Parvovirus Minute Virus of Mice Induces a DNA Damage Response That Facilitates Viral Replication

PubMed Central

Adeyemi, Richard O.; Landry, Sebastien; Davis, Meredith E.; Weitzman, Matthew D.; Pintel, David J.

2010-01-01

Infection by DNA viruses can elicit DNA damage responses (DDRs) in host cells. In some cases the DDR presents a block to viral replication that must be overcome, and in other cases the infecting agent exploits the DDR to facilitate replication. We find that low multiplicity infection with the autonomous parvovirus minute virus of mice (MVM) results in the activation of a DDR, characterized by the phosphorylation of H2AX, Nbs1, RPA32, Chk2 and p53. These proteins are recruited to MVM replication centers, where they co-localize with the main viral replication protein, NS1. The response is seen in both human and murine cell lines following infection with either the MVMp or MVMi strains. Replication of the virus is required for DNA damage signaling. Damage response proteins, including the ATM kinase, accumulate in viral-induced replication centers. Using mutant cell lines and specific kinase inhibitors, we show that ATM is the main transducer of the signaling events in the normal murine host. ATM inhibitors restrict MVM replication and ameliorate virus-induced cell cycle arrest, suggesting that DNA damage signaling facilitates virus replication, perhaps in part by promoting cell cycle arrest. Thus it appears that MVM exploits the cellular DNA damage response machinery early in infection to enhance its replication in host cells. PMID:20949077

Tombusviruses upregulate phospholipid biosynthesis via interaction between p33 replication protein and yeast lipid sensor proteins during virus replication in yeast

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

Barajas, Daniel; Xu, Kai; Sharma, Monika

Positive-stranded RNA viruses induce new membranous structures and promote membrane proliferation in infected cells to facilitate viral replication. In this paper, the authors show that a plant-infecting tombusvirus upregulates transcription of phospholipid biosynthesis genes, such as INO1, OPI3 and CHO1, and increases phospholipid levels in yeast model host. This is accomplished by the viral p33 replication protein, which interacts with Opi1p FFAT domain protein and Scs2p VAP protein. Opi1p and Scs2p are phospholipid sensor proteins and they repress the expression of phospholipid genes. Accordingly, deletion of OPI1 transcription repressor in yeast has a stimulatory effect on TBSV RNA accumulation andmore » enhanced tombusvirus replicase activity in an in vitro assay. Altogether, the presented data convincingly demonstrate that de novo lipid biosynthesis is required for optimal TBSV replication. Overall, this work reveals that a (+)RNA virus reprograms the phospholipid biosynthesis pathway in a unique way to facilitate its replication in yeast cells. - Highlights: • Tombusvirus p33 replication protein interacts with FFAT-domain host protein. • Tombusvirus replication leads to upregulation of phospholipids. • Tombusvirus replication depends on de novo lipid synthesis. • Deletion of FFAT-domain host protein enhances TBSV replication. • TBSV rewires host phospholipid synthesis.« less

[Research Progress on Antiviral Activity of Interferon-induced Transmembrane Proteins].

PubMed

Chen, Yongkun; Zhu, Wenfei; Shu, Yuelong

2016-03-01

Interferon-induced Transmembrane Proteins (IFITMs) were identified through small interference RNA (siRNA) screening method in 1980s. The antiviral properties of the IFITMs were firstly discovered in 1996. Recently, its antiviral effect and mechanism have become a research hotspot. Many studies have shown that IFITM can inhibit the replication of multiple pathogenic viruses, including influenza A virus (IAV), Human Immunodeficiency Virus (HIV-1), hepatitis C virus (HCV), Ebola virus (EBOV), West Nile virus and so on. IFITMs inhibit the replication of virus in the early stage of the viral life cycle, which occurred before the release of viral genomes into the cytosol. Recent studies indicate that IFITM proteins could block viral replication by mediate viral membrane fusion. However, the mechanism is still under investigation. Here we review the discovery and characterization of the IFITM proteins, elucidate their antiviral activities and the potential mechanisms.

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.

Karyopherin Alpha 6 Is Required for Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus.

PubMed

Yang, Liping; Wang, Rong; Yang, Shixing; Ma, Zexu; Lin, Shaoli; Nan, Yuchen; Li, Qisheng; Tang, Qiyi; Zhang, Yan-Jin

2018-05-01

Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin α6 (KPNA6; also known as importin α7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1β but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1β and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV. IMPORTANCE Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin α6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1β depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1β nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics. Copyright © 2018 American Society for Microbiology.

Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system.

PubMed

Ikegami, Tetsuro; Peters, C J; Makino, Shinji

2005-05-01

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-alpha/beta) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-alpha/beta production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.

Reverse Genetics System Demonstrates that Rotavirus Nonstructural Protein NSP6 Is Not Essential for Viral Replication in Cell Culture.

PubMed

Komoto, Satoshi; Kanai, Yuta; Fukuda, Saori; Kugita, Masanori; Kawagishi, Takahiro; Ito, Naoto; Sugiyama, Makoto; Matsuura, Yoshiharu; Kobayashi, Takeshi; Taniguchi, Koki

2017-11-01

The use of overlapping open reading frames (ORFs) to synthesize more than one unique protein from a single mRNA has been described for several viruses. Segment 11 of the rotavirus genome encodes two nonstructural proteins, NSP5 and NSP6. The NSP6 ORF is present in the vast majority of rotavirus strains, and therefore the NSP6 protein would be expected to have a function in viral replication. However, there is no direct evidence of its function or requirement in the viral replication cycle yet. Here, taking advantage of a recently established plasmid-only-based reverse genetics system that allows rescue of recombinant rotaviruses entirely from cloned cDNAs, we generated NSP6-deficient viruses to directly address its significance in the viral replication cycle. Viable recombinant NSP6-deficient viruses could be engineered. Single-step growth curves and plaque formation of the NSP6-deficient viruses confirmed that NSP6 expression is of limited significance for RVA replication in cell culture, although the NSP6 protein seemed to promote efficient virus growth. IMPORTANCE Rotavirus is one of the most important pathogens of severe diarrhea in young children worldwide. The rotavirus genome, consisting of 11 segments of double-stranded RNA, encodes six structural proteins (VP1 to VP4, VP6, and VP7) and six nonstructural proteins (NSP1 to NSP6). Although specific functions have been ascribed to each of the 12 viral proteins, the role of NSP6 in the viral replication cycle remains unknown. In this study, we demonstrated that the NSP6 protein is not essential for viral replication in cell culture by using a recently developed plasmid-only-based reverse genetics system. This reverse genetics approach will be successfully applied to answer questions of great interest regarding the roles of rotaviral proteins in replication and pathogenicity, which can hardly be addressed by conventional approaches. Copyright © 2017 American Society for Microbiology.

Identification of rep-associated factors in herpes simplex virus type 1-induced adeno-associated virus type 2 replication compartments.

PubMed

Nicolas, Armel; Alazard-Dany, Nathalie; Biollay, Coline; Arata, Loredana; Jolinon, Nelly; Kuhn, Lauriane; Ferro, Myriam; Weller, Sandra K; Epstein, Alberto L; Salvetti, Anna; Greco, Anna

2010-09-01

Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase delta was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

PubMed

Polpitiya Arachchige, Sachith; Henke, Wyatt; Pramanik, Ankita; Kalamvoki, Maria; Stephens, Edward B

2018-01-15

Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways. IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4 + T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell. Copyright © 2018 American Society for Microbiology.

Development of a Novel Anti-HIV-1 Agent from within: Effect of Chimeric Vpr-Containing Protease Cleavage Site Residues on Virus Replication

NASA Astrophysics Data System (ADS)

Serio, D.; Rizvi, T. A.; Cartas, M.; Kalyanaraman, V. S.; Weber, I. T.; Koprowski, H.; Srinivasan, A.

1997-04-01

Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag-Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection.

Chicken Interferon-induced Protein with Tetratricopeptide Repeats 5 Antagonizes Replication of RNA Viruses.

PubMed

Santhakumar, Diwakar; Rohaim, Mohammed Abdel Mohsen Shahaat; Hussein, Hussein A; Hawes, Pippa; Ferreira, Helena Lage; Behboudi, Shahriar; Iqbal, Munir; Nair, Venugopal; Arns, Clarice W; Munir, Muhammad

2018-05-01

The intracellular actions of interferon (IFN)-regulated proteins, including IFN-induced proteins with tetratricopeptide repeats (IFITs), attribute a major component of the protective antiviral host defense. Here we applied genomics approaches to annotate the chicken IFIT locus and currently identified a single IFIT (chIFIT5) gene. The profound transcriptional level of this effector of innate immunity was mapped within its unique cis-acting elements. This highly virus- and IFN-responsive chIFIT5 protein interacted with negative sense viral RNA structures that carried a triphosphate group on its 5' terminus (ppp-RNA). This interaction reduced the replication of RNA viruses in lentivirus-mediated IFIT5-stable chicken fibroblasts whereas CRISPR/Cas9-edited chIFIT5 gene knockout fibroblasts supported the replication of RNA viruses. Finally, we generated mosaic transgenic chicken embryos stably expressing chIFIT5 protein or knocked-down for endogenous chIFIT5 gene. Replication kinetics of RNA viruses in these transgenic chicken embryos demonstrated the antiviral potential of chIFIT5 in ovo. Taken together, these findings propose that IFIT5 specifically antagonize RNA viruses by sequestering viral nucleic acids in chickens, which are unique in innate immune sensing and responses to viruses of both poultry and human health significance.

Ebolaviruses: New roles for old proteins.

PubMed

Cantoni, Diego; Rossman, Jeremy S

2018-05-01

In 2014, the world witnessed the largest Ebolavirus outbreak in recorded history. The subsequent humanitarian effort spurred extensive research, significantly enhancing our understanding of ebolavirus replication and pathogenicity. The main functions of each ebolavirus protein have been studied extensively since the discovery of the virus in 1976; however, the recent expansion of ebolavirus research has led to the discovery of new protein functions. These newly discovered roles are revealing new mechanisms of virus replication and pathogenicity, whilst enhancing our understanding of the broad functions of each ebolavirus viral protein (VP). Many of these new functions appear to be unrelated to the protein's primary function during virus replication. Such new functions range from bystander T-lymphocyte death caused by VP40-secreted exosomes to new roles for VP24 in viral particle formation. This review highlights the newly discovered roles of ebolavirus proteins in order to provide a more encompassing view of ebolavirus replication and pathogenicity.

Friendly fire: redirecting herpes simplex virus-1 for therapeutic applications.

PubMed

Advani, S J; Weichselbaum, R R; Whitley, R J; Roizman, B

2002-09-01

Herpes simplex virus-1 (HSV-1) is a relatively large double-stranded DNA virus encoding at least 89 proteins with well characterized disease pathology. An understanding of the functions of viral proteins together with the ability to genetically engineer specific viral mutants has led to the development of attenuated HSV-1 for gene therapy. This review highlights the progress in creating attenuated genetically engineered HSV-1 mutants that are either replication competent (viral non-essential gene deleted) or replication defective (viral essential gene deleted). The choice between a replication-competent or -defective virus is based on the end-goal of the therapeutic intervention. Replication-competent HSV-1 mutants have primarily been employed as antitumor oncolytic viruses, with the lytic nature of the virus harnessed to destroy tumor cells selectively. In replacement gene therapy, replication-defective viruses have been utilized as delivery vectors. The advantages of HSV-1 vectors are that they infect quiescent and dividing cells efficiently and can encode for relatively large transgenes.

Lipids and RNA virus replication.

PubMed

Konan, Kouacou V; Sanchez-Felipe, Lorena

2014-12-01

Most viruses rely heavily on their host machinery to successfully replicate their genome and produce new virus particles. Recently, the interaction of positive-strand RNA viruses with the lipid biosynthetic and transport machinery has been the subject of intense investigation. In this review, we will discuss the contribution of various host lipids and related proteins in RNA virus replication and maturation. Copyright © 2014 Elsevier B.V. All rights reserved.

Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase.

PubMed

Murata, Takayuki; Isomura, Hiroki; Yamashita, Yoriko; Toyama, Shigenori; Sato, Yoshitaka; Nakayama, Sanae; Kudoh, Ayumi; Iwahori, Satoko; Kanda, Teru; Tsurumi, Tatsuya

2009-06-20

The Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial.

Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

PubMed

Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

2018-01-01

Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell endomembrane system to produce a membranous replication organelle (RO). The underlying mechanisms are far from being elucidated fully. In this report, we provide evidence that HCV RNA replication depends on functional lipid transport along the endosomal-lysosomal pathway that is mediated by several lipid transfer proteins, such as the Niemann-Pick type C1 (NPC1) protein. Pharmacological inhibition of NPC1 function reduced viral replication, impaired the transport of cholesterol to the viral replication organelle, and altered organelle morphology. Besides NPC1, our study reports the importance of additional endosomal and lysosomal lipid transfer proteins required for viral replication, thus contributing to our understanding of how HCV manipulates their function in order to generate a membranous replication organelle. These results might have implications for the biogenesis of replication organelles of other positive-strand RNA viruses. Copyright © 2017 American Society for Microbiology.

2′-5′-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1

PubMed Central

Dhar, Jayeeta; Cuevas, Rolando A.; Goswami, Ramansu; Zhu, Jianzhong

2015-01-01

2′-5′-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. PMID:26178980

Activation of DNA damage repair pathways by murine polyomavirus

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

Heiser, Katie; Nicholas, Catherine; Garcea, Robert

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling.more » ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. -- Highlights: •Murine polyomavirus activates and recruits DNA damage repair (DDR) proteins to replication centers. •Large T-antigen mediates recruitment of DDR proteins to viral replication centers. •Inhibition or knockout of CHK1, CHK2, DNA-PK or H2AX do not affect viral titers. •Inhibition of ATR activity reduces viral titers, but not viral DNA accumulation.« less

Replication of alfalfa mosaic virus RNA 3 with movement and coat protein genes replaced by corresponding genes of Prunus necrotic ringspot ilarvirus.

PubMed

Sánchez-Navarro, J A; Reusken, C B; Bol, J F; Pallás, V

1997-12-01

Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) are tripartite positive-strand RNA plant viruses that encode functionally similar translation products. Although the two viruses are phylogenetically closely related, they infect a very different range of natural hosts. The coat protein (CP) gene, the movement protein (MP) gene or both genes in AMV RNA 3 were replaced by the corresponding genes of PNRSV. The chimeric viruses were tested for heterologous encapsidation, replication in protoplasts from plants transformed with AMV replicase genes P1 and P2 (P12 plants) and for cell-to-cell transport in P12 plants. The chimeric viruses exhibited basic competence for encapsidation and replication in P12 protoplasts and for a low level of cell-to-cell movement in P12 plants. The potential involvement of the MP gene in determining host specificity in ilarviruses is discussed.

Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes

PubMed Central

Mühlberger, Elke; Lötfering, Beate; Klenk, Hans-Dieter; Becker, Stephan

1998-01-01

This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the order Mononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication. PMID:9765419

Myxoma Virus Protein M029 Is a Dual Function Immunomodulator that Inhibits PKR and Also Conscripts RHA/DHX9 to Promote Expanded Host Tropism and Viral Replication

PubMed Central

Rahman, Masmudur M.; Liu, Jia; Chan, Winnie M.; Rothenburg, Stefan; McFadden, Grant

2013-01-01

Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells. PMID:23853588

Intracellular Transport of Plant Viruses: Finding the Door out of the Cell

PubMed Central

Schoelz, James E.; Harries, Phillip A.; Nelson, Richard S.

2011-01-01

Plant viruses are a class of plant pathogens that specialize in movement from cell to cell. As part of their arsenal for infection of plants, every virus encodes a movement protein (MP), a protein dedicated to enlarging the pore size of plasmodesmata (PD) and actively transporting the viral nucleic acid into the adjacent cell. As our knowledge of intercellular transport has increased, it has become apparent that viruses must also use an active mechanism to target the virus from their site of replication within the cell to the PD. Just as viruses are too large to fit through an unmodified plasmodesma, they are also too large to be freely diffused through the cytoplasm of the cell. Evidence has accumulated now for the involvement of other categories of viral proteins in intracellular movement in addition to the MP, including viral proteins originally associated with replication or gene expression. In this review, we will discuss the strategies that viruses use for intracellular movement from the replication site to the PD, in particular focusing on the role of host membranes for intracellular transport and the coordinated interactions between virus proteins within cells that are necessary for successful virus spread. PMID:21896501

The Rubella virus capsid is an anti-apoptotic protein that attenuates the pore-forming ability of Bax.

