected the ts phenotype. Our study revealed that MP-12 is a unique vaccine carrying ts mutations in the L, M, and S segments. Rift Valley fever (RVF) is a mosquito-borne viral disease endemic to Africa, characterized by high rates of abortion in ruminants and severe diseases in humans. Vaccination is important to prevent the spread of disease, and a live-attenuated MP-12 vaccine is currently the only vaccine with a conditional license in the United States. This study determined the temperature sensitivity (ts) of MP-12 vaccine to understand virologic characteristics. Our study revealed that MP-12 vaccine contains ts mutations independently in the L, M, and S segments and that MP-12 displays a restrictive replication at 38°C. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

A universal mammalian vaccine cell line substrate.

PubMed

Murray, Jackelyn; Todd, Kyle V; Bakre, Abhijeet; Orr-Burks, Nichole; Jones, Les; Wu, Weilin; Tripp, Ralph A

2017-01-01

Using genome-wide small interfering RNA (siRNA) screens for poliovirus, influenza A virus and rotavirus, we validated the top 6 gene hits PV, RV or IAV to search for host genes that when knocked-down (KD) enhanced virus permissiveness and replication over wild type Vero cells or HEp-2 cells. The enhanced virus replication was tested for 12 viruses and ranged from 2-fold to >1000-fold. There were variations in virus-specific replication (strain differences) across the cell lines examined. Some host genes (CNTD2, COQ9, GCGR, NDUFA9, NEU2, PYCR1, SEC16G, SVOPL, ZFYVE9, and ZNF205) showed that KD resulted in enhanced virus replication. These findings advance platform-enabling vaccine technology, the creation of diagnostic cells substrates, and are informative about the host mechanisms that affect virus replication in mammalian cells.

Lethal mutagenesis: targeting the mutator phenotype in cancer.

PubMed

Fox, Edward J; Loeb, Lawrence A

2010-10-01

The evolution of cancer and RNA viruses share many similarities. Both exploit high levels of genotypic diversity to enable extensive phenotypic plasticity and thereby facilitate rapid adaptation. In order to accumulate large numbers of mutations, we have proposed that cancers express a mutator phenotype. Similar to cancer cells, many viral populations, by replicating their genomes with low fidelity, carry a substantial mutational load. As high levels of mutation are potentially deleterious, the viral mutation frequency is thresholded at a level below which viral populations equilibrate in a traditional mutation-selection balance, and above which the population is no longer viable, i.e., the population undergoes an error catastrophe. Because their mutation frequencies are fine-tuned just below this error threshold, viral populations are susceptible to further increases in mutational load and, recently this phenomenon has been exploited therapeutically by a concept that has been termed lethal mutagenesis. Here we review the application of lethal mutagenesis to the treatment of HIV and discuss how lethal mutagenesis may represent a novel therapeutic approach for the treatment of solid cancers. Copyright © 2010 Elsevier Ltd. All rights reserved.

Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype.

PubMed

Yao, Likun; Chen, Jing; Wu, Xiaotong; Jia, Shunji; Meng, Anming

2017-11-01

Cell Division Cycle 6 (Cdc6) is a component of pre-replicative complex (preRC) forming on DNA replication origins in eukaryotes. Recessive mutations in ORC1, ORC4, ORC6, CDT1 or CDC6 of the preRC in human cause Meier-Gorlin syndrome (MGS) that is characterized by impaired post-natal growth, short stature and microcephaly. However, vertebrate models of MGS have not been reported. Through N-ethyl-N-nitrosourea mutagenesis and Cas9 knockout, we generate several cdc6 mutant lines in zebrafish. Loss-of-function mutations of cdc6, as manifested by cdc6tsu4305 and cdc6tsu7cd mutants, lead to embryonic lethality due to cell cycle arrest at the S phase and extensive apoptosis. Embryos homozygous for a cdc6 hypomorphic mutation, cdc6tsu21cd, develop normally during embryogenesis. Later on, compared with their wild-type (WT) siblings, cdc6tsu21cd mutant fish show growth retardation, and their body weight and length in adulthood are greatly reduced, which resemble human MGS. Surprisingly, cdc6tsu21cd mutant fish become males with a short life and fail to mate with WT females, suggesting defective reproduction. Overexpression of Cdc6 mutant forms, which mimic human CDC6(T323R) mutation found in a MGS patient, in zebrafish cdc6tsu4305 mutant embryos partially represses cell death phenotype, suggesting that the human CDC6(T323R) mutation is a hypomorph. cdc6tsu21cd mutant fish will be useful to detect more tissue defects and develop medical treatment strategies for MGS patients. © The Author 2017. Published by Oxford University Press.

The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

PubMed Central

Julias, J G; Kim, T; Arnold, G; Pathak, V K

1997-01-01

It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis. PMID:9151812

Systematic reconstruction of autism biology from massive genetic mutation profiles

PubMed Central

Zhang, Chaolin; Jiang, Yong-hui

2018-01-01

Autism spectrum disorder (ASD) affects 1% of world population and has become a pressing medical and social problem worldwide. As a paradigmatic complex genetic disease, ASD has been intensively studied and thousands of gene mutations have been reported. Because these mutations rarely recur, it is difficult to (i) pinpoint the fewer disease-causing versus majority random events and (ii) replicate or verify independent studies. A coherent and systematic understanding of autism biology has not been achieved. We analyzed 3392 and 4792 autism-related mutations from two large-scale whole-exome studies across multiple resolution levels, that is, variants (single-nucleotide), genes (protein-coding unit), and pathways (molecular module). These mutations do not recur or replicate at the variant level, but significantly and increasingly do so at gene and pathway levels. Genetic association reveals a novel gene + pathway dual-hit model, where the mutation burden becomes less relevant. In multiple independent analyses, hundreds of variants or genes repeatedly converge to several canonical pathways, either novel or literature-supported. These pathways define recurrent and systematic ASD biology, distinct from previously reported gene groups or networks. They also present a catalog of novel ASD risk factors including 118 variants and 72 genes. At a subpathway level, most variants disrupt the pathway-related gene functions, and in the same gene, they tend to hit residues extremely close to each other and in the same domain. Multiple interacting variants spotlight key modules, including the cAMP (adenosine 3′,5′-monophosphate) second-messenger system and mGluR (metabotropic glutamate receptor) signaling regulation by GRKs (G protein–coupled receptor kinases). At a superpathway level, distinct pathways further interconnect and converge to three biology themes: synaptic function, morphology, and plasticity. PMID:29651456

Systematic reconstruction of autism biology from massive genetic mutation profiles.

PubMed

Luo, Weijun; Zhang, Chaolin; Jiang, Yong-Hui; Brouwer, Cory R

2018-04-01

Autism spectrum disorder (ASD) affects 1% of world population and has become a pressing medical and social problem worldwide. As a paradigmatic complex genetic disease, ASD has been intensively studied and thousands of gene mutations have been reported. Because these mutations rarely recur, it is difficult to (i) pinpoint the fewer disease-causing versus majority random events and (ii) replicate or verify independent studies. A coherent and systematic understanding of autism biology has not been achieved. We analyzed 3392 and 4792 autism-related mutations from two large-scale whole-exome studies across multiple resolution levels, that is, variants (single-nucleotide), genes (protein-coding unit), and pathways (molecular module). These mutations do not recur or replicate at the variant level, but significantly and increasingly do so at gene and pathway levels. Genetic association reveals a novel gene + pathway dual-hit model, where the mutation burden becomes less relevant. In multiple independent analyses, hundreds of variants or genes repeatedly converge to several canonical pathways, either novel or literature-supported. These pathways define recurrent and systematic ASD biology, distinct from previously reported gene groups or networks. They also present a catalog of novel ASD risk factors including 118 variants and 72 genes. At a subpathway level, most variants disrupt the pathway-related gene functions, and in the same gene, they tend to hit residues extremely close to each other and in the same domain. Multiple interacting variants spotlight key modules, including the cAMP (adenosine 3',5'-monophosphate) second-messenger system and mGluR (metabotropic glutamate receptor) signaling regulation by GRKs (G protein-coupled receptor kinases). At a superpathway level, distinct pathways further interconnect and converge to three biology themes: synaptic function, morphology, and plasticity.