PubMed

Ilkow, Carolina S; Goping, Ing Swie; Hobman, Tom C

2011-02-01

Apoptosis is an important mechanism by which virus-infected cells are eliminated from the host. Accordingly, many viruses have evolved strategies to prevent or delay apoptosis in order to provide a window of opportunity in which virus replication, assembly and egress can take place. Interfering with apoptosis may also be important for establishment and/or maintenance of persistent infections. Whereas large DNA viruses have the luxury of encoding accessory proteins whose primary function is to undermine programmed cell death pathways, it is generally thought that most RNA viruses do not encode these types of proteins. Here we report that the multifunctional capsid protein of Rubella virus is a potent inhibitor of apoptosis. The main mechanism of action was specific for Bax as capsid bound Bax and prevented Bax-induced apoptosis but did not bind Bak nor inhibit Bak-induced apoptosis. Intriguingly, interaction with capsid protein resulted in activation of Bax in the absence of apoptotic stimuli, however, release of cytochrome c from mitochondria and concomitant activation of caspase 3 did not occur. Accordingly, we propose that binding of capsid to Bax induces the formation of hetero-oligomers that are incompetent for pore formation. Importantly, data from reverse genetic studies are consistent with a scenario in which the anti-apoptotic activity of capsid protein is important for virus replication. If so, this would be among the first demonstrations showing that blocking apoptosis is important for replication of an RNA virus. Finally, it is tempting to speculate that other slowly replicating RNA viruses employ similar mechanisms to avoid killing infected cells.

Coat as a Dagger: The Use of Capsid Proteins to Perforate Membranes during Non-Enveloped DNA Viruses Trafficking

PubMed Central

Bilkova, Eva; Forstova, Jitka; Abrahamyan, Levon

2014-01-01

To get access to the replication site, small non-enveloped DNA viruses have to cross the cell membrane using a limited number of capsid proteins, which also protect the viral genome in the extracellular environment. Most of DNA viruses have to reach the nucleus to replicate. The capsid proteins involved in transmembrane penetration are exposed or released during endosomal trafficking of the virus. Subsequently, the conserved domains of capsid proteins interact with cellular membranes and ensure their efficient permeabilization. This review summarizes our current knowledge concerning the role of capsid proteins of small non-enveloped DNA viruses in intracellular membrane perturbation in the early stages of infection. PMID:25055856

p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

NASA Astrophysics Data System (ADS)

Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

1995-02-01

Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

PA-X protein decreases replication and pathogenicity of swine influenza virus in cultured cells and mouse models.

PubMed

Gong, Xiao-Qian; Sun, Ying-Feng; Ruan, Bao-Yang; Liu, Xiao-Min; Wang, Qi; Yang, Hai-Ming; Wang, Shuai-Yong; Zhang, Peng; Wang, Xiu-Hui; Shan, Tong-Ling; Tong, Wu; Zhou, Yan-Jun; Li, Guo-Xin; Zheng, Hao; Tong, Guang-Zhi; Yu, Hai

2017-06-01

Swine influenza viruses have been circulating in pigs throughout world and might be potential threats to human health. PA-X protein is a newly discovered protein produced from the PA gene by ribosomal frameshifting and the effects of PA-X on the 1918 H1N1, the pandemic 2009 H1N1, the highly pathogenic avian H5N1 and the avian H9N2 influenza viruses have been reported. However, the role of PA-X in the pathogenesis of swine influenza virus is still unknown. In this study, we rescued the H1N1 wild-type (WT) classical swine influenza virus (A/Swine/Guangdong/1/2011 (H1N1)) and H1N1 PA-X deficient virus containing mutations at the frameshift motif, and compared their replication properties and pathogenicity of swine influenza virus in vitro and in vivo. Our results show that the expression of PA-X inhibits virus replication and polymerase activity in cultured cells and decreases virulence in mouse models. Therefore, our study demonstrates that PA-X protein acts as a negative virulence regulator for classical H1N1 swine influenza virus and decreases virulence by inhibiting viral replication and polymerase activity, deepening our understanding of the pathogenesis of swine influenza virus. Copyright © 2017 Elsevier B.V. All rights reserved.

N-Myc Interactor Inhibits Prototype Foamy Virus by Sequestering Viral Tas Protein in the Cytoplasm

PubMed Central

Hu, Xiaomei; Yang, Wei; Liu, Ruikang; Geng, Yunqi; Qiao, Wentao

2014-01-01

ABSTRACT Foamy viruses (FVs) are complex retroviruses that establish lifelong persistent infection without evident pathology. However, the roles of cellular factors in FV latency are poorly understood. This study revealed that N-Myc interactor (Nmi) could inhibit the replication of prototype foamy virus (PFV). Overexpression of Nmi reduced PFV replication, whereas its depletion by small interfering RNA increased PFV replication. The Nmi-mediated impairment of PFV replication resulted from the diminished transactivation by PFV Tas of the viral long terminal repeat (LTR) and an internal promoter (IP). Nmi was determined to interact with Tas and abrogate its function by sequestration in the cytoplasm. In addition, human and bovine Nmi proteins were found to inhibit the replication of bovine foamy virus (BFV) and PFV. Together, these results indicate that Nmi inhibits both human and bovine FVs by interfering with the transactivation function of Tas and may have a role in the host defense against FV infection. IMPORTANCE From this study, we report that the N-Myc interactor (Nmi), an interferon-induced protein, can interact with the regulatory protein Tas of the prototype foamy virus and sequester it in the cytoplasm. The results of this study suggest that Nmi plays an important role in maintaining foamy virus latency and may reveal a new pathway in the interferon-mediated antiviral barrier against viruses. These findings are important for understanding virus-host relationships not only with FVs but potentially for other retroviruses as well. PMID:24719420

The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus.

PubMed

Brass, Abraham L; Huang, I-Chueh; Benita, Yair; John, Sinu P; Krishnan, Manoj N; Feeley, Eric M; Ryan, Bethany J; Weyer, Jessica L; van der Weyden, Louise; Fikrig, Erol; Adams, David J; Xavier, Ramnik J; Farzan, Michael; Elledge, Stephen J

2009-12-24

Influenza viruses exploit host cell machinery to replicate, resulting in epidemics of respiratory illness. In turn, the host expresses antiviral restriction factors to defend against infection. To find host cell modifiers of influenza A H1N1 viral infection, we used a functional genomic screen and identified over 120 influenza A virus-dependency factors with roles in endosomal acidification, vesicular trafficking, mitochondrial metabolism, and RNA splicing. We discovered that the interferon-inducible transmembrane proteins IFITM1, 2, and 3 restrict an early step in influenza A viral replication. The IFITM proteins confer basal resistance to influenza A virus but are also inducible by interferons type I and II and are critical for interferon's virustatic actions. Further characterization revealed that the IFITM proteins inhibit the early replication of flaviviruses, including dengue virus and West Nile virus. Collectively this work identifies a family of antiviral restriction factors that mediate cellular innate immunity to at least three major human pathogens. Copyright 2009 Elsevier Inc. All rights reserved.

Ebolaviruses: New roles for old proteins

PubMed Central

2018-01-01

In 2014, the world witnessed the largest Ebolavirus outbreak in recorded history. The subsequent humanitarian effort spurred extensive research, significantly enhancing our understanding of ebolavirus replication and pathogenicity. The main functions of each ebolavirus protein have been studied extensively since the discovery of the virus in 1976; however, the recent expansion of ebolavirus research has led to the discovery of new protein functions. These newly discovered roles are revealing new mechanisms of virus replication and pathogenicity, whilst enhancing our understanding of the broad functions of each ebolavirus viral protein (VP). Many of these new functions appear to be unrelated to the protein’s primary function during virus replication. Such new functions range from bystander T-lymphocyte death caused by VP40-secreted exosomes to new roles for VP24 in viral particle formation. This review highlights the newly discovered roles of ebolavirus proteins in order to provide a more encompassing view of ebolavirus replication and pathogenicity. PMID:29723187

Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1.

PubMed

Ishikawa-Sasaki, Kumiko; Nagashima, Shigeo; Taniguchi, Koki; Sasaki, Jun

2018-04-15

Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs. IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi virus (AiV), a picornavirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions. Copyright © 2018 American Society for Microbiology.

T135I substitution in the nonstructural protein 2C enhances foot-and-mouth disease virus replication.

PubMed

Yuan, Tiangang; Wang, Haiwei; Li, Chen; Yang, Decheng; Zhou, Guohui; Yu, Li

2017-12-01

The foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays an important role in viral replication, virulence, and host range. It has been shown that deletions of 10 or 19-20 amino acids in the C-terminal half of 3A attenuate serotype O and C FMDVs, which replicate poorly in bovine cells but normally in porcine-derived cells, and the C-terminal half of 3A is not essential for serotype Asia1 FMDV replication in BHK-21 cells. In this study, we constructed a 3A deletion FMDV mutant based on a serotype O FMDV, the wild-type virus O/YS/CHA/05, with a 60-amino acid deletion in the 3A protein sequence, between residues 84 and 143. The rescued virus O/YS/CHA/05-Δ3A exhibited slower growth kinetics and formed smaller plaques compared to O/YS/CHA/05 in both BHK-21 and IBRS-2 cells, indicating that the 60-amino acid deletion in the 3A protein impaired FMDV replication. After 14 passages in BHK-21 cells, the replication capacity of the passaged virus O/YS/CHA/05-Δ3A-P14 returned to a level similar to the wild-type virus, suggesting that amino acid substitutions responsible for the enhanced replication capacity occurred in the genome of O/YS/CHA/05-Δ3A-P14. By sequence analysis, two amino acid substitutions, P153L in VP1 and T135I in 2C, were found in the O/YS/CHA/05-Δ3A-P14 genome compared to the O/YS/CHA/05-Δ3A genome. Subsequently, the amino acid substitutions VP1 P153L and 2C T135I were separately introduced into O/YS/CHA/05-Δ3A to rescue mutant viruses for examining their growth kinetics. Results showed that the 2C T135I instead of the VP1 P153L enhanced the virus replication capacity. The 2C T135I substitution also improved the replication of the wild-type virus, indicating that the effect of 2C T135I substitution on FMDV replication is not associated with the 3A deletion. Furthermore, our results showed that the T135I substitution in the nonstructural protein 2C enhanced O/YS/CHA/05 replication through promoting viral RNA synthesis.

HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

PubMed Central

Moisy, Dorothée; Avilov, Sergiy V.; Jacob, Yves; Laoide, Brid M.; Ge, Xingyi; Baudin, Florence; Jestin, Jean-Luc

2012-01-01

Influenza virus has evolved replication strategies that hijack host cell pathways. To uncover interactions between viral macromolecules and host proteins, we applied a phage display strategy. A library of human cDNA expression products displayed on filamentous phages was submitted to affinity selection for influenza viral ribonucleoproteins (vRNPs). High-mobility-group box (HMGB) proteins were found to bind to the nucleoprotein (NP) component of vRNPs. HMGB1 and HMGB2 bind directly to the purified NP in the absence of viral RNA, and the HMG box A domain is sufficient to bind the NP. We show that HMGB1 associates with the viral NP in the nuclei of infected cells, promotes viral growth, and enhances the activity of the viral polymerase. The presence of a functional HMGB1 DNA-binding site is required to enhance influenza virus replication. Glycyrrhizin, which reduces HMGB1 binding to DNA, inhibits influenza virus polymerase activity. Our data show that the HMGB1 protein can play a significant role in intranuclear replication of influenza viruses, thus extending previous findings on the bornavirus and on a number of DNA viruses. PMID:22696656

Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells.

PubMed

Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J; Méndez, Ernesto; Arias, Carlos F

2015-10-01

Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells

PubMed Central

Murillo, Andrea; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Méndez, Ernesto

2015-01-01

ABSTRACT Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication. PMID:26246569

Long-term protection against SHIV89.6P replication in HIV-1 Tat vaccinated cynomolgus monkeys.

PubMed

Maggiorella, Maria Teresa; Baroncelli, Silvia; Michelini, Zuleika; Fanales-Belasio, Emanuele; Moretti, Sonia; Sernicola, Leonardo; Cara, Andrea; Negri, Donatella R M; Buttò, Stefano; Fiorelli, Valeria; Tripiciano, Antonella; Scoglio, Arianna; Caputo, Antonella; Borsetti, Alessandra; Ridolfi, Barbara; Bona, Roberta; ten Haaft, Peter; Macchia, Iole; Leone, Pasqualina; Pavone-Cossut, Maria Rosaria; Nappi, Filomena; Ciccozzi, Massimo; Heeney, Jonathan; Titti, Fausto; Cafaro, Aurelio; Ensoli, Barbara

2004-09-03

Vaccination with a biologically active Tat protein or tat DNA contained infection with the highly pathogenic SHIV89.6P virus, preventing CD4 T-cell decline and disease onset. Here we show that protection was prolonged, since neither CD4 T-cell decline nor active virus replication was observed in all vaccinated animals that controlled virus replication up to week 104 after the challenge. In contrast, virus persisted and replicated in peripheral blood mononuclear cells and lymph nodes of infected animals, two of which died. Tat-specific antibody, CD4 and CD8 T-cell responses were high and stable only in the animals controlling the infection. In contrast, Gag-specific antibody production and CD4 and CD8 T-cell responses were consistently and persistently positive only in the monkeys that did not control primary virus replication. These results indicate that vaccination with Tat protein or DNA induced long-term memory Tat-specific immune responses and controlled primary infection at its early stages allowing a long-term containment of virus replication and spread in blood and tissues.

2'-5'-Oligoadenylate Synthetase-Like Protein Inhibits Respiratory Syncytial Virus Replication and Is Targeted by the Viral Nonstructural Protein 1.

PubMed

Dhar, Jayeeta; Cuevas, Rolando A; Goswami, Ramansu; Zhu, Jianzhong; Sarkar, Saumendra N; Barik, Sailen

2015-10-01

2'-5'-Oligoadenylate synthetase-like protein (OASL) is an interferon-inducible antiviral protein. Here we describe differential inhibitory activities of human OASL and the two mouse OASL homologs against respiratory syncytial virus (RSV) replication. Interestingly, nonstructural protein 1 (NS1) of RSV promoted proteasome-dependent degradation of specific OASL isoforms. We conclude that OASL acts as a cellular antiviral protein and that RSV NS1 suppresses this function to evade cellular innate immunity and allow virus growth. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

The nucleolar phosphoprotein B23 targets Newcastle disease virus matrix protein to the nucleoli and facilitates viral replication.

PubMed

Duan, Zhiqiang; Chen, Jian; Xu, Haixu; Zhu, Jie; Li, Qunhui; He, Liang; Liu, Huimou; Hu, Shunlin; Liu, Xiufan

2014-03-01

The cellular nucleolar proteins are reported to facilitate the replication cycles of some human and animal viruses by interaction with viral proteins. In this study, a nucleolar phosphoprotein B23 was identified to interact with Newcastle disease virus (NDV) matrix (M) protein. We found that NDV M protein accumulated in the nucleolus by binding B23 early in infection, but resulted in the redistribution of B23 from the nucleoli to the nucleoplasm later in infection. In vitro binding studies utilizing deletion mutants indicated that amino acids 30-60 of M and amino acids 188-245 of B23 were required for binding. Furthermore, knockdown of B23 by siRNA or overexpression of B23 or M-binding B23-derived polypeptides remarkably reduced cytopathic effect and inhibited NDV replication. Collectively, we show that B23 facilitates NDV replication by targeting M to the nucleolus, demonstrating for the first time a direct role for nucleolar protein B23 in a paramyxovirus replication process. Copyright © 2014 Elsevier Inc. All rights reserved.

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement.

PubMed

Cho, Sang-Yun; Cho, Won Kyong; Sohn, Seong-Han; Kim, Kook-Hyung

2012-01-06

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP. Copyright © 2011 Elsevier Inc. All rights reserved.

Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation.

PubMed

Tran, Anh T; Cortens, John P; Du, Qiujiang; Wilkins, John A; Coombs, Kevin M

2013-01-01

Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.

Influenza Virus Induces Apoptosis via BAD-Mediated Mitochondrial Dysregulation

PubMed Central

Tran, Anh T.; Cortens, John P.; Du, Qiujiang; Wilkins, John A.

2013-01-01

Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication. PMID:23135712

Quantitative Proteomic Analysis of the Influenza A Virus Nonstructural Proteins NS1 and NS2 during Natural Cell Infection Identifies PACT as an NS1 Target Protein and Antiviral Host Factor

PubMed Central

Tawaratsumida, Kazuki; Phan, Van; Hrincius, Eike R.; High, Anthony A.; Webby, Richard; Redecke, Vanessa

2014-01-01

ABSTRACT Influenza A virus (IAV) replication depends on the interaction of virus proteins with host factors. The viral nonstructural protein 1 (NS1) is essential in this process by targeting diverse cellular functions, including mRNA splicing and translation, cell survival, and immune defense, in particular the type I interferon (IFN-I) response. In order to identify host proteins targeted by NS1, we established a replication-competent recombinant IAV that expresses epitope-tagged forms of NS1 and NS2, which are encoded by the same gene segment, allowing purification of NS proteins during natural cell infection and analysis of interacting proteins by quantitative mass spectrometry. We identified known NS1- and NS2-interacting proteins but also uncharacterized proteins, including PACT, an important cofactor for the IFN-I response triggered by the viral RNA-sensor RIG-I. We show here that NS1 binds PACT during virus replication and blocks PACT/RIG-I-mediated activation of IFN-I, which represents a critical event for the host defense. Protein interaction and interference with IFN-I activation depended on the functional integrity of the highly conserved RNA binding domain of NS1. A mutant virus with deletion of NS1 induced high levels of IFN-I in control cells, as expected; in contrast, shRNA-mediated knockdown of PACT compromised IFN-I activation by the mutant virus, but not wild-type virus, a finding consistent with the interpretation that PACT (i) is essential for IAV recognition and (ii) is functionally compromised by NS1. Together, our data describe a novel approach to identify virus-host protein interactions and demonstrate that NS1 interferes with PACT, whose function is critical for robust IFN-I production. IMPORTANCE Influenza A virus (IAV) is an important human pathogen that is responsible for annual epidemics and occasional devastating pandemics. Viral replication and pathogenicity depends on the interference of viral factors with components of the host defense system, particularly the type I interferon (IFN-I) response. The viral NS1 protein is known to counteract virus recognition and IFN-I production, but the molecular mechanism is only partially defined. We used a novel proteomic approach to identify host proteins that are bound by NS1 during virus replication and identified the protein PACT, which had previously been shown to be involved in virus-mediated IFN-I activation. We find that NS1 prevents PACT from interacting with an essential component of the virus recognition pathway, RIG-I, thereby disabling efficient IFN-I production. These observations provide an important piece of information on how IAV efficiently counteracts the host immune defense. PMID:24899174

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

Measles virus: Background and oncolytic virotherapy.

PubMed

Bhattacharjee, Sankhajit; Yadava, Pramod Kumar

2018-03-01

Measles is a highly transmissible disease caused by measles virus and remains a major cause of child mortality in developing countries. Measles virus nucleoprotein (N) encapsidates the RNA genome of the virus for providing protection from host cell endonucleases and for specific recognition of viral RNA as template for transcription and replication. This protein is over-expressed at the time of viral replication. The C-terminal of N protein is intrinsically disordered, which enables this protein to interact with several host cell proteins. It was previously proved in our laboratory that N expressing human cancerous cells undergo programmed cell death because of reactive oxygen species (ROS) generation as well as Caspase 3 activation. The phosphoprotein (P) along with N protein enclosed viral genomic RNA forming a ribonucleoprotein complex (RNP). It also establishes interaction with the large protein (L) i.e. viral RNA dependent RNA polymerase to ensure viral replication within host cells. The host cell receptors of this virus are CD46, SLAM/CD150 and PVRL4. Measles virus is latently oncotropic in nature and possesses oncolytic property by syncytia formation. We try to highlight the application of this property in developing a virotherapeutic vehicle.

Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA.

PubMed Central

Prats, A C; Sarih, L; Gabus, C; Litvak, S; Keith, G; Darlix, J L

1988-01-01

Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5' end of the RNA genome. An RSV mutant with a Val-Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way. Images PMID:2458920

Small finger protein of avian and murine retroviruses has nucleic acid annealing activity and positions the replication primer tRNA onto genomic RNA.

PubMed

Prats, A C; Sarih, L; Gabus, C; Litvak, S; Keith, G; Darlix, J L

1988-06-01

Retrovirus virions carry a diploid genome associated with a large number of small viral finger protein molecules which are required for encapsidation. Our present results show that finger protein p12 of Rous sarcoma virus (RSV) and p10 of murine leukaemia virus (MuLV) positions replication primer tRNA on the replication initiation site (PBS) at the 5' end of the RNA genome. An RSV mutant with a Val-Pro insertion in the finger motif of p12 is able to partially encapsidate genomic RNA but is not infectious because mutated p12 is incapable of positioning the replication primer, tRNATrp. Since all known replication competent retroviruses, and the plant virus CaMV, code for finger proteins analogous to RSV p12 or MuLV p10, the initial stage of reverse transcription in avian, mammalian and human retroviruses and in CaMV is probably controlled in an analogous way.

Role of Bunyamwera Orthobunyavirus NSs protein in infection of mosquito cells.

PubMed

Szemiel, Agnieszka M; Failloux, Anna-Bella; Elliott, Richard M

2012-01-01

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line. To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus. Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.

Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

PubMed Central

Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

2017-01-01

ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time-controlled windows for HSV-1 replication. High Rep protein levels in S/G2 phase support AAV2 replication and inhibit HSV-1 replication. Conversely, low Rep protein levels in G1 phase permit HSV-1 replication but are insufficient for AAV2 replication. This allows both viruses to productively replicate in distinct sets of dividing cells. PMID:28515305

Sterol Binding by the Tombusviral Replication Proteins Is Essential for Replication in Yeast and Plants.

PubMed

Xu, Kai; Nagy, Peter D

2017-04-01

Membranous structures derived from various organelles are important for replication of plus-stranded RNA viruses. Although the important roles of co-opted host proteins in RNA virus replication have been appreciated for a decade, the equally important functions of cellular lipids in virus replication have been gaining full attention only recently. Previous work with Tomato bushy stunt tombusvirus (TBSV) in model host yeast has revealed essential roles for phosphatidylethanolamine and sterols in viral replication. To further our understanding of the role of sterols in tombusvirus replication, in this work we showed that the TBSV p33 and p92 replication proteins could bind to sterols in vitro The sterol binding by p33 is supported by cholesterol recognition/interaction amino acid consensus (CRAC) and CARC-like sequences within the two transmembrane domains of p33. Mutagenesis of the critical Y amino acids within the CRAC and CARC sequences blocked TBSV replication in yeast and plant cells. We also showed the enrichment of sterols in the detergent-resistant membrane (DRM) fractions obtained from yeast and plant cells replicating TBSV. The DRMs could support viral RNA synthesis on both the endogenous and exogenous templates. A lipidomic approach showed the lack of enhancement of sterol levels in yeast and plant cells replicating TBSV. The data support the notion that the TBSV replication proteins are associated with sterol-rich detergent-resistant membranes in yeast and plant cells. Together, the results obtained in this study and the previously published results support the local enrichment of sterols around the viral replication proteins that is critical for TBSV replication. IMPORTANCE One intriguing aspect of viral infections is their dependence on efficient subcellular assembly platforms serving replication, virion assembly, or virus egress via budding out of infected cells. These assembly platforms might involve sterol-rich membrane microdomains, which are heterogeneous and highly dynamic nanoscale structures usurped by various viruses. Here, we demonstrate that TBSV p33 and p92 replication proteins can bind to sterol in vitro Mutagenesis analysis of p33 within the CRAC and CARC sequences involved in sterol binding shows the important connection between the abilities of p33 to bind to sterol and to support TBSV replication in yeast and plant cells. Together, the results further strengthen the model that cellular sterols are essential as proviral lipids during viral replication. Copyright © 2017 American Society for Microbiology.

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8

PubMed Central

Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R.; Kolb, Gaëlle; Wiley, Michael R.; Jozwick, Lucas; Kuhn, Jens H.; Palacios, Gustavo; Radoshitzky, Sheli R.; J. Le Grice, Stuart F.; Johnson, Reed F.

2016-01-01

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA–RNA and RNA–protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2′-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3′ stem-loop (nucleotides 1868–1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication. PMID:27651462

The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

PubMed

Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

2018-02-01

Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

Cell-Specific Establishment of Poliovirus Resistance to an Inhibitor Targeting a Cellular Protein

PubMed Central

Viktorova, Ekaterina G.; Nchoutmboube, Jules; Ford-Siltz, Lauren A.

2015-01-01

ABSTRACT It is hypothesized that targeting stable cellular factors involved in viral replication instead of virus-specific proteins may raise the barrier for development of resistant mutants, which is especially important for highly adaptable small (+)RNA viruses. However, contrary to this assumption, the accumulated evidence shows that these viruses easily generate mutants resistant to the inhibitors of cellular proteins at least in some systems. We investigated here the development of poliovirus resistance to brefeldin A (BFA), an inhibitor of the cellular protein GBF1, a guanine nucleotide exchange factor for the small cellular GTPase Arf1. We found that while resistant viruses can be easily selected in HeLa cells, they do not emerge in Vero cells, in spite that in the absence of the drug both cultures support robust virus replication. Our data show that the viral replication is much more resilient to BFA than functioning of the cellular secretory pathway, suggesting that the role of GBF1 in the viral replication is independent of its Arf activating function. We demonstrate that the level of recruitment of GBF1 to the replication complexes limits the establishment and expression of a BFA resistance phenotype in both HeLa and Vero cells. Moreover, the BFA resistance phenotype of poliovirus mutants is also cell type dependent in different cells of human origin and results in a fitness loss in the form of reduced efficiency of RNA replication in the absence of the drug. Thus, a rational approach to the development of host-targeting antivirals may overcome the superior adaptability of (+)RNA viruses. IMPORTANCE Compared to the number of viral diseases, the number of available vaccines is miniscule. For some viruses vaccine development has not been successful after multiple attempts, and for many others vaccination is not a viable option. Antiviral drugs are needed for clinical practice and public health emergencies. However, viruses are highly adaptable and can easily generate mutants resistant to practically any compounds targeting viral proteins. An alternative approach is to target stable cellular factors recruited for the virus-specific functions. In the present study, we analyzed the factors permitting and restricting the establishment of the resistance of poliovirus, a small (+)RNA virus, to brefeldin A (BFA), a drug targeting a cellular component of the viral replication complex. We found that the emergence and replication potential of resistant mutants is cell type dependent and that BFA resistance reduces virus fitness. Our data provide a rational approach to the development of antiviral therapeutics targeting host factors. PMID:25653442

Activation of DNA Damage Repair Pathways by Murine Polyomavirus

PubMed Central

Heiser, Katie; Nicholas, Catherine; Garcea, Robert L.

2016-01-01

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. PMID:27529739

Inhibition of avian tumor virus replication by CCCH-type zinc finger antiviral protein

PubMed Central

Zhu, Mingjun; Ma, Xiaoqian; Cui, Xiyao; Zhou, Jing; Li, Chengui; Huang, Libo; Shang, Yingli; Cheng, Ziqiang

2017-01-01

CCCH type zinc finger antiviral protein (ZAP) is a host restriction factor that inhibits the replication of a variety of viruses in mammals. However, little is known about its antiviral activity on avian tumor virus. Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, induces myelocytomas and various other tumors in meat and egg type chickens. Here, we identified a chicken ZAP (chZAP) that increased at early stage, and subsequently decreased after infection of ALV-J in DF-1 cells, indicating the inducible feature of the endogenous chZAP. To demonstrate the inhibitory effect on ALV-J replication by chZAP, we expressed exogenous chZAP by lentivirus based vectors in DF-1 cells that infected by ALV-J. The result showed that overexpression of chZAP significantly inhibited ALV-J replication at both mRNA level and protein level. Consequently, knockdown of endogenous chZAP by RNAi facilitated ALV-J replication in DF-1 cells. Further, we demonstrated that chZAP interacts with SU protein (encode by gp85 gene) of ALV-J in cytoplasm. Taken together, our results demonstrated that chZAP inhibits ALV-J by both mRNA and protein pathway and it may shed light on a novel antiviral approach in poultry. PMID:28938603

The Nucleocapsid Protein of Coronaviruses Acts as a Viral Suppressor of RNA Silencing in Mammalian Cells.

PubMed

Cui, Lei; Wang, Haiying; Ji, Yanxi; Yang, Jie; Xu, Shan; Huang, Xingyu; Wang, Zidao; Qin, Lei; Tien, Po; Zhou, Xi; Guo, Deyin; Chen, Yu

2015-09-01

RNA interference (RNAi) is a process of eukaryotic posttranscriptional gene silencing that functions in antiviral immunity in plants, nematodes, and insects. However, recent studies provided strong supports that RNAi also plays a role in antiviral mechanism in mammalian cells. To combat RNAi-mediated antiviral responses, many viruses encode viral suppressors of RNA silencing (VSR) to facilitate their replication. VSRs have been widely studied for plant and insect viruses, but only a few have been defined for mammalian viruses currently. We identified a novel VSR from coronaviruses, a group of medically important mammalian viruses including Severe acute respiratory syndrome coronavirus (SARS-CoV), and showed that the nucleocapsid protein (N protein) of coronaviruses suppresses RNAi triggered by either short hairpin RNAs or small interfering RNAs in mammalian cells. Mouse hepatitis virus (MHV) is closely related to SARS-CoV in the family Coronaviridae and was used as a coronavirus replication model. The replication of MHV increased when the N proteins were expressed in trans, while knockdown of Dicer1 or Ago2 transcripts facilitated the MHV replication in mammalian cells. These results support the hypothesis that RNAi is a part of the antiviral immunity responses in mammalian cells. IMPORTANCE RNAi has been well known to play important antiviral roles from plants to invertebrates. However, recent studies provided strong supports that RNAi is also involved in antiviral response in mammalian cells. An important indication for RNAi-mediated antiviral activity in mammals is the fact that a number of mammalian viruses encode potent suppressors of RNA silencing. Our results demonstrate that coronavirus N protein could function as a VSR through its double-stranded RNA binding activity. Mutational analysis of N protein allowed us to find out the critical residues for the VSR activity. Using the MHV-A59 as the coronavirus replication model, we showed that ectopic expression of SARS-CoV N protein could promote MHV replication in RNAi-active cells but not in RNAi-depleted cells. These results indicate that coronaviruses encode a VSR that functions in the replication cycle and provide further evidence to support that RNAi-mediated antiviral response exists in mammalian cells.

Rabies viruses leader RNA interacts with host Hsc70 and inhibits virus replication

PubMed Central

Zhang, Ran; Liu, Chuangang; Cao, Yunzi; Jamal, Muhammad; Chen, Xi; Zheng, Jinfang; Li, Liang; You, Jing; Zhu, Qi; Liu, Shiyong; Dai, Jinxia; Cui, Min; Fu, Zhen F.; Cao, Gang

2017-01-01

Viruses have been shown to be equipped with regulatory RNAs to evade host defense system. It has long been known that rabies virus (RABV) transcribes a small regulatory RNA, leader RNA (leRNA), which mediates the transition from viral RNA transcription to replication. However, the detailed molecular mechanism remains enigmatic. In the present study, we determined the genetic architecture of RABV leRNA and demonstrated its inhibitory effect on replication of wild-type rabies, DRV-AH08. The RNA immunoprecipitation results suggest that leRNA inhibits RABV replication via interfering the binding of RABV nucleoprotein with genomic RNA. Furthermore, we identified heat shock cognate 70 kDa protein (Hsc70) as a leRNA host cellular interacting protein, of which the expression level was dynamically regulated by RABV infection. Notably, our data suggest that Hsc70 was involved in suppressing RABV replication by leader RNA. Finally, our experiments imply that leRNA might be potentially useful as a novel drug in rabies post-exposure prophylaxis. Together, this study suggested leRNA in concert with its host interacting protein Hsc70, dynamically down-regulate RABV replication. PMID:28388579

Generation of Recombinant Polioviruses Harboring RNA Affinity Tags in the 5′ and 3′ Noncoding Regions of Genomic RNAs

PubMed Central

Flather, Dylan; Cathcart, Andrea L.; Cruz, Casey; Baggs, Eric; Ngo, Tuan; Gershon, Paul D.; Semler, Bert L.

2016-01-01

Despite being intensely studied for more than 50 years, a complete understanding of the enterovirus replication cycle remains elusive. Specifically, only a handful of cellular proteins have been shown to be involved in the RNA replication cycle of these viruses. In an effort to isolate and identify additional cellular proteins that function in enteroviral RNA replication, we have generated multiple recombinant polioviruses containing RNA affinity tags within the 3′ or 5′ noncoding region of the genome. These recombinant viruses retained RNA affinity sequences within the genome while remaining viable and infectious over multiple passages in cell culture. Further characterization of these viruses demonstrated that viral protein production and growth kinetics were unchanged or only slightly altered relative to wild type poliovirus. However, attempts to isolate these genetically-tagged viral genomes from infected cells have been hindered by high levels of co-purification of nonspecific proteins and the limited matrix-binding efficiency of RNA affinity sequences. Regardless, these recombinant viruses represent a step toward more thorough characterization of enterovirus ribonucleoprotein complexes involved in RNA replication. PMID:26861382

The Unstructured Paramyxovirus Nucleocapsid Protein Tail Domain Modulates Viral Pathogenesis through Regulation of Transcriptase Activity.