TBX6 Null Variants and a Common Hypomorphic Allele in Congenital Scoliosis

PubMed Central

Wu, N.; Ming, X.; Xiao, J.; Wu, Z.; Chen, X.; Shinawi, M.; Shen, Y.; Yu, G.; Liu, J.; Xie, H.; Gucev, Z.S.; Liu, S.; Yang, N.; Al-Kateb, H.; Chen, J.; Zhang, Jian; Hauser, N.; Zhang, T.; Tasic, V.; Liu, P.; Su, X.; Pan, X.; Liu, C.; Wang, L.; Shen, Joseph; Shen, Jianxiong; Chen, Y.; Zhang, T.; Zhang, Jianguo; Choy, K.W.; Wang, Jun; Wang, Q.; Li, S.; Zhou, W.; Guo, J.; Wang, Y.; Zhang, C.; Zhao, H.; An, Y.; Zhao, Y.; Wang, Jiucun; Liu, Z.; Zuo, Y.; Tian, Y.; Weng, X.; Sutton, V.R.; Wang, H.; Ming, Y.; Kulkarni, S.; Zhong, T.P.; Giampietro, P.F.; Dunwoodie, S.L.; Cheung, S.W.; Zhang, X.; Jin, L.; Lupski, J.R.; Qiu, G.; Zhang, F.

2015-01-01

BACKGROUND Congenital scoliosis is a common type of vertebral malformation. Genetic susceptibility has been implicated in congenital scoliosis. METHODS We evaluated 161 Han Chinese persons with sporadic congenital scoliosis, 166 Han Chinese controls, and 2 pedigrees, family members of which had a 16p11.2 deletion, using comparative genomic hybridization, quantitative polymerase-chain-reaction analysis, and DNA sequencing. We carried out tests of replication using an additional series of 76 Han Chinese persons with congenital scoliosis and a multi-center series of 42 persons with 16p11.2 deletions. RESULTS We identified a total of 17 heterozygous TBX6 null mutations in the 161 persons with sporadic congenital scoliosis (11%); we did not observe any null mutations in TBX6 in 166 controls (P<3.8×10−6). These null alleles include copy-number variants (12 instances of a 16p11.2 deletion affecting TBX6) and single-nucleotide variants (1 nonsense and 4 frame-shift mutations). However, the discordant intrafamilial phenotypes of 16p11.2 deletion carriers suggest that heterozygous TBX6 null mutation is insufficient to cause congenital scoliosis. We went on to identify a common TBX6 haplotype as the second risk allele in all 17 carriers of TBX6 null mutations (P<1.1×10−6). Replication studies involving additional persons with congenital scoliosis who carried a deletion affecting TBX6 confirmed this compound inheritance model. In vitro functional assays suggested that the risk haplotype is a hypomorphic allele. Hemivertebrae are characteristic of TBX6-associated congenital scoliosis. CONCLUSIONS Compound inheritance of a rare null mutation and a hypomorphic allele of TBX6 accounted for up to 11% of congenital scoliosis cases in the series that we analyzed. PMID:25564734

Zebrafish cdc6 hypomorphic mutation causes Meier-Gorlin syndrome-like phenotype

PubMed Central

Yao, Likun; Chen, Jing; Wu, Xiaotong; Jia, Shunji; Meng, Anming

2017-01-01

Abstract Cell Division Cycle 6 (Cdc6) is a component of pre-replicative complex (preRC) forming on DNA replication origins in eukaryotes. Recessive mutations in ORC1, ORC4, ORC6, CDT1 or CDC6 of the preRC in human cause Meier-Gorlin syndrome (MGS) that is characterized by impaired post-natal growth, short stature and microcephaly. However, vertebrate models of MGS have not been reported. Through N-ethyl-N-nitrosourea mutagenesis and Cas9 knockout, we generate several cdc6 mutant lines in zebrafish. Loss-of-function mutations of cdc6, as manifested by cdc6tsu4305 and cdc6tsu7cd mutants, lead to embryonic lethality due to cell cycle arrest at the S phase and extensive apoptosis. Embryos homozygous for a cdc6 hypomorphic mutation, cdc6tsu21cd, develop normally during embryogenesis. Later on, compared with their wild-type (WT) siblings, cdc6tsu21cd mutant fish show growth retardation, and their body weight and length in adulthood are greatly reduced, which resemble human MGS. Surprisingly, cdc6tsu21cd mutant fish become males with a short life and fail to mate with WT females, suggesting defective reproduction. Overexpression of Cdc6 mutant forms, which mimic human CDC6(T323R) mutation found in a MGS patient, in zebrafish cdc6tsu4305 mutant embryos partially represses cell death phenotype, suggesting that the human CDC6(T323R) mutation is a hypomorph. cdc6tsu21cd mutant fish will be useful to detect more tissue defects and develop medical treatment strategies for MGS patients. PMID:28985365