PubMed

Thakkar, Vidhi D; Cox, Robert M; Sawatsky, Bevan; da Fontoura Budaszewski, Renata; Sourimant, Julien; Wabbel, Katrin; Makhsous, Negar; Greninger, Alexander L; von Messling, Veronika; Plemper, Richard K

2018-04-15

The paramyxovirus replication machinery comprises the viral large (L) protein and phosphoprotein (P-protein) in addition to the nucleocapsid (N) protein, which encapsidates the single-stranded RNA genome. Common to paramyxovirus N proteins is a C-terminal tail (Ntail). The mechanistic role and relevance for virus replication of the structurally disordered central Ntail section are unknown. Focusing initially on members of the Morbillivirus genus, a series of measles virus (MeV) and canine distemper virus (CDV) N proteins were generated with internal deletions in the unstructured tail section. N proteins with large tail truncations remained bioactive in mono- and polycistronic minireplicon assays and supported efficient replication of recombinant viruses. Bioactivity of Ntail mutants extended to N proteins derived from highly pathogenic Nipah virus. To probe an effect of Ntail truncations on viral pathogenesis, recombinant CDVs were analyzed in a lethal CDV/ferret model of morbillivirus disease. The recombinant viruses displayed different stages of attenuation ranging from ameliorated clinical symptoms to complete survival of infected animals, depending on the molecular nature of the Ntail truncation. Reinfection of surviving animals with pathogenic CDV revealed robust protection against a lethal challenge. The highly attenuated virus was genetically stable after ex vivo passaging and recovery from infected animals. Mechanistically, gradual viral attenuation coincided with stepwise altered viral transcriptase activity in infected cells. These results identify the central Ntail section as a determinant for viral pathogenesis and establish a novel platform to engineer gradual virus attenuation for next-generation paramyxovirus vaccine design. IMPORTANCE Investigating the role of the paramyxovirus N protein tail domain (Ntail) in virus replication, we demonstrated in this study that the structurally disordered central Ntail region is a determinant for viral pathogenesis. We show that internal deletions in this Ntail region of up to 55 amino acids in length are compatible with efficient replication of recombinant viruses in cell culture but result in gradual viral attenuation in a lethal canine distemper virus (CDV)/ferret model. Mechanistically, we demonstrate a role of the intact Ntail region in the regulation of viral transcriptase activity. Recombinant viruses with Ntail truncations induce protective immunity against lethal challenge of ferrets with pathogenic CDV. This identification of the unstructured central Ntail domain as a nonessential paramyxovirus pathogenesis factor establishes a foundation for harnessing Ntail truncations for vaccine engineering against emerging and reemerging members of the paramyxovirus family. Copyright © 2018 American Society for Microbiology.

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells.

PubMed

Nain, Minu; Mukherjee, Sriparna; Karmakar, Sonali Porey; Paton, Adrienne W; Paton, James C; Abdin, M Z; Basu, Anirban; Kalia, Manjula; Vrati, Sudhanshu

2017-03-15

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is the leading cause of viral encephalitis in Southeast Asia with potential to become a global pathogen. Here, we identify glucose-regulated protein 78 (GRP78) as an important host protein for virus entry and replication. Using the plasma membrane fractions from mouse neuronal (Neuro2a) cells, mass spectroscopy analysis identified GRP78 as a protein interacting with recombinant JEV envelope protein domain III. GRP78 was found to be expressed on the plasma membranes of Neuro2a cells, mouse primary neurons, and human epithelial Huh-7 cells. Antibodies against GRP78 significantly inhibited JEV entry in all three cell types, suggesting an important role of the protein in virus entry. Depletion of GRP78 by small interfering RNA (siRNA) significantly blocked JEV entry into Neuro2a cells, further supporting its role in virus uptake. Immunofluorescence studies showed extensive colocalization of GRP78 with JEV envelope protein in virus-infected cells. This interaction was also confirmed by immunoprecipitation studies. Additionally, GRP78 was shown to have an important role in JEV replication, as treatment of cells post-virus entry with subtilase cytotoxin that specifically cleaved GRP78 led to a substantial reduction in viral RNA replication and protein synthesis, resulting in significantly reduced extracellular virus titers. Our results indicate that GRP78, an endoplasmic reticulum chaperon of the HSP70 family, is a novel host factor involved at multiple steps of the JEV life cycle and could be a potential therapeutic target. IMPORTANCE Recent years have seen a rapid spread of mosquito-borne diseases caused by flaviviruses. The flavivirus family includes West Nile, dengue, Japanese encephalitis, and Zika viruses, which are major threats to public health with potential to become global pathogens. JEV is the major cause of viral encephalitis in several parts of Southeast Asia, affecting a predominantly pediatric population with a high mortality rate. This study is focused on identification of crucial host factors that could be targeted to cripple virus infection and ultimately lead to development of effective antivirals. We have identified a cellular protein, GRP78, that plays a dual role in virus entry and virus replication, two crucial steps of the virus life cycle, and thus is a novel host factor that could be a potential therapeutic target. Copyright © 2017 American Society for Microbiology.

Cellular Restriction Factors of Feline Immunodeficiency Virus

PubMed Central

Zielonka, Jörg; Münk, Carsten

2011-01-01

Lentiviruses are known for their narrow cell- and species-tropisms, which are determined by cellular proteins whose absence or presence either support viral replication (dependency factors, cofactors) or inhibit viral replication (restriction factors). Similar to Human immunodeficiency virus type 1 (HIV-1), the cat lentivirus Feline immunodeficiency virus (FIV) is sensitive to recently discovered cellular restriction factors from non-host species that are able to stop viruses from replicating. Of particular importance are the cellular proteins APOBEC3, TRIM5α and tetherin/BST-2. In general, lentiviruses counteract or escape their species’ own variant of the restriction factor, but are targeted by the orthologous proteins of distantly related species. Most of the knowledge regarding lentiviral restriction factors has been obtained in the HIV-1 system; however, much less is known about their effects on other lentiviruses. We describe here the molecular mechanisms that explain how FIV maintains its replication in feline cells, but is largely prevented from cross-species infections by cellular restriction factors. PMID:22069525

The molecular biology of Bluetongue virus replication.

PubMed

Patel, Avnish; Roy, Polly

2014-03-01

The members of Orbivirus genus within the Reoviridae family are arthropod-borne viruses which are responsible for high morbidity and mortality in ruminants. Bluetongue virus (BTV) which causes disease in livestock (sheep, goat, cattle) has been in the forefront of molecular studies for the last three decades and now represents the best understood orbivirus at a molecular and structural level. The complex nature of the virion structure has been well characterised at high resolution along with the definition of the virus encoded enzymes required for RNA replication; the ordered assembly of the capsid shell as well as the protein and genome sequestration required for it; and the role of host proteins in virus entry and virus release. More recent developments of Reverse Genetics and Cell-Free Assembly systems have allowed integration of the accumulated structural and molecular knowledge to be tested at meticulous level, yielding higher insight into basic molecular virology, from which the rational design of safe efficacious vaccines has been possible. This article is centred on the molecular dissection of BTV with a view to understanding the role of each protein in the virus replication cycle. These areas are important in themselves for BTV replication but they also indicate the pathways that related viruses, which includes viruses that are pathogenic to man and animals, might also use providing an informed starting point for intervention or prevention. Copyright © 2014 Elsevier B.V. All rights reserved.

Epstein-Barr virus origin of lytic replication mediates association of replicating episomes with promyelocytic leukaemia protein nuclear bodies and replication compartments.

PubMed

Amon, Wolfgang; White, Robert E; Farrell, Paul J

2006-05-01

Epstein-Barr virus (EBV) establishes a latent persistence from which it can be reactivated to undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the viral origin of lytic replication (ori lyt) is required in cis for late-gene expression. During the lytic cycle, the viral genome forms replication compartments, which are usually adjacent to promyelocytic leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain-enhanced green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline operator sequence array. ori lyt mediated the production of plasmid replication compartments that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were visualized in the same cells and replicated in similar regions of the nucleus, further supporting the validity of the plasmids for studying late-gene regulation.

Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

PubMed Central

Walsh, Derek; Mathews, Michael B.; Mohr, Ian

2013-01-01

Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus. PMID:23209131

Expression of the lef5 gene from Spodoptera exigua multiple nucleopolyhedrovirus contributes to the baculovirus stability in cell culture.

PubMed

Martínez-Solís, María; Jakubowska, Agata K; Herrero, Salvador

2017-10-01

Baculoviruses are a broad group of viruses infecting insects, predominately of the order Lepidoptera. They are used worldwide as biological insecticides and as expression vectors to produce recombinant proteins. Baculoviruses replicate in their host, although several cell lines have been developed for in vitro replication. Nevertheless, replication of baculoviruses in cell culture involves the generation of defective viruses with a decrease in productivity and virulence. Transcriptional studies of the Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infective process revealed differences in the expression patterns when the virus replicated under in vitro (Se301 cells) or in vivo (S. exigua larvae) conditions. The late expression factor 5 (lef5) gene was found to be highly overexpressed when the virus replicates in larvae. To test the possible role of lef5 expression in viral stability, recombinant AcMNPV expressing the lef5 gene from SeMNPV (Se-lef5) was generated and its stability was monitored during successive infection passages in Sf21 cells by evaluating the loss of several essential and non-essential genes. The gfp transgene was more stable in those viruses expressing the Se-LEF5 protein and the GFP-defective viruses were accumulated at a lower level when compared to its control viruses, confirming the positive influence of lef5 in viral stability during the multiplication process. This work describes for the first time a viral factor involved in transgene stability when baculoviruses replicate in cell culture, opening new ways to facilitate the in vitro production of recombinant proteins using baculovirus.

Morphological and biochemical characterization of the membranous hepatitis C virus replication compartment.

PubMed

Paul, David; Hoppe, Simone; Saher, Gesine; Krijnse-Locker, Jacomine; Bartenschlager, Ralf

2013-10-01

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses.

Morphological and Biochemical Characterization of the Membranous Hepatitis C Virus Replication Compartment

PubMed Central

Hoppe, Simone; Saher, Gesine; Krijnse-Locker, Jacomine

2013-01-01

Like all other positive-strand RNA viruses, hepatitis C virus (HCV) induces rearrangements of intracellular membranes that are thought to serve as a scaffold for the assembly of the viral replicase machinery. The most prominent membranous structures present in HCV-infected cells are double-membrane vesicles (DMVs). However, their composition and role in the HCV replication cycle are poorly understood. To gain further insights into the biochemcial properties of HCV-induced membrane alterations, we generated a functional replicon containing a hemagglutinin (HA) affinity tag in nonstructural protein 4B (NS4B), the supposed scaffold protein of the viral replication complex. By using HA-specific affinity purification we isolated NS4B-containing membranes from stable replicon cells. Complementing biochemical and electron microscopy analyses of purified membranes revealed predominantly DMVs, which contained viral proteins NS3 and NS5A as well as enzymatically active viral replicase capable of de novo synthesis of HCV RNA. In addition to viral factors, co-opted cellular proteins, such as vesicle-associated membrane protein-associated protein A (VAP-A) and VAP-B, that are crucial for viral RNA replication, as well as cholesterol, a major structural lipid of detergent-resistant membranes, are highly enriched in DMVs. Here we describe the first isolation and biochemical characterization of HCV-induced DMVs. The results obtained underline their central role in the HCV replication cycle and suggest that DMVs are sites of viral RNA replication. The experimental approach described here is a powerful tool to more precisely define the molecular composition of membranous replication factories induced by other positive-strand RNA viruses, such as picorna-, arteri- and coronaviruses. PMID:23885072

Identification and Molecular Characterization of the Chloroplast Targeting Domain of Turnip yellow mosaic virus Replication Proteins

PubMed Central

Moriceau, Lucille; Jomat, Lucile; Bressanelli, Stéphane; Alcaide-Loridan, Catherine; Jupin, Isabelle

2017-01-01

Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus infecting plants. The TYMV 140K replication protein is a key organizer of viral replication complex (VRC) assembly, being responsible for recruitment of the viral polymerase and for targeting the VRCs to the chloroplast envelope where viral replication takes place. However, the structural requirements determining the subcellular localization and membrane association of this essential viral protein have not yet been defined. In this study, we investigated determinants for the in vivo chloroplast targeting of the TYMV 140K replication protein. Subcellular localization studies of deletion mutants identified a 41-residue internal sequence as the chloroplast targeting domain (CTD) of TYMV 140K; this sequence is sufficient to target GFP to the chloroplast envelope. The CTD appears to be located in the C-terminal extension of the methyltransferase domain—a region shared by 140K and its mature cleavage product 98K, which behaves as an integral membrane protein during infection. We predicted the CTD to fold into two amphipathic α-helices—a folding that was confirmed in vitro by circular dichroism spectroscopy analyses of a synthetic peptide. The importance for subcellular localization of the integrity of these amphipathic helices, and the function of 140K/98K, was demonstrated by performing amino acid substitutions that affected chloroplast targeting, membrane association and viral replication. These results establish a short internal α-helical peptide as an unusual signal for targeting proteins to the chloroplast envelope membrane, and provide new insights into membrane targeting of viral replication proteins—a universal feature of positive-strand RNA viruses. PMID:29312393

Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection

PubMed Central

Cui, Hongguang

2016-01-01

ABSTRACT The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage. IMPORTANCE Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature proteins, with the majority of them having been at least partially functionally characterized. However, the functional role of a small protein named 6K1 remains obscure. In this study, we showed that deletion of 6K1 or a short motif/region of 6K1 in the full-length cDNA clones of plum pox virus abolishes viral replication and that mutation of the N- or C-terminal cleavage sites of 6K1 to prevent its release from the polyprotein greatly attenuates or completely inhibits viral replication, suggesting its important role in potyviral infection. We report that 6K1 forms punctate structures and targets the replication vesicles in PPV-infected plant leaf cells at the early infection stage. Our data reveal that 6K1 is an important viral protein of the potyviral replication complex. PMID:26962227

A small stem-loop structure of the Ebola virus trailer is essential for replication and interacts with heat-shock protein A8.

PubMed

Sztuba-Solinska, Joanna; Diaz, Larissa; Kumar, Mia R; Kolb, Gaëlle; Wiley, Michael R; Jozwick, Lucas; Kuhn, Jens H; Palacios, Gustavo; Radoshitzky, Sheli R; J Le Grice, Stuart F; Johnson, Reed F

2016-11-16

Ebola virus (EBOV) is a single-stranded negative-sense RNA virus belonging to the Filoviridae family. The leader and trailer non-coding regions of the EBOV genome likely regulate its transcription, replication, and progeny genome packaging. We investigated the cis-acting RNA signals involved in RNA-RNA and RNA-protein interactions that regulate replication of eGFP-encoding EBOV minigenomic RNA and identified heat shock cognate protein family A (HSC70) member 8 (HSPA8) as an EBOV trailer-interacting host protein. Mutational analysis of the trailer HSPA8 binding motif revealed that this interaction is essential for EBOV minigenome replication. Selective 2'-hydroxyl acylation analyzed by primer extension analysis of the secondary structure of the EBOV minigenomic RNA indicates formation of a small stem-loop composed of the HSPA8 motif, a 3' stem-loop (nucleotides 1868-1890) that is similar to a previously identified structure in the replicative intermediate (RI) RNA and a panhandle domain involving a trailer-to-leader interaction. Results of minigenome assays and an EBOV reverse genetic system rescue support a role for both the panhandle domain and HSPA8 motif 1 in virus replication. Published by Oxford University Press on behalf of Nucleic Acids Research 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Archaeal Viruses: Diversity, Replication, and Structure.

PubMed

Dellas, Nikki; Snyder, Jamie C; Bolduc, Benjamin; Young, Mark J

2014-11-01

The Archaea-and their viruses-remain the most enigmatic of life's three domains. Once thought to inhabit only extreme environments, archaea are now known to inhabit diverse environments. Even though the first archaeal virus was described over 40 years ago, only 117 archaeal viruses have been discovered to date. Despite this small number, these viruses have painted a portrait of enormous morphological and genetic diversity. For example, research centered around the various steps of the archaeal virus life cycle has led to the discovery of unique mechanisms employed by archaeal viruses during replication, maturation, and virion release. In many instances, archaeal virus proteins display very low levels of sequence homology to other proteins listed in the public database, and therefore, structural characterization of these proteins has played an integral role in functional assignment. These structural studies have not only provided insights into structure-function relationships but have also identified links between viruses across all three domains of life.

Vaccinia Virus C9 Ankyrin Repeat/F-Box Protein Is a Newly Identified Antagonist of the Type I Interferon-Induced Antiviral State.

PubMed

Liu, Ruikang; Moss, Bernard

2018-05-01

Type I interferons (IFNs) induce expression of more than 300 cellular genes that provide protection against viruses and other pathogens. For survival, viruses evolved defenses to prevent the IFN response or counteract the IFN-induced antiviral state. However, because viruses and cells coevolved, the dynamic relationship between virus and host is difficult to discern. In the present study, we demonstrated that vaccinia virus with a large deletion near the left end of the genome had a diminished ability to replicate in cells that had been pretreated with beta interferon (IFN-β), suggesting that one or more of the missing 17 open reading frames (ORFs) encode an antagonist of the IFN-induced antiviral state. By systematically deleting groups of ORFs and then individual ORFs, the C9L gene was shown to be required for IFN resistance. Replication of the C9L deletion mutant (vΔC9) was impaired in human cells that had been pretreated with IFN-β. Expression of viral early genes occurred, but subsequent events, including genome uncoating, genome replication, and postreplicative gene expression, were inhibited. Expression of the C9 protein occurred prior to genome replication, consistent with an early role in counteracting the IFN-induced antiviral state. C9 contains six ankyrin repeat motifs and a near C-terminal F-box. Mass spectrometry and immunoblotting identified host proteins that copurified with a functional epitope-tagged C9. The most abundant proteins were components of the SCF (CUL1, SKP1, F-box) and signalosome/deneddylation complexes, which interact with each other, suggesting a possible role in proteolysis of one or more interferon-induced proteins. IMPORTANCE Poxviruses comprise a family of large DNA viruses that replicate in the cytoplasm of vertebrate and insect hosts and cause human and zoonotic diseases. In most cases the primary infection is moderated by innate immune defenses. Vertebrates, including fish, amphibians, reptiles, birds, and mammals, all produce type I interferon homologs. In humans, interferon stimulates the synthesis of more than 300 proteins thought to have roles in host defense. Conversely, viruses have evolved means to thwart the host defenses. We are attempting to deconstruct the established virus-host relationship in order to better understand the molecular mechanisms involved. In the present study, we identified a vaccinia virus gene that prevents interferon-mediated inhibition of very early stages of viral replication and is conserved in orthopoxviruses. The viral protein was shown to interact with host proteins involved in proteolysis, suggesting that vaccinia virus may subvert the cellular apparatus for its own defense. Copyright © 2018 American Society for Microbiology.

Suppression of the pelo protein by Wolbachia and its effect on dengue virus in Aedes aegypti

PubMed Central

Asad, Sultan; Hussain, Mazhar; Hugo, Leon; Zhang, Guangmei; Watterson, Daniel

2018-01-01

The endosymbiont Wolbachia is known to block replication of several important arboviruses, including dengue virus (DENV), in the mosquito vector Aedes aegypti. So far, the exact mechanism of this viral inhibition is not fully understood. A recent study in Drosophila melanogaster has demonstrated an interaction between the pelo gene and Drosophila C virus. In this study, we explored the possible involvement of the pelo protein, that is involved in protein translation, in Wolbachia-mediated antiviral response and mosquito-DENV interaction. We found that pelo is upregulated during DENV replication and its silencing leads to reduced DENV virion production suggesting that it facilities DENV replication. However, in the presence of Wolbachia, specifically in female mosquitoes, the pelo protein is downregulated and its subcellular localization is altered, which could contribute to reduction in DENV replication in Ae. aegypti. In addition, we show that the microRNA aae-miR-2940-5p, whose abundance is highly enriched in Wolbachia-infected mosquitoes, might mediate regulation of pelo. Our data reveals identification of pelo as a host factor that is positively involved in DENV replication, and its suppression in the presence of Wolbachia may contribute to virus blocking exhibited by the endosymbiont. PMID:29641562

Y-box-binding protein 1 interacts with hepatitis C virus NS3/4A and influences the equilibrium between viral RNA replication and infectious particle production.

PubMed

Chatel-Chaix, Laurent; Melançon, Pierre; Racine, Marie-Ève; Baril, Martin; Lamarre, Daniel

2011-11-01

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.

Satellite RNAs of plant viruses: structures and biological effects.

PubMed Central

Roossinck, M J; Sleat, D; Palukaitis, P

1992-01-01

Plant viruses often contain parasites of their own, referred to as satellites. Satellite RNAs are dependent on their associated (helper) virus for both replication and encapsidation. Satellite RNAs vary from 194 to approximately 1,500 nucleotides (nt). The larger satellites (900 to 1,500 nt) contain open reading frames and express proteins in vitro and in vivo, whereas the smaller satellites (194 to 700 nt) do not appear to produce functional proteins. The smaller satellites contain a high degree of secondary structure involving 49 to 73% of their sequences, with the circular satellites containing more base pairing than the linear satellites. Many of the smaller satellites produce multimeric forms during replication. There are various models to account for their formation and role in satellite replication. Some of these smaller satellites encode ribozymes and are able to undergo autocatalytic cleavage. The enzymology of satellite replication is poorly understood, as is the replication of their helper viruses. In many cases the coreplication of satellites suppresses the replication of the helper virus genome. This is usually paralleled by a reduction in the disease induced by the helper virus; however, there are notable exceptions in which the satellite exacerbates the pathogenicity of the helper virus, albeit on only a limited number of hosts. The ameliorative satellites are being assessed as biocontrol agents of virus-induced disease. In greenhouse studies, satellites have been known to "spontaneously" appear in virus cultures. The possible origin of satellites will be briefly considered. PMID:1620065

Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

PubMed

Kaul, Artur; Stauffer, Sarah; Berger, Carola; Pertel, Thomas; Schmitt, Jennifer; Kallis, Stephanie; Zayas, Margarita; Lopez, Margarita Zayas; Lohmann, Volker; Luban, Jeremy; Bartenschlager, Ralf

2009-08-01

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

Hepatitis C Virus-Induced Cytoplasmic Organelles Use the Nuclear Transport Machinery to Establish an Environment Conducive to Virus Replication

PubMed Central

Neufeldt, Christopher J.; Joyce, Michael A.; Levin, Aviad; Steenbergen, Rineke H.; Pang, Daniel; Shields, Justin; Tyrrell, D. Lorne J.; Wozniak, Richard W.

2013-01-01

Hepatitis C virus (HCV) infection induces formation of a membranous web structure in the host cell cytoplasm where the viral genome replicates and virions assemble. The membranous web is thought to concentrate viral components and hide viral RNA from pattern recognition receptors. We have uncovered a role for nuclear pore complex proteins (Nups) and nuclear transport factors (NTFs) in the membranous web. We show that HCV infection leads to increased levels of cytoplasmic Nups that accumulate at sites enriched for HCV proteins. Moreover, we detected interactions between specific HCV proteins and both Nups and NTFs. We hypothesize that cytoplasmically positioned Nups facilitate formation of the membranous web and contribute to the compartmentalization of viral replication. Accordingly, we show that transport cargo proteins normally targeted to the nucleus are capable of entering regions of the membranous web, and that depletion of specific Nups or Kaps inhibits HCV replication and assembly. PMID:24204278

Genome-Wide Screen Reveals Valosin-Containing Protein Requirement for Coronavirus Exit from Endosomes

PubMed Central

Wong, Hui Hui; Kumar, Pankaj; Tay, Felicia Pei Ling; Moreau, Dimitri

2015-01-01

ABSTRACT Coronaviruses are RNA viruses with a large zoonotic reservoir and propensity for host switching, representing a real threat for public health, as evidenced by severe acute respiratory syndrome (SARS) and the emerging Middle East respiratory syndrome (MERS). Cellular factors required for their replication are poorly understood. Using genome-wide small interfering RNA (siRNA) screening, we identified 83 novel genes supporting infectious bronchitis virus (IBV) replication in human cells. Thirty of these hits can be placed in a network of interactions with viral proteins and are involved in RNA splicing, membrane trafficking, and ubiquitin conjugation. In addition, our screen reveals an unexpected role for valosin-containing protein (VCP/p97) in early steps of infection. Loss of VCP inhibits a previously uncharacterized degradation of the nucleocapsid N protein. This inhibition derives from virus accumulation in early endosomes, suggesting a role for VCP in the maturation of virus-loaded endosomes. The several host factors identified in this study may provide avenues for targeted therapeutics. IMPORTANCE Coronaviruses are RNA viruses representing a real threat for public health, as evidenced by SARS and the emerging MERS. However, cellular factors required for their replication are poorly understood. Using genome-wide siRNA screening, we identified novel genes supporting infectious bronchitis virus (IBV) replication in human cells. The several host factors identified in this study may provide directions for future research on targeted therapeutics. PMID:26311884

Activation of DNA damage repair pathways by murine polyomavirus.

PubMed

Heiser, Katie; Nicholas, Catherine; Garcea, Robert L

2016-10-01

Nuclear replication of DNA viruses activates DNA damage repair (DDR) pathways, which are thought to detect and inhibit viral replication. However, many DNA viruses also depend on these pathways in order to optimally replicate their genomes. We investigated the relationship between murine polyomavirus (MuPyV) and components of DDR signaling pathways including CHK1, CHK2, H2AX, ATR, and DNAPK. We found that recruitment and retention of DDR proteins at viral replication centers was independent of H2AX, as well as the viral small and middle T-antigens. Additionally, infectious virus production required ATR kinase activity, but was independent of CHK1, CHK2, or DNAPK signaling. ATR inhibition did not reduce the total amount of viral DNA accumulated, but affected the amount of virus produced, indicating a defect in virus assembly. These results suggest that MuPyV may utilize a subset of DDR proteins or non-canonical DDR signaling pathways in order to efficiently replicate and assemble. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle

PubMed Central

Londrigan, Sarah L.; Short, Kirsty R.; Ma, Joel; Gillespie, Leah; Rockman, Steven P.; Brooks, Andrew G.

2015-01-01

ABSTRACT Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle. IMPORTANCE Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages. PMID:26423941

Infection of Mouse Macrophages by Seasonal Influenza Viruses Can Be Restricted at the Level of Virus Entry and at a Late Stage in the Virus Life Cycle.

PubMed

Londrigan, Sarah L; Short, Kirsty R; Ma, Joel; Gillespie, Leah; Rockman, Steven P; Brooks, Andrew G; Reading, Patrick C

2015-12-01

Airway epithelial cells are susceptible to infection with seasonal influenza A viruses (IAV), resulting in productive virus replication and release. Macrophages (MΦ) are also permissive to IAV infection; however, virus replication is abortive. Currently, it is unclear how productive infection of MΦ is impaired or the extent to which seasonal IAV replicate in MΦ. Herein, we compared mouse MΦ and epithelial cells for their ability to support genomic replication and transcription, synthesis of viral proteins, assembly of virions, and release of infectious progeny following exposure to genetically defined IAV. We confirm that seasonal IAV differ in their ability to utilize cell surface receptors for infectious entry and that this represents one level of virus restriction. Following virus entry, we demonstrate synthesis of all eight segments of genomic viral RNA (vRNA) and mRNA, as well as seven distinct IAV proteins, in IAV-infected mouse MΦ. Although newly synthesized hemagglutinin (HA) and neuraminidase (NA) glycoproteins are incorporated into the plasma membrane and expressed at the cell surface, electron microscopy confirmed that virus assembly was defective in IAV-infected MΦ, defining a second level of restriction late in the virus life cycle. Seasonal influenza A viruses (IAV) and highly pathogenic avian influenza viruses (HPAI) infect macrophages, but only HPAI replicate productively in these cells. Herein, we demonstrate that impaired virus uptake into macrophages represents one level of restriction limiting infection by seasonal IAV. Following uptake, seasonal IAV do not complete productive replication in macrophages, representing a second level of restriction. Using murine macrophages, we demonstrate that productive infection is blocked late in the virus life cycle, such that virus assembly is defective and newly synthesized virions are not released. These studies represent an important step toward identifying host-encoded factors that block replication of seasonal IAV, but not HPAI, in macrophages. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Functional analysis of human foamy virus accessory reading frames.

PubMed Central

Baunach, G; Maurer, B; Hahn, H; Kranz, M; Rethwilm, A

1993-01-01

Foamy viruses belong to the retroviruses which possess a complex genome structure. The human foamy virus (HFV) isolate bears three open reading frames (the so-called bel genes) in the 3' region of the genome which have been reported to give rise to possibly six different proteins via alternative splicing (W. Muranyi and R. M. Flügel, J. Virol. 65:727-735, 1991). In order to analyze the requirements of these proteins for HFV replication in vitro, we constructed a set of single and combinatory bel gene mutants of an infectious molecular clone of HFV. The mutant which lacked the transacting activator, bel-1, was found to be replication incompetent. All other mutants replicated equally well and gave rise to comparable titers of infectious cell-free virus. When HFV proviruses were put under the control of a heterologous promoter (simian virus 40), none of the accessory gene products was found to be required for expression of structural (gag) proteins. There was no evidence for a posttranscriptional regulatory protein that is present in other complex retroviruses. Images PMID:8394455

hnRNP A2/B1 interacts with influenza A viral protein NS1 and inhibits virus replication potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nuclear export

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

Wang, Yimeng; Zhou, Jianhong; Du, Yuchun, E-mail: ydu@uark.edu

The NS1 protein of influenza viruses is a major virulence factor and exerts its function through interacting with viral/cellular RNAs and proteins. In this study, we identified heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) as an interacting partner of NS1 proteins by a proteomic method. Knockdown of hnRNP A2/B1 by small interfering RNA (siRNA) resulted in higher levels of NS vRNA, NS1 mRNA, and NS1 protein in the virus-infected cells. In addition, we demonstrated that hnRNP A2/B1 proteins are associated with NS1 and NS2 mRNAs and that knockdown of hnRNP A2/B1 promotes transport of NS1 mRNA from the nucleus to themore » cytoplasm in the infected cells. Lastly, we showed that knockdown of hnRNP A2/B1 leads to enhanced virus replication. Our results suggest that hnRNP A2/B1 plays an inhibitory role in the replication of influenza A virus in host cells potentially through suppressing NS1 RNA/protein levels and NS1 mRNA nucleocytoplasmic translocation. - Highlights: • Cellular protein hnRNP A2/B1 interacts with influenza viral protein NS1. • hnRNP A2/B1 suppresses the levels of NS1 protein, vRNA and mRNA in infected cells. • hnRNP A2/B1 protein is associated with NS1 and NS2 mRNAs. • hnRNP A2/B1 inhibits the nuclear export of NS1 mRNAs. • hnRNP A2/B1 inhibits influenza virus replication.« less

Ectromelia virus upregulates the expression of heat shock protein 70 to promote viral replication.

PubMed

Cheng, Wenyu; Jia, Huaijie; Wang, Xiaoxia; He, Xiaobing; Jin, Qiwang; Cao, Jingxin; Jing, Zhizhong

2018-08-01

The ectromelia virus (ECTV) is a mouse specific Orthopoxvirus that causes lethal infection in some mouse strains. ECTV infection of these mouse strains has been used as a valuable model for understanding the interplay between Orthopoxvirus species and their hosts, including variola virus in humans. Although poxviruses encode numerous proteins required for DNA and RNA synthesis, and are less dependent on host functions than other DNA viruses, a detailed understanding of the host factors required for the replication of poxviruses is lacking. Heat shock protein 70 (Hsp70) isoforms have been reported to serve various roles in the replication cycle of numerous viruses. In the present study, microarray and reverse transcription‑quantitative polymerase chain reaction analysis were conducted to investigate the host gene expression profiles following ECTV infection in mice and cell cultures. The results indicated that one Hsp70 isoform, Hsp70 member 1B (Hspa1b), was highly upregulated during ECTV infection in vitro and in vivo. Subsequently, overexpression of Hspa1b protein and small interfering RNA‑mediated gene silencing of Hspa1b revealed that Hspa1b is required for efficient replication of ECTV. Furthermore, the results demonstrated that ECTV replication may be significantly suppressed by two chemical Hspa1b inhibitors: Quercetin and VER155008. In conclusion, the present study clearly demonstrated that ECTV infection upregulates the expression of Hspa1b in order to promote its replication. The dependence on Hsp70 may be used as a novel therapeutic target for the treatment of Orthopoxvirus infection.

Eilat virus host range restriction is present at multiple levels of the virus life cycle.

PubMed

Nasar, Farooq; Gorchakov, Rodion V; Tesh, Robert B; Weaver, Scott C

2015-01-15

Most alphaviruses are mosquito-borne and exhibit a broad host range, infecting many different vertebrates, including birds, rodents, equids, humans, and nonhuman primates. This ability of most alphaviruses to infect arthropods and vertebrates is essential for their maintenance in nature. Recently, a new alphavirus, Eilat virus (EILV), was described, and in contrast to all other mosquito-borne viruses, it is unable to replicate in vertebrate cell lines. Investigations into the nature of its host range restriction showed the inability of genomic EILV RNA to replicate in vertebrate cells. Here, we investigated whether the EILV host range restriction is present at the entry level and further explored the viral factors responsible for the lack of genomic RNA replication. Utilizing Sindbis virus (SINV) and EILV chimeras, we show that the EILV vertebrate host range restriction is also manifested at the entry level. Furthermore, the EILV RNA replication restriction is independent of the 3' untranslated genome region (UTR). Complementation experiments with SINV suggested that RNA replication is restricted by the inability of the EILV nonstructural proteins to form functional replicative complexes. These data demonstrate that the EILV host range restriction is multigenic, involving at least one gene from both nonstructural protein (nsP) and structural protein (sP) open reading frames (ORFs). As EILV groups phylogenetically within the mosquito-borne virus clade of pathogenic alphaviruses, our findings have important evolutionary implications for arboviruses. Our work explores the nature of host range restriction of the first "mosquito-only alphavirus," EILV. EILV is related to pathogenic mosquito-borne viruses (Eastern equine encephalitis virus [EEEV], Western equine encephalitis virus [WEEV], Venezuelan equine encephalitis virus [VEEV], and Chikungunya virus [CHIKV]) that cause severe disease in humans. Our data demonstrate that EILV is restricted both at entry and genomic RNA replication levels in vertebrate cells. These findings have important implications for arbovirus evolution and will help elucidate the viral factors responsible for the broad host range of pathogenic mosquito-borne alphaviruses, facilitate vaccine development, and inform potential strategies to reduce/prevent alphavirus transmission. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A Kinome-Wide Small Interfering RNA Screen Identifies Proviral and Antiviral Host Factors in Severe Acute Respiratory Syndrome Coronavirus Replication, Including Double-Stranded RNA-Activated Protein Kinase and Early Secretory Pathway Proteins

PubMed Central

de Wilde, Adriaan H.; Wannee, Kazimier F.; Scholte, Florine E. M.; Goeman, Jelle J.; ten Dijke, Peter; Snijder, Eric J.