High-resolution mapping, characterization, and optimization of autonomously replicating sequences in yeast

PubMed Central

Liachko, Ivan; Youngblood, Rachel A.; Keich, Uri; Dunham, Maitreya J.

2013-01-01

DNA replication origins are necessary for the duplication of genomes. In addition, plasmid-based expression systems require DNA replication origins to maintain plasmids efficiently. The yeast autonomously replicating sequence (ARS) assay has been a valuable tool in dissecting replication origin structure and function. However, the dearth of information on origins in diverse yeasts limits the availability of efficient replication origin modules to only a handful of species and restricts our understanding of origin function and evolution. To enable rapid study of origins, we have developed a sequencing-based suite of methods for comprehensively mapping and characterizing ARSs within a yeast genome. Our approach finely maps genomic inserts capable of supporting plasmid replication and uses massively parallel deep mutational scanning to define molecular determinants of ARS function with single-nucleotide resolution. In addition to providing unprecedented detail into origin structure, our data have allowed us to design short, synthetic DNA sequences that retain maximal ARS function. These methods can be readily applied to understand and modulate ARS function in diverse systems. PMID:23241746

Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Malkinson, Mertyn; Winocour, Ernest

The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM.more » AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host.« less

Baicalin benefits the anti-HBV therapy via inhibiting HBV viral RNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Hai, E-mail: HHai3552@sina.cn

Background: Although current antiviral treatments (nucleoside analogs, NAs) for chronic hepatitis B virus (HBV) infection are effective in suppressing HBV-DNA replication, their clinical outcomes can be compromised by the increasing drug resistance and the inefficiency in promoting HBsAg/HBeAg seroconversion. Objectives: In this study, we will explore possible effects and mechanism of a natural product baicalin (BA) with the anti-HBV efficacy of entecavir (ETV), a first-line anti-HBV drug, in HBV-DNA, HBsAg/HBeAg seroconversion and drug-resistance. Methods: The co-effects of BA and ETV were conducted in wild-type/NA-resistance mutant HBV cell lines and DHBV-infected duckling models. HBV-DNA/RNAs, HBsAg/HBeAg, host factors (hepatocyte nuclear factors) weremore » explored for possible anti-HBV mechanism. Results and discussion: BA could significantly enhance and reduced HBsAg and HBeAg in hepG2.2.15, a wild-type HBV cell line. Co-treatment of BA and ETV had a more dramatic effect in NA-resistant HBV{sup rtM204V/rtLl80M} transfected hepG2 cells. Our study further revealed that BA mainly inhibited the production of HBV RNAs (3.5, 2.4, 2.1 kb), the templates for viral proteins and HBV-DNA synthesis. BA blocked HBV RNAs transcription possibly by down-regulating transcription and expression of HBV replication dependent hepatocyte nuclear factors (HNF1α and HNF4α). Thus, BA may benefit the anti-HBV therapy via inhibiting HBV viral RNAs. - Highlights: • Baicalin benefits the anti-HBV therapy. • Baicalin enhances ETV antiviral efficacy and overcomes NA-resistant HBV mutation. • The anti-HBV effect of baicalin is achieved by inhibiting HBV RNAs. • Baicalin down-regulates HBV replication-dependent host factors HNF 1α and HNF 4α.« less

Hepatitis C Virus Nucleotide Inhibitors PSI-352938 and PSI-353661 Exhibit a Novel Mechanism of Resistance Requiring Multiple Mutations within Replicon RNA▿†

PubMed Central

Lam, Angela M.; Espiritu, Christine; Bansal, Shalini; Micolochick Steuer, Holly M.; Zennou, Veronique; Otto, Michael J.; Furman, Phillip A.