2015-01-01

ABSTRACT To identify host factors relevant for severe acute respiratory syndrome-coronavirus (SARS-CoV) replication, we performed a small interfering RNA (siRNA) library screen targeting the human kinome. Protein kinases are key regulators of many cellular functions, and the systematic knockdown of their expression should provide a broad perspective on factors and pathways promoting or antagonizing coronavirus replication. In addition to 40 proteins that promote SARS-CoV replication, our study identified 90 factors exhibiting an antiviral effect. Pathway analysis grouped subsets of these factors in specific cellular processes, including the innate immune response and the metabolism of complex lipids, which appear to play a role in SARS-CoV infection. Several factors were selected for in-depth validation in follow-up experiments. In cells depleted for the β2 subunit of the coatomer protein complex (COPB2), the strongest proviral hit, we observed reduced SARS-CoV protein expression and a >2-log reduction in virus yield. Knockdown of the COPB2-related proteins COPB1 and Golgi-specific brefeldin A-resistant guanine nucleotide exchange factor 1 (GBF1) also suggested that COPI-coated vesicles and/or the early secretory pathway are important for SARS-CoV replication. Depletion of the antiviral double-stranded RNA-activated protein kinase (PKR) enhanced virus replication in the primary screen, and validation experiments confirmed increased SARS-CoV protein expression and virus production upon PKR depletion. In addition, cyclin-dependent kinase 6 (CDK6) was identified as a novel antiviral host factor in SARS-CoV replication. The inventory of pro- and antiviral host factors and pathways described here substantiates and expands our understanding of SARS-CoV replication and may contribute to the identification of novel targets for antiviral therapy. IMPORTANCE Replication of all viruses, including SARS-CoV, depends on and is influenced by cellular pathways. Although substantial progress has been made in dissecting the coronavirus replicative cycle, our understanding of the host factors that stimulate (proviral factors) or restrict (antiviral factors) infection remains far from complete. To study the role of host proteins in SARS-CoV infection, we set out to systematically identify kinase-regulated processes that influence virus replication. Protein kinases are key regulators in signal transduction, controlling a wide variety of cellular processes, and many of them are targets of approved drugs and other compounds. Our screen identified a variety of hits and will form the basis for more detailed follow-up studies that should contribute to a better understanding of SARS-CoV replication and coronavirus-host interactions in general. The identified factors could be interesting targets for the development of host-directed antiviral therapy to treat infections with SARS-CoV or other pathogenic coronaviruses. PMID:26041291

Promotion of Hendra Virus Replication by MicroRNA 146a

PubMed Central

Marsh, Glenn A.; Jenkins, Kristie A.; Gantier, Michael P.; Tizard, Mark L.; Middleton, Deborah; Lowenthal, John W.; Haining, Jessica; Izzard, Leonard; Gough, Tamara J.; Deffrasnes, Celine; Stambas, John; Robinson, Rachel; Heine, Hans G.; Pallister, Jackie A.; Foord, Adam J.; Bean, Andrew G.; Wang, Lin-Fa

2013-01-01

Hendra virus is a highly pathogenic zoonotic paramyxovirus in the genus Henipavirus. Thirty-nine outbreaks of Hendra virus have been reported since its initial identification in Queensland, Australia, resulting in seven human infections and four fatalities. Little is known about cellular host factors impacting Hendra virus replication. In this work, we demonstrate that Hendra virus makes use of a microRNA (miRNA) designated miR-146a, an NF-κB-responsive miRNA upregulated by several innate immune ligands, to favor its replication. miR-146a is elevated in the blood of ferrets and horses infected with Hendra virus and is upregulated by Hendra virus in human cells in vitro. Blocking miR-146a reduces Hendra virus replication in vitro, suggesting a role for this miRNA in Hendra virus replication. In silico analysis of miR-146a targets identified ring finger protein (RNF)11, a member of the A20 ubiquitin editing complex that negatively regulates NF-κB activity, as a novel component of Hendra virus replication. RNA interference-mediated silencing of RNF11 promotes Hendra virus replication in vitro, suggesting that increased NF-κB activity aids Hendra virus replication. Furthermore, overexpression of the IκB superrepressor inhibits Hendra virus replication. These studies are the first to demonstrate a host miRNA response to Hendra virus infection and suggest an important role for host miRNAs in Hendra virus disease. PMID:23345523

Hepatitis A Virus Genome Organization and Replication Strategy.

PubMed

McKnight, Kevin L; Lemon, Stanley M

2018-04-02

Hepatitis A virus (HAV) is a positive-strand RNA virus classified in the genus Hepatovirus of the family Picornaviridae It is an ancient virus with a long evolutionary history and multiple features of its capsid structure, genome organization, and replication cycle that distinguish it from other mammalian picornaviruses. HAV proteins are produced by cap-independent translation of a single, long open reading frame under direction of an inefficient, upstream internal ribosome entry site (IRES). Genome replication occurs slowly and is noncytopathic, with transcription likely primed by a uridylated protein primer as in other picornaviruses. Newly produced quasi-enveloped virions (eHAV) are released from cells in a nonlytic fashion in a unique process mediated by interactions of capsid proteins with components of the host cell endosomal sorting complexes required for transport (ESCRT) system. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.

Structure and function of the Zika virus full-length NS5 protein

DOE PAGES

Zhao, Baoyu; Yi, Guanghui; Du, Fenglei; ...

2017-03-27

The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions tomore » those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Altogether, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.« less

Host and viral RNA-binding proteins involved in membrane targeting, replication and intercellular movement of plant RNA virus genomes

PubMed Central

Hyodo, Kiwamu; Kaido, Masanori; Okuno, Tetsuro

2014-01-01

Many plant viruses have positive-strand RNA [(+)RNA] as their genome. Therefore, it is not surprising that RNA-binding proteins (RBPs) play important roles during (+)RNA virus infection in host plants. Increasing evidence demonstrates that viral and host RBPs play critical roles in multiple steps of the viral life cycle, including translation and replication of viral genomic RNAs, and their intra- and intercellular movement. Although studies focusing on the RNA-binding activities of viral and host proteins, and their associations with membrane targeting, and intercellular movement of viral genomes have been limited to a few viruses, these studies have provided important insights into the molecular mechanisms underlying the replication and movement of viral genomic RNAs. In this review, we briefly overview the currently defined roles of viral and host RBPs whose RNA-binding activity have been confirmed experimentally in association with their membrane targeting, and intercellular movement of plant RNA virus genomes. PMID:25071804

Role of MAPK/MNK1 signaling in virus replication.

PubMed

Kumar, Ram; Khandelwal, Nitin; Thachamvally, Riyesh; Tripathi, Bhupendra Nath; Barua, Sanjay; Kashyap, Sudhir Kumar; Maherchandani, Sunil; Kumar, Naveen

2018-06-01

Viruses are obligate intracellular parasites; they heavily depend on the host cell machinery to effectively replicate and produce new progeny virus particles. Following viral infection, diverse cell signaling pathways are initiated by the cells, with the major goal of establishing an antiviral state. However, viruses have been shown to exploit cellular signaling pathways for their own effective replication. Genome-wide siRNA screens have also identified numerous host factors that either support (proviral) or inhibit (antiviral) virus replication. Some of the host factors might be dispensable for the host but may be critical for virus replication; therefore such cellular factors may serve as targets for development of antiviral therapeutics. Mitogen activated protein kinase (MAPK) is a major cell signaling pathway that is known to be activated by diverse group of viruses. MAPK interacting kinase 1 (MNK1) has been shown to regulate both cap-dependent and internal ribosomal entry sites (IRES)-mediated mRNA translation. In this review we have discuss the role of MAPK in virus replication, particularly the role of MNK1 in replication and translation of viral genome. Copyright © 2018 Elsevier B.V. All rights reserved.

Apoptosis induced in an early step of African swine fever virus entry into vero cells does not require virus replication.

PubMed

Carrascosa, Angel L; Bustos, María J; Nogal, María L; González de Buitrago, Gonzalo; Revilla, Yolanda

2002-03-15

Permissive Vero cells develop apoptosis, as characterized by DNA fragmentation, caspases activation, cytosolic release of mitochondrial cytochrome c, and flow cytometric analysis of DNA content, upon infection with African swine fever virus (ASFV). To determine the step in virus replication that triggers apoptosis, we used UV-inactivated virus, inhibitors of protein and nucleic acid synthesis, and lysosomotropic drugs that block virus uncoating. ASFV-induced apoptosis was accompanied by caspase-3 activation, which was detected even in the presence of either cytosine arabinoside or cycloheximide, indicating that viral DNA replication and protein synthesis were not required to activate the apoptotic process. The activation of caspase-3 was released from chloroquine inhibition 2 h after virus absorption, while the infection with UV-inactivated ASFV did not induce the activation of the caspase cascade. We conclude that ASFV induces apoptosis in the infected cell by an intracellular pathway probably triggered during the process of virus uncoating.

RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

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

Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.

2015-01-15

Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore bemore » added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.« less

B23/nucleophosmin interacts with bovine immunodeficiency virus Rev protein and facilitates viral replication.

PubMed

Passos-Castilho, Ana Maria; Marchand, Claude; Archambault, Denis

2018-02-01

The bovine immunodeficiency virus (BIV) Rev shuttling protein contains nuclear/nucleolar localization signals and nuclear import/export mechanisms that are novel among lentivirus Rev proteins. Several viral proteins localize to the nucleolus, which may play a role in processes that are essential to the outcome of viral replication. Although BIV Rev localizes to the nucleoli of transfected/infected cells and colocalizes with one of its major proteins, nucleophosmin (NPM1, also known as B23), the role of the nucleolus and B23 in BIV replication remains to be determined. Here, we demonstrate for the first time that BIV Rev interacts with nucleolar phosphoprotein B23 in cells. Using small interfering RNA (siRNA) technology, we show that depletion of B23 expression inhibits virus production by BIV-infected cells, indicating that B23 plays an important role in BIV replication. The interaction between Rev and B23 may represent a potential new target for the development of antiviral drugs against lentiviruses. Copyright © 2017 Elsevier Inc. All rights reserved.

Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection

PubMed Central

Taguwa, Shuhei; Maringer, Kevin; Li, Xiaokai; Bernal-Rubio, Dabeiba; Rauch, Jennifer N.; Gestwicki, Jason E.; Andino, Raul; Fernandez-Sesma, Ana; Frydman, Judith

2015-01-01

Summary Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals. PMID:26582131

PARP12 suppresses Zika virus infection through PARP-dependent degradation of NS1 and NS3 viral proteins.

PubMed

Li, Lili; Zhao, Hui; Liu, Ping; Li, Chunfeng; Quanquin, Natalie; Ji, Xue; Sun, Nina; Du, Peishuang; Qin, Cheng-Feng; Lu, Ning; Cheng, Genhong

2018-06-19

Zika virus infection stimulates a type I interferon (IFN) response in host cells, which suppresses viral replication. Type I IFNs exert antiviral effects by inducing the expression of hundreds of IFN-stimulated genes (ISGs). To screen for antiviral ISGs that restricted Zika virus replication, we individually knocked out 21 ISGs in A549 lung cancer cells and identified PARP12 as a strong inhibitor of Zika virus replication. Our findings suggest that PARP12 mediated the ADP-ribosylation of NS1 and NS3, nonstructural viral proteins that are involved in viral replication and modulating host defense responses. This modification of NS1 and NS3 triggered their proteasome-mediated degradation. These data increase our understanding of the antiviral activity of PARP12 and suggest a molecular basis for the potential development of therapeutics against Zika virus. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

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

Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.

2010-09-30

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granularmore » bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.« less

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

Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription.

PubMed

Assenberg, R; Delmas, O; Morin, B; Graham, S C; De Lamballerie, X; Laubert, C; Coutard, B; Grimes, J M; Neyts, J; Owens, R J; Brandt, B W; Gorbalenya, A; Tucker, P; Stuart, D I; Canard, B; Bourhy, H

2010-08-01

Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication. Copyright 2010 Elsevier B.V. All rights reserved.

The ubiquitin-proteasome system is required for African swine fever replication.

PubMed

Barrado-Gil, Lucía; Galindo, Inmaculada; Martínez-Alonso, Diego; Viedma, Sergio; Alonso, Covadonga

2017-01-01

Several viruses manipulate the ubiquitin-proteasome system (UPS) to initiate a productive infection. Determined viral proteins are able to change the host's ubiquitin machinery and some viruses even encode their own ubiquitinating or deubiquitinating enzymes. African swine fever virus (ASFV) encodes a gene homologous to the E2 ubiquitin conjugating (UBC) enzyme. The viral ubiquitin-conjugating enzyme (UBCv1) is expressed throughout ASFV infection and accumulates at late times post infection. UBCv is also present in the viral particle suggesting that the ubiquitin-proteasome pathway could play an important role at early ASFV infection. We determined that inhibition of the final stage of the ubiquitin-proteasome pathway blocked a post-internalization step in ASFV replication in Vero cells. Under proteasome inhibition, ASF viral genome replication, late gene expression and viral production were severely reduced. Also, ASFV enhanced proteasome activity at late times and the accumulation of polyubiquitinated proteins surrounding viral factories. Core-associated and/or viral proteins involved in DNA replication may be targets for the ubiquitin-proteasome pathway that could possibly assist virus uncoating at final core breakdown and viral DNA release. At later steps, polyubiquitinated proteins at viral factories could exert regulatory roles in cell signaling.

Critical Role of HAX-1 in Promoting Avian Influenza Virus Replication in Lung Epithelial Cells

PubMed Central

He, Ganlin; Cardona, Carol J.

2018-01-01

The PB1-F2 protein of influenza A virus has been considered a virulence factor, but its function in inducing apoptosis may be of disadvantage to viral replication. Host mechanisms to regulate PB1-F2-induced apoptosis remain unknown. We generated a PB1-F2-deficient avian influenza virus (AIV) H9N2 and found that the mutant virus replicated less efficiently in human lung epithelial cells. The PB1-F2-deficient virus produced less apoptotic cells, indicating that PB1-F2 of the H9N2 virus promotes apoptosis, occurring at the early stage of infection, in the lung epithelial cells. To understand how host cells regulate PB1-F2-induced apoptosis, we explored to identify cellular proteins interacting with PB1-F2 and found that HCLS1-associated protein X-1 (HAX-1), located mainly in the mitochondria as an apoptotic inhibitor, interacted with PB1-F2. Increased procaspase-9 activations, induced by PB1-F2, could be suppressed by HAX-1. In HAX-1 knockdown A549 cells, the replication of AIV H9N2 was suppressed in parallel to the activation of caspase-3 activation, which increased at the early stage of infection. We hypothesize that HAX-1 promotes AIV replication by interacting with PB1-F2, resulting in the suppression of apoptosis, prolonged cell survival, and enhancement of viral replication. Our data suggest that HAX-1 may be a promoting factor for AIV H9N2 replication through desensitizing PB1-F2 from its apoptotic induction in human lung epithelial cells. PMID:29576744

Dynamic and nucleolin-dependent localization of human cytomegalovirus UL84 to the periphery of viral replication compartments and nucleoli.

PubMed

Bender, Brian J; Coen, Donald M; Strang, Blair L

2014-10-01

Protein-protein and protein-nucleic acid interactions within subcellular compartments are required for viral genome replication. To understand the localization of the human cytomegalovirus viral replication factor UL84 relative to other proteins involved in viral DNA synthesis and to replicating viral DNA in infected cells, we created a recombinant virus expressing a FLAG-tagged version of UL84 (UL84FLAG) and used this virus in immunofluorescence assays. UL84FLAG localization differed at early and late times of infection, transitioning from diffuse distribution throughout the nucleus to exclusion from the interior of replication compartments, with some concentration at the periphery of replication compartments with newly labeled DNA and the viral DNA polymerase subunit UL44. Early in infection, UL84FLAG colocalized with the viral single-stranded DNA binding protein UL57, but colocalization became less prominent as infection progressed. A portion of UL84FLAG also colocalized with the host nucleolar protein nucleolin at the peripheries of both replication compartments and nucleoli. Small interfering RNA (siRNA)-mediated knockdown of nucleolin resulted in a dramatic elimination of UL84FLAG from replication compartments and other parts of the nucleus and its accumulation in the cytoplasm. Reciprocal coimmunoprecipitation of viral proteins from infected cell lysates revealed association of UL84, UL44, and nucleolin. These results indicate that UL84 localization during infection is dynamic, which is likely relevant to its functions, and suggest that its nuclear and subnuclear localization is highly dependent on direct or indirect interactions with nucleolin. Importance: The protein-protein interactions among viral and cellular proteins required for replication of the human cytomegalovirus (HCMV) DNA genome are poorly understood. We sought to understand how an enigmatic HCMV protein critical for virus replication, UL84, localizes relative to other viral and cellular proteins required for HCMV genome replication and replicating viral DNA. We found that UL84 localizes with viral proteins, viral DNA, and the cellular nucleolar protein nucleolin in the subnuclear replication compartments in which viral DNA replication occurs. Unexpectedly, we also found localization of UL84 with nucleolin in nucleoli and showed that the presence of nucleolin is involved in localization of UL84 to the nucleus. These results add to previous work showing the importance of nucleolin in replication compartment architecture and viral DNA synthesis and are relevant to understanding UL84 function. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

The Low-pH Resistance of Neuraminidase Is Essential for the Replication of Influenza A Virus in Duck Intestine following Infection via the Oral Route.

PubMed

Fujimoto, Yoshikazu; Ito, Hiroshi; Ono, Etsuro; Kawaoka, Yoshihiro; Ito, Toshihiro

2016-04-01

Influenza A viruses are known to primarily replicate in duck intestine following infection via the oral route, but the specific role of neuraminidase (NA) for the intestinal tropism of influenza A viruses has been unclear. A reassortant virus (Dk78/Eng62N2) did not propagate in ducks infected via the oral route. To generate variant viruses that grow well in ducks via the oral route, we isolated viruses that effectively replicate in intestinal mucosal cells by passaging Dk78/Eng62N2 in duck via rectal-route infection. This procedure led to the isolation of a variant virus from the duck intestine. This virus was propagated using embryonated chicken eggs and inoculated into a duck via the oral route, which led to the isolation of Dk-rec6 from the duck intestine. Experimental infections with mutant viruses generated by using reverse genetics indicated that the paired mutation of residues 356 and 431 in NA was necessary for the viral replication in duck intestine. The NA assay revealed that the activity of Dk78/Eng62N2 almost disappeared after pH 3 treatment, whereas that of Dk-rec6 was maintained. Furthermore, to identify the amino acid residues associated with the low-pH resistance, we measured the activities of mutant NA proteins transiently expressed in 293 cells after pH 3 treatment. All mutant NA proteins that possessed proline at position 431 showed higher activities than NA proteins that possessed glutamine at this position. These findings indicate that the low-pH resistance of NA plays an important role in the ability of influenza A virus to replicate in duck intestine. Neuraminidase (NA) activity facilitates the release of viruses from cells and, as such, is important for the replicative efficiency of influenza A virus. Ducks are believed to serve as the principal natural reservoir for influenza A virus; however, the key properties of NA for viral infection in duck are not well understood. In this study, we identify amino acid residues in NA that contribute to viral replication in ducks via the natural route of infection and demonstrate that maintenance of NA activity under low-pH conditions is associated with the biological properties of the virus. These findings provide insights into the mechanisms of replication of influenza A virus in ducks. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Roles of viroplasm-like structures formed by nonstructural protein NSs in infection with severe fever with thrombocytopenia syndrome virus.

PubMed

Wu, Xiaodong; Qi, Xian; Liang, Mifang; Li, Chuan; Cardona, Carol J; Li, Dexin; Xing, Zheng

2014-06-01

Severe fever with thrombocytopenia syndrome (SFTS) virus is an emerging bunyavirus that causes a hemorrhagic fever with a high mortality rate. The virus is likely tick-borne and replicates primarily in hemopoietic cells, which may lead to disregulation of proinflammatory cytokine induction and loss of leukocytes and platelets. The viral genome contains L, M, and S segments encoding a viral RNA polymerase, glycoproteins G(n) and G(c), nucleoprotein (NP), and a nonstructural S segment (NSs) protein. NSs protein is involved in the regulation of host innate immune responses and suppression of IFNβ-promoter activities. In this article, we demonstrate that NSs protein can form viroplasm-like structures (VLSs) in infected and transfected cells. NSs protein molecules interact with one another, interact with NP, and were associated with viral RNA in infected cells, suggesting that NSs protein may be involved in viral replication. Furthermore, we observed that NSs-formed VLS colocalized with lipid droplets and that inhibitors of fatty acid biosynthesis decreased VLS formation or viral replication in transfected and infected cells. Finally, we have demonstrated that viral dsRNAs were also localized in VLS in infected cells, suggesting that NSs-formed VLS may be implicated in the replication of SFTS bunyavirus. These findings identify a novel function of nonstructural NSs in SFTSV-infected cells where it is a scaffolding component in a VLS functioning as a virus replication factory. This function is in addition to the role of NSs protein in modulating host responses that will broaden our understanding of viral pathogenesis of phleboviruses. © FASEB.

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

TRIM79α, an interferon-stimulated gene product, restricts tick-borne encephalitis virus replication by degrading the viral RNA polymerase

PubMed Central

Taylor, R. Travis; Lubick, Kirk J.; Robertson, Shelly J.; Broughton, James P.; Bloom, Marshall E.; Bresnahan, Wade A.; Best, Sonja M.

2011-01-01

In response to virus infection, type I interferons (IFNs) induce several genes, most of whose functions are largely unknown. Here we show that the tripartite motif (TRIM) protein, TRIM79α, is an IFN-stimulated gene (ISG) product that specifically targets tick-borne encephalitis virus (TBEV), a Flavivirus that causes encephalitides in humans. TRIM79α restricts TBEV replication by mediating lysosome-dependent degradation of the flavivirus NS5 protein, an RNA-dependent RNA polymerase essential for virus replication. NS5 degradation was specific to tick-borne flaviviruses as TRIM79α did not recognize NS5 from West Nile virus (WNV) or inhibit WNV replication. In the absence of TRIM79α, IFN-β was less effective in inhibiting tick-borne flavivirus infection of mouse macrophages, highlighting the importance of a single virus-specific ISG in establishing an antiviral state. The specificity of TRIM79α for TBEV reveals a remarkable ability of the innate IFN response to discriminate between closely related flaviviruses. PMID:21925107

Analysis of rubella virus capsid protein-mediated enhancement of replicon replication and mutant rescue.

PubMed

Tzeng, Wen-Pin; Matthews, Jason D; Frey, Teryl K

2006-04-01

The rubella virus capsid protein (C) has been shown to complement a lethal deletion (termed deltaNotI) in P150 replicase protein. To investigate this phenomenon, we generated two lines of Vero cells that stably expressed either C (C-Vero cells) or C lacking the eight N-terminal residues (Cdelta8-Vero cells), a construct previously shown to be unable to complement DeltaNotI. In C-Vero cells but not Vero or Cdelta8-Vero cells, replication of a wild-type (wt) replicon expressing the green fluorescent protein (GFP) reporter gene (RUBrep/GFP) was enhanced, and replication of a replicon with deltaNotI (RUBrep/GFP-deltaNotI) was rescued. Surprisingly, replicons with deleterious mutations in the 5' and 3' cis-acting elements were also rescued in C-Vero cells. Interestingly, the Cdelta8 construct localized to the nucleus while the C construct localized in the cytoplasm, explaining the lack of enhancement and rescue in Cdelta8-Vero cells since rubella virus replication occurs in the cytoplasm. Enhancement and rescue in C-Vero cells were at a basic step in the replication cycle, resulting in a substantial increase in the accumulation of replicon-specific RNAs. There was no difference in translation of the nonstructural proteins in C-Vero and Vero cells transfected with the wt and mutant replicons, demonstrating that enhancement and rescue were not due to an increase in the efficiency of translation of the transfected replicon transcripts. In replicon-transfected C-Vero cells, C and the P150 replicase protein associated by coimmunoprecipitation, suggesting that C might play a role in RNA replication, which could explain the enhancement and rescue phenomena. A unifying model that accounts for enhancement of wt replicon replication and rescue of diverse mutations by the rubella virus C protein is proposed.

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin.

PubMed

Song, Tao; Fang, Liurong; Wang, Dang; Zhang, Ruoxi; Zeng, Songlin; An, Kang; Chen, Huanchun; Xiao, Shaobo

2016-06-16

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has heavily impacted the global swine industry. The PRRSV nonstructural protein 2 (nsp2) plays crucial roles in viral replication and host immune regulation, most likely by interacting with viral or cellular proteins that have not yet been identified. In this study, a quantitative interactome approach based on immunoprecipitation and stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify nsp2-interacting proteins in PRRSV-infected cells with an nsp2-specific monoclonal antibody. Nine viral proteins and 62 cellular proteins were identified as potential nsp2-interacting partners. Our data demonstrate that the PRRSV nsp1α, nsp1β, and nucleocapsid proteins all interact directly with nsp2. Nsp2-interacting cellular proteins were classified into different functional groups and an interactome network of nsp2 was generated. Interestingly, cellular vimentin, a known receptor for PRRSV, forms a complex with nsp2 by using viral nucleocapsid protein as an intermediate. Taken together, the nsp2 interactome under the condition of virus infection clarifies a role of nsp2 in PRRSV replication and immune evasion. Viral proteins must interact with other virus-encoded proteins and/or host cellular proteins to function, and interactome analysis is an ideal approach for identifying such interacting proteins. In this study, we used the quantitative interactome methodology to identify the viral and cellular proteins that potentially interact with the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) under virus infection conditions, thus providing a rich source of potential viral and cellular interaction partners for PRRSV nsp2. Based on the interactome data, we further demonstrated that PRRSV nsp2 and nucleocapsid protein together with cellular vimentin, form a complex that may be essential for viral attachment and replication, which partly explains the role of nsp2 in PRRSV replication and immune evasion. Copyright © 2016 Elsevier B.V. All rights reserved.

Construction of Nef-positive doxycycline-dependent HIV-1 variants using bicistronic expression elements

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

Velden, Yme U. van der; Kleibeuker, Wendy; Harwig, Alex

Conditionally replicating HIV-1 variants that can be switched on and off at will are attractive tools for HIV research. We previously developed a genetically modified HIV-1 variant that replicates exclusively when doxycycline (dox) is administered. The nef gene in this HIV-rtTA variant was replaced with the gene encoding the dox-dependent rtTA transcriptional activator. Because loss of Nef expression compromises virus replication in primary cells and precludes studies on Nef function, we tested different approaches to restore Nef production in HIV-rtTA. Strategies that involved translation via an EMCV or synthetic internal ribosome entry site (IRES) failed because these elements were incompatiblemore » with efficient virus replication. Fusion protein approaches with the FMDV 2A peptide and human ubiquitin were successful and resulted in genetically-stable Nef-expressing HIV-rtTA strains that replicate more efficiently in primary T-cells and human immune system (HIS) mice than Nef-deficient variants, thus confirming the positive effect of Nef on in vivo virus replication. - Highlights: • Different approaches to encode additional proteins in the HIV-1 genome were tested. • IRES translation elements are incompatible with efficient HIV-1 replication. • Ubiquitin and 2A fusion protein approaches allow efficient HIV-1 replication. • Doxycycline-controlled HIV-1 variants that encode all viral proteins were developed. • Nef stimulates HIV-rtTA replication in primary cells and human immune system mice.« less

Plum Pox Virus 6K1 Protein Is Required for Viral Replication and Targets the Viral Replication Complex at the Early Stage of Infection.

PubMed

Cui, Hongguang; Wang, Aiming

2016-05-15

The potyviral RNA genome encodes two polyproteins that are proteolytically processed by three viral protease domains into 11 mature proteins. Extensive molecular studies have identified functions for the majority of the viral proteins. For example, 6K2, one of the two smallest potyviral proteins, is an integral membrane protein and induces the endoplasmic reticulum (ER)-originated replication vesicles that target the chloroplast for robust viral replication. However, the functional role of 6K1, the other smallest protein, remains uncharacterized. In this study, we developed a series of recombinant full-length viral cDNA clones derived from a Canadian Plum pox virus (PPV) isolate. We found that deletion of any of the short motifs of 6K1 (each of which ranged from 5 to 13 amino acids), most of the 6K1 sequence (but with the conserved sequence of the cleavage sites being retained), or all of the 6K1 sequence in the PPV infectious clone abolished viral replication. The trans expression of 6K1 or the cis expression of a dislocated 6K1 failed to rescue the loss-of-replication phenotype, suggesting the temporal and spatial requirement of 6K1 for viral replication. Disruption of the N- or C-terminal cleavage site of 6K1, which prevented the release of 6K1 from the polyprotein, either partially or completely inhibited viral replication, suggesting the functional importance of the mature 6K1. We further found that green fluorescent protein-tagged 6K1 formed punctate inclusions at the viral early infection stage and colocalized with chloroplast-bound viral replicase elements 6K2 and NIb. Taken together, our results suggest that 6K1 is required for viral replication and is an important viral element of the viral replication complex at the early infection stage. Potyviruses account for more than 30% of known plant viruses and consist of many agriculturally important viruses. The genomes of potyviruses encode two polyproteins that are proteolytically processed into 11 mature proteins, with the majority of them having been at least partially functionally characterized. However, the functional role of a small protein named 6K1 remains obscure. In this study, we showed that deletion of 6K1 or a short motif/region of 6K1 in the full-length cDNA clones of plum pox virus abolishes viral replication and that mutation of the N- or C-terminal cleavage sites of 6K1 to prevent its release from the polyprotein greatly attenuates or completely inhibits viral replication, suggesting its important role in potyviral infection. We report that 6K1 forms punctate structures and targets the replication vesicles in PPV-infected plant leaf cells at the early infection stage. Our data reveal that 6K1 is an important viral protein of the potyviral replication complex. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Adaptor protein complexes-1 and 3 are involved at distinct stages of flavivirus life-cycle

PubMed Central

Agrawal, Tanvi; Schu, Peter; Medigeshi, Guruprasad R.

2013-01-01

Intracellular protein trafficking pathways are hijacked by viruses at various stages of viral life-cycle. Heterotetrameric adaptor protein complexes (APs) mediate vesicular trafficking at distinct intracellular sites and are essential for maintaining the organellar homeostasis. In the present study, we studied the effect of AP-1 and AP-3 deficiency on flavivirus infection in cells functionally lacking these proteins. We show that AP-1 and AP-3 participate in flavivirus life-cycle at distinct stages. AP-3-deficient cells showed delay in initiation of Japanese encephalitis virus and dengue virus RNA replication, which resulted in reduction of infectious virus production. AP-3 was found to colocalize with RNA replication compartments in infected wild-type cells. AP-1 deficiency affected later stages of dengue virus infection where increased intracellular accumulation of infectious virus was observed. Therefore, our results propose a novel role for AP-1 and AP-3 at distinct stages of infection of some of the RNA viruses. PMID:23657274

Adaptor protein complexes-1 and 3 are involved at distinct stages of flavivirus life-cycle.

PubMed

Agrawal, Tanvi; Schu, Peter; Medigeshi, Guruprasad R

2013-01-01

Intracellular protein trafficking pathways are hijacked by viruses at various stages of viral life-cycle. Heterotetrameric adaptor protein complexes (APs) mediate vesicular trafficking at distinct intracellular sites and are essential for maintaining the organellar homeostasis. In the present study, we studied the effect of AP-1 and AP-3 deficiency on flavivirus infection in cells functionally lacking these proteins. We show that AP-1 and AP-3 participate in flavivirus life-cycle at distinct stages. AP-3-deficient cells showed delay in initiation of Japanese encephalitis virus and dengue virus RNA replication, which resulted in reduction of infectious virus production. AP-3 was found to colocalize with RNA replication compartments in infected wild-type cells. AP-1 deficiency affected later stages of dengue virus infection where increased intracellular accumulation of infectious virus was observed. Therefore, our results propose a novel role for AP-1 and AP-3 at distinct stages of infection of some of the RNA viruses.

Chimpanzee GB virus C and GB virus A E2 envelope glycoproteins contain a peptide motif that inhibits human immunodeficiency virus type 1 replication in human CD4+ T-cells

PubMed Central

McLinden, James H.; Stapleton, Jack T.; Klinzman, Donna; Murthy, Krishna K.; Chang, Qing; Kaufman, Thomas M.; Bhattarai, Nirjal

2013-01-01

GB virus type C (GBV-C) is a lymphotropic virus that can cause persistent infection in humans. GBV-C is not associated with any disease, but is associated with reduced mortality in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Related viruses have been isolated from chimpanzees (GBV-Ccpz) and from New World primates (GB virus type A, GBV-A). These viruses are also capable of establishing persistent infection. We determined the nucleotide sequence encoding the envelope glycoprotein (E2) of two GBV-Ccpz isolates obtained from the sera of captive chimpanzees. The deduced GBV-Ccpz E2 protein differed from human GBV-C by 31 % at the amino acid level. Similar to human GBV-C E2, expression of GBV-Ccpz E2 in a tet-off human CD4+ Jurkat T-cell line significantly inhibited the replication of diverse HIV-1 isolates. This anti-HIV-replication effect of GBV-Ccpz E2 protein was reversed by maintaining cells in doxycycline to reduce E2 expression. Previously, we found a 17 aa region within human GBV-C E2 that was sufficient to inhibit HIV-1. Although GBV-Ccpz E2 differed by 3 aa differences in this region, the chimpanzee GBV-C 17mer E2 peptide inhibited HIV-1 replication. Similarly, the GBV-A peptide that aligns with this GBV-C E2 region inhibited HIV-1 replication despite sharing only 5 aa with the human GBV-C E2 sequence. Thus, despite amino acid differences, the peptide region on both the GBV-Ccpz and the GBV-A E2 protein inhibit HIV-1 replication similar to human GBV-C. Consequently, GBV-Ccpz or GBV-A infection of non-human primates may provide an animal model to study GB virus–HIV interactions. PMID:23288422

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses.