2011-01-01

PSI-352938, a cyclic phosphate nucleotide, and PSI-353661, a phosphoramidate nucleotide, are prodrugs of β-d-2′-deoxy-2′-α-fluoro-2′-β-C-methylguanosine-5′-monophosphate. Both compounds are metabolized to the same active 5′-triphosphate, PSI-352666, which serves as an alternative substrate inhibitor of the NS5B RNA-dependent RNA polymerase during HCV replication. PSI-352938 and PSI-353661 retained full activity against replicons containing the S282T substitution, which confers resistance to certain 2′-substituted nucleoside/nucleotide analogs. PSI-352666 was also similarly active against both wild-type and S282T NS5B polymerases. In order to identify mutations that confer resistance to these compounds, in vitro selection studies were performed using HCV replicon cells. While no resistant genotype 1a or 1b replicons could be selected, cells containing genotype 2a JFH-1 replicons cultured in the presence of PSI-352938 or PSI-353661 developed resistance to both compounds. Sequencing of the NS5B region identified a number of amino acid changes, including S15G, R222Q, C223Y/H, L320I, and V321I. Phenotypic evaluation of these mutations indicated that single amino acid changes were not sufficient to significantly reduce the activity of PSI-352938 and PSI-353661. Instead, a combination of three amino acid changes, S15G/C223H/V321I, was required to confer a high level of resistance. No cross-resistance exists between the 2′-F-2′-C-methylguanosine prodrugs and other classes of HCV inhibitors, including 2′-modified nucleoside/-tide analogs such as PSI-6130, PSI-7977, INX-08189, and IDX-184. Finally, we determined that in genotype 1b replicons, the C223Y/H mutation failed to support replication, and although the A15G/C223H/V321I triple mutation did confer resistance to PSI-352938 and PSI-353661, this mutant replicated at only about 10% efficiency compared to the wild type. PMID:21957306

Poliovirus Polymerase Leu420 Facilitates RNA Recombination and Ribavirin Resistance

PubMed Central

Kempf, Brian J.; Peersen, Olve B.

2016-01-01

ABSTRACT RNA recombination is important in the formation of picornavirus species groups and the ongoing evolution of viruses within species groups. In this study, we examined the structure and function of poliovirus polymerase, 3Dpol, as it relates to RNA recombination. Recombination occurs when nascent RNA products exchange one viral RNA template for another during RNA replication. Because recombination is a natural aspect of picornavirus replication, we hypothesized that some features of 3Dpol may exist, in part, to facilitate RNA recombination. Furthermore, we reasoned that alanine substitution mutations that disrupt 3Dpol-RNA interactions within the polymerase elongation complex might increase and/or decrease the magnitudes of recombination. We found that an L420A mutation in 3Dpol decreased the frequency of RNA recombination, whereas alanine substitutions at other sites in 3Dpol increased the frequency of recombination. The 3Dpol Leu420 side chain interacts with a ribose in the nascent RNA product 3 nucleotides from the active site of the polymerase. Notably, the L420A mutation that reduced recombination also rendered the virus more susceptible to inhibition by ribavirin, coincident with the accumulation of ribavirin-induced G→A and C→U mutations in viral RNA. We conclude that 3Dpol Leu420 is critically important for RNA recombination and that RNA recombination contributes to ribavirin resistance. IMPORTANCE Recombination contributes to the formation of picornavirus species groups and the emergence of circulating vaccine-derived polioviruses (cVDPVs). The recombinant viruses that arise in nature are occasionally more fit than either parental strain, especially when the two partners in recombination are closely related, i.e., members of characteristic species groups, such as enterovirus species groups A to H or rhinovirus species groups A to C. Our study shows that RNA recombination requires conserved features of the viral polymerase. Furthermore, a polymerase mutation that disables recombination renders the virus more susceptible to the antiviral drug ribavirin, suggesting that recombination contributes to ribavirin resistance. Elucidating the molecular mechanisms of RNA replication and recombination may help mankind achieve and maintain poliovirus eradication. PMID:27412593

HIV-1-encoded antisense RNA suppresses viral replication for a prolonged period

PubMed Central

2012-01-01

Background Recent evidence proposes a novel concept that mammalian natural antisense RNAs play important roles in cellular homeostasis by regulating the expression of several genes. Identification and characterization of retroviral antisense RNA would provide new insights into mechanisms of replication and pathogenesis. HIV-1 encoded-antisense RNAs have been reported, although their structures and functions remain to be studied. We have tried to identify and characterize antisense RNAs of HIV-1 and their function in viral infection. Results Characterization of transcripts of HEK293T cells that were transiently transfected with an expression plasmid with HIV-1NL4–3 DNA in the antisense orientation showed that various antisense transcripts can be expressed. By screening and characterizing antisense RNAs in HIV-1NL4–3-infected cells, we defined the primary structure of a major form of HIV-1 antisense RNAs, which corresponds to a variant of previously reported ASP mRNA. This 2.6 kb RNA was transcribed from the U3 region of the 3′ LTR and terminated at the env region in acutely or chronically infected cell lines and acutely infected human peripheral blood mononuclear cells. Reporter assays clearly demonstrated that the HIV-1 LTR harbours promoter activity in the reverse orientation. Mutation analyses suggested the involvement of NF-κΒ binding sites in the regulation of antisense transcription. The antisense RNA was localized in the nuclei of the infected cells. The expression of this antisense RNA suppressed HIV-1 replication for more than one month. Furthermore, the specific knockdown of this antisense RNA enhanced HIV-1 gene expression and replication. Conclusions The results of the present study identified an accurate structure of the major form of antisense RNAs expressed from the HIV-1NL4–3 provirus and demonstrated its nuclear localization. Functional studies collectively demonstrated a new role of the antisense RNA in viral replication. Thus, we suggest a novel viral mechanism that self-limits HIV-1 replication and provides new insight into the viral life cycle. PMID:22569184

Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase.

PubMed

Yamauchi, Shota; Takeuchi, Kenji; Chihara, Kazuyasu; Sun, Xuedong; Honjoh, Chisato; Yoshiki, Hatsumi; Hotta, Hak; Sada, Kiyonao

2015-09-04

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr(330) (Tyr(2306) in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr(330). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication.

PubMed

Aye, Seaim Lwin; Fujiwara, Kei; Ueki, Asuka; Doi, Nobuhide

2018-05-05

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5' to 3' exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol. Copyright © 2018 Elsevier Inc. All rights reserved.

Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli.

PubMed

Petit, M A; Bedale, W; Osipiuk, J; Lu, C; Rajagopalan, M; McInerney, P; Goodman, M F; Echols, H

1994-09-23

Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response. Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria. The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M. F., Echols, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10777-10781). In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro. We treated purified and inactive UmuC with Hsp70 and Hsp60. After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not. However, lesion bypass activity was recovered upon further treatment with Hsp60. The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis.

Xeroderma pigmentosum variant cells are less likely than normal cells to incorporate dAMP opposite photoproducts during replication of UV-irradiated plasmids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Y.C.; Maher, V.M.; McCormich, J.J.

1991-09-01

Xeroderma pigmentosum (XP) variant patients show the clinical characteristics of the disease, with increased frequencies of skin cancer, but their cells have a normal, or nearly normal, rate of nucleotide excision repair of UV-induced DNA damage and are only slightly more sensitive than normal cells to the cytotoxic effect of UV radiation. However, they are significantly more sensitive to its mutagenic effect. To examine the mechanisms responsible for this hypermutability, the authors transfected an XP variant cell line with a UV-irradiated (at 254 nm) shuttle vector carrying the {sup F} gene as a target for mutations, allowed replication of themore » plasmid, determined the frequency and spectrum of mutations induced, and compared the results with those obtained previously when irradiated plasmids carrying the same target gene replicated in a normal cell line. The frequency of mutants increased linearly with dose, but with a slope 5 times steeper than that seen with normal cells. Sequence analysis of the {sup F} gene showed that 52 of 53 independent mutants generated in the XP variant cells contained base substitutions, with 62 of 64 of the substitutions involving a dipyrimidine.« less