PubMed

Whitt, Michael A; Geisbert, Thomas W; Mire, Chad E

2016-01-01

There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.

Inhibition of HIV replication by pokeweed antiviral protein targeted to CD4+ cells by monoclonal antibodies

NASA Astrophysics Data System (ADS)

Zarling, Joyce M.; Moran, Patricia A.; Haffar, Omar; Sias, Joan; Richman, Douglas D.; Spina, Celsa A.; Myers, Dorothea E.; Kuebelbeck, Virginia; Ledbetter, Jeffrey A.; Uckun, Fatih M.

1990-09-01

FUNCTIONAL impairment and selective depletion of CD4+ T cells, the hallmark of AIDS, are at least partly caused by human immunodeficiency virus (HIV-1) type 1 binding to the CD4 molecule and infecting CD4+ cells1,2. It may, therefore, be of therapeutic value to target an antiviral agent to CD4+ cells to prevent infection and to inhibit HIV-1 production in patients' CD4+ cells which contain proviral DNA3,4. We report here that HIV-1 replication in normal primary CD4+ T cells can be inhibited by pokeweed antiviral protein, a plant protein of relative molecular mass 30,000 (ref. 5), which inhibits replication of certain plant RNA viruses6-8, and of herpes simplex virus, poliovirus and influenza virus9-11. Targeting pokeweed antiviral protein to CD4+ T cells by conjugating it to monoclonal antibodies reactive with CDS, CD7 or CD4 expressed on CD4+ cells, increased its anti-HIV potency up to 1,000-fold. HIV-1 replication is inhibited at picomolar concentrations of conjugates of pokeweed antiviral protein and monoclonal antibodies, which do not inhibit proliferation of normal CD4+ T cells or CD4-dependent responses. These conjugates inhibit HIV-1 protein synthesis and also strongly inhibit HIV-1 production in activated CD4+ T cells from infected patients.

Replicative Functions of Minute Virus of Mice NS1 Protein Are Regulated In Vitro by Phosphorylation through Protein Kinase C

PubMed Central

Nüesch, Jürg P. F.; Dettwiler, Sabine; Corbau, Romuald; Rommelaere, Jean

1998-01-01

NS1, the major nonstructural protein of the parvovirus minute virus of mice, is a multifunctional phosphoprotein which is involved in cytotoxicity, transcriptional regulation, and initiation of viral DNA replication. For coordination of these various functions during virus propagation, NS1 has been proposed to be regulated by posttranslational modifications, in particular phosphorylation. Recent in vitro studies (J. P. F. Nüesch, R. Corbau, P. Tattersall, and J. Rommelaere, J. Virol. 72:8002–8012, 1998) provided evidence that distinct NS1 activities, notably the intrinsic helicase function, are modulated by the phosphorylation state of the protein. In order to study the dependence of the initiation of viral DNA replication on NS1 phosphorylation and to identify the protein kinases involved, we established an in vitro replication system that is devoid of endogenous protein kinases and is based on plasmid substrates containing the minimal left-end origins of replication. Cellular components necessary to drive NS1-dependent rolling-circle replication (RCR) were freed from endogenous serine/threonine protein kinases by affinity chromatography, and the eukaryotic DNA polymerases were replaced by the bacteriophage T4 DNA polymerase. While native NS1 (NS1P) supported RCR under these conditions, dephosphorylated NS1 (NS1O) was impaired. Using fractionated HeLa cell extracts, we identified two essential protein components which are able to phosphorylate NS1O, are enriched in protein kinase C (PKC), and, when present together, reactivate NS1O for replication. One of these components, containing atypical PKC, was sufficient to restore NS1O helicase activity. The requirement of NS1O reactivation for characteristic PKC cofactors such as Ca2+/phosphatidylserine or phorbol esters strongly suggests the involvement of this protein kinase family in regulation of NS1 replicative functions in vitro. PMID:9811734

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

Cellular Hsp27 interacts with classical swine fever virus NS5A protein and negatively regulates viral replication by the NF-κB signaling pathway.

PubMed

Ling, Shifeng; Luo, Mingyang; Jiang, Shengnan; Liu, Jiayu; Ding, Chunying; Zhang, Qinghuan; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

2018-05-01

Classical swine fever virus (CSFV) nonstructural protein NS5A is a multifunctional protein functioning in regulation of viral genome replication, protein translation and assembly by interaction with viral or host proteins. Here, heat shock protein 27 (Hsp27) has been identified as a novel binding partner of NS5A by using His tag "pull down" coupled with shotgun LC-MS/MS, with interaction of both proteins further confirmed by co-immunoprecipitation and laser confocal assays. In PK-15 cells, silencing of Hsp27 expression by siRNA enhanced CSFV replication, and upregulation of Hsp27 inhibited viral proliferation. Additionally, we have shown that overexpression of Hsp27 increased NF-κB signaling induced by TNFα. Blocking NF-κB signaling in PK-15 cells overexpressing Hsp27 by ammonium pyrrolidinedithiocarbamate (PDTC) eliminated the inhibition of CSFV replication by Hsp27. These findings clearly demonstrate that the inhibition of CSFV replication by Hsp27 is mediated via the NF-κB signaling pathway. Copyright © 2018 Elsevier Inc. All rights reserved.

A Unique N-Terminal Sequence in the Carnation Italian ringspot virus p36 Replicase-Associated Protein Interacts with the Host Cell ESCRT-I Component Vps23

PubMed Central

Richardson, Lynn G. L.; Clendening, Eric A.; Sheen, Hyukho; Gidda, Satinder K.; White, K. Andrew

2014-01-01

ABSTRACT Like most positive-strand RNA viruses, infection by plant tombusviruses results in extensive rearrangement of specific host cell organelle membranes that serve as the sites of viral replication. The tombusvirus Tomato bushy stunt virus (TBSV) replicates within spherules derived from the peroxisomal boundary membrane, a process that involves the coordinated action of various viral and cellular factors, including constituents of the endosomal sorting complex required for transport (ESCRT). ESCRT is comprised of a series of protein subcomplexes (i.e., ESCRT-0 -I, -II, and -III) that normally participate in late endosome biogenesis and some of which are also hijacked by certain enveloped retroviruses (e.g., HIV) for viral budding from the plasma membrane. Here we show that the replication of Carnation Italian ringspot virus (CIRV), a tombusvirus that replicates at mitochondrial membranes also relies on ESCRT. In plant cells, CIRV recruits the ESCRT-I protein, Vps23, to mitochondria through an interaction that involves a unique region in the N terminus of the p36 replicase-associated protein that is not conserved in TBSV or other peroxisome-targeted tombusviruses. The interaction between p36 and Vps23 also involves the Vps23 C-terminal steadiness box domain and not its N-terminal ubiquitin E2 variant domain, which in the case of TBSV (and enveloped retroviruses) mediates the interaction with ESCRT. Overall, these results provide evidence that CIRV uses a unique N-terminal sequence for the recruitment of Vps23 that is distinct from those used by TBSV and certain mammalian viruses for ESCRT recruitment. Characterization of this novel interaction with Vps23 contributes to our understanding of how CIRV may have evolved to exploit key differences in the plant ESCRT machinery. IMPORTANCE Positive-strand RNA viruses replicate their genomes in association with specific host cell membranes. To accomplish this, cellular components responsible for membrane biogenesis and modeling are appropriated by viral proteins and redirected to assemble membrane-bound viral replicase complexes. The diverse pathways leading to the formation of these replication structures are poorly understood. We have determined that the cellular ESCRT system that is normally responsible for mediating late endosome biogenesis is also involved in the replication of the tombusvirus Carnation Italian ringspot virus (CIRV) at mitochondria. Notably, CIRV recruits ESCRT to the mitochondrial outer membrane via an interaction between a unique motif in the viral protein p36 and the ESCRT component Vps23. Our findings provide new insights into tombusvirus replication and the virus-induced remodeling of plant intracellular membranes, as well as normal ESCRT assembly in plants. PMID:24672030

A Physical Interaction Network of Dengue Virus and Human Proteins*

PubMed Central

Khadka, Sudip; Vangeloff, Abbey D.; Zhang, Chaoying; Siddavatam, Prasad; Heaton, Nicholas S.; Wang, Ling; Sengupta, Ranjan; Sahasrabudhe, Sudhir; Randall, Glenn; Gribskov, Michael; Kuhn, Richard J.; Perera, Rushika; LaCount, Douglas J.

2011-01-01

Dengue virus (DENV), an emerging mosquito-transmitted pathogen capable of causing severe disease in humans, interacts with host cell factors to create a more favorable environment for replication. However, few interactions between DENV and human proteins have been reported to date. To identify DENV-human protein interactions, we used high-throughput yeast two-hybrid assays to screen the 10 DENV proteins against a human liver activation domain library. From 45 DNA-binding domain clones containing either full-length viral genes or partially overlapping gene fragments, we identified 139 interactions between DENV and human proteins, the vast majority of which are novel. These interactions involved 105 human proteins, including six previously implicated in DENV infection and 45 linked to the replication of other viruses. Human proteins with functions related to the complement and coagulation cascade, the centrosome, and the cytoskeleton were enriched among the DENV interaction partners. To determine if the cellular proteins were required for DENV infection, we used small interfering RNAs to inhibit their expression. Six of 12 proteins targeted (CALR, DDX3X, ERC1, GOLGA2, TRIP11, and UBE2I) caused a significant decrease in the replication of a DENV replicon. We further showed that calreticulin colocalized with viral dsRNA and with the viral NS3 and NS5 proteins in DENV-infected cells, consistent with a direct role for calreticulin in DENV replication. Human proteins that interacted with DENV had significantly higher average degree and betweenness than expected by chance, which provides additional support for the hypothesis that viruses preferentially target cellular proteins that occupy central position in the human protein interaction network. This study provides a valuable starting point for additional investigations into the roles of human proteins in DENV infection. PMID:21911577

The cold adapted and temperature sensitive influenza A/Ann Arbor/6/60 virus, the master donor virus for live attenuated influenza vaccines, has multiple defects in replication at the restrictive temperature

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

Chan, Winnie; Zhou, Helen; Kemble, George

2008-10-25

We have previously determined that the temperature sensitive (ts) and attenuated (att) phenotypes of the cold adapted influenza A/Ann Arbor/6/60 strain (MDV-A), the master donor virus for the live attenuated influenza A vaccines (FluMist), are specified by the five amino acids in the PB1, PB2 and NP gene segments. To understand how these loci control the ts phenotype of MDV-A, replication of MDV-A at the non-permissive temperature (39 deg. C) was compared with recombinant wild-type A/Ann Arbor/6/60 (rWt). The mRNA and protein synthesis of MDV-A in the infected MDCK cells were not significantly reduced at 39 deg. C during amore » single-step replication, however, vRNA synthesis was reduced and the nuclear-cytoplasmic export of viral RNP (vRNP) was blocked. In addition, the virions released from MDV-A infected cells at 39 deg. C exhibited irregular morphology and had a greatly reduced amount of the M1 protein incorporated. The reduced M1 protein incorporation and vRNP export blockage correlated well with the virus ts phenotype because these defects could be partially alleviated by removing the three ts loci from the PB1 gene. The virions and vRNPs isolated from the MDV-A infected cells contained a higher level of heat shock protein 70 (Hsp70) than those of rWt, however, whether Hsp70 is involved in thermal inhibition of MDV-A replication remains to be determined. Our studies demonstrate that restrictive replication of MDV-A at the non-permissive temperature occurs in multiple steps of the virus replication cycle.« less

Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

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

Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan

Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase activemore » site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.« less

Accessory genes confer a high replication rate to virulent feline immunodeficiency virus.

PubMed

Troyer, Ryan M; Thompson, Jesse; Elder, John H; VandeWoude, Sue

2013-07-01

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.

A physical interaction between viral replicase and capsid protein is required for genome-packaging specificity in an RNA virus.

PubMed

Seo, Jang-Kyun; Kwon, Sun-Jung; Rao, A L N

2012-06-01

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity.

A Physical Interaction between Viral Replicase and Capsid Protein Is Required for Genome-Packaging Specificity in an RNA Virus

PubMed Central

Seo, Jang-Kyun; Kwon, Sun-Jung

2012-01-01

Genome packaging is functionally coupled to replication in RNA viruses pathogenic to humans (Poliovirus), insects (Flock house virus [FHV]), and plants (Brome mosaic virus [BMV]). However, the underlying mechanism is not fully understood. We have observed previously that in FHV and BMV, unlike ectopically expressed capsid protein (CP), packaging specificity results from RNA encapsidation by CP that has been translated from mRNA produced from replicating genomic RNA. Consequently, we hypothesize that a physical interaction with replicase increases the CP specificity for packaging viral RNAs. We tested this hypothesis by evaluating the molecular interaction between replicase protein and CP using a FHV-Nicotiana benthamiana system. Bimolecular fluorescence complementation in conjunction with fluorescent cellular protein markers and coimmunoprecipitation assays demonstrated that FHV replicase (protein A) and CP physically interact at the mitochondrial site of replication and that this interaction requires the N-proximal region from either amino acids 1 to 31 or amino acids 32 to 50 of the CP. In contrast to the mitochondrial localization of CP derived from FHV replication, ectopic expression displayed a characteristic punctate pattern on the endoplasmic reticulum (ER). This pattern was altered to relocalize the CP throughout the cytoplasm when the C-proximal hydrophobic domain was deleted. Analysis of the packaging phenotypes of the CP mutants defective either in protein A-CP interactions or ER localization suggested that synchronization between protein A-CP interaction and its subcellular localization is imperative to confer packaging specificity. PMID:22438552

WDR5 Facilitates Human Cytomegalovirus Replication by Promoting Capsid Nuclear Egress.

PubMed

Yang, Bo; Liu, Xi-Juan; Yao, Yongxuan; Jiang, Xuan; Wang, Xian-Zhang; Yang, Hong; Sun, Jin-Yan; Miao, Yun; Wang, Wei; Huang, Zhen-Li; Wang, Yanyi; Tang, Qiyi; Rayner, Simon; Britt, William J; McVoy, Michael A; Luo, Min-Hua; Zhao, Fei

2018-05-01

WD repeat-containing protein 5 (WDR5) is essential for assembling the VISA-associated complex to induce a type I interferon antiviral response to Sendai virus infection. However, the roles of WDR5 in DNA virus infections are not well described. Here, we report that human cytomegalovirus exploits WDR5 to facilitate capsid nuclear egress. Overexpression of WDR5 in fibroblasts slightly enhanced the infectious virus yield. However, WDR5 knockdown dramatically reduced infectious virus titers with only a small decrease in viral genome replication or gene expression. Further investigation of late steps of viral replication found that WDR5 knockdown significantly impaired formation of the viral nuclear egress complex and induced substantially fewer infoldings of the inner nuclear membrane. In addition, fewer capsids were associated with these infoldings, and there were fewer capsids in the cytoplasm. Restoration of WDR5 partially reversed these effects. These results suggest that WDR5 knockdown impairs the nuclear egress of capsids, which in turn decreases virus titers. These findings reveal an important role for a host factor whose function(s) is usurped by a viral pathogen to promote efficient replication. Thus, WDR5 represents an interesting regulatory mechanism and a potential antiviral target. IMPORTANCE Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 170 open reading frames and exploits numerous cellular factors to facilitate its replication. HCMV infection increases protein levels of WD repeat-containing protein 5 (WDR5) during infection, overexpression of WDR5 enhances viral replication, and knockdown of WDR5 dramatically attenuates viral replication. Our results indicate that WDR5 promotes the nuclear egress of viral capsids, the depletion of WDR5 resulting in a significant decrease in production of infectious virions. This is the first report that WDR5 favors HCMV, a DNA virus, replication and highlights a novel target for antiviral therapy. Copyright © 2018 American Society for Microbiology.

Non-structural protein 1 of influenza viruses inhibits rapid mRNA degradation mediated by double-stranded RNA-binding protein, staufen1.

PubMed

Cho, Hana; Ahn, Sang Ho; Kim, Kyoung Mi; Kim, Yoon Ki

2013-07-11

Although non-structural protein 1 (NS1) of influenza viruses is not essential for virulence, this protein is involved in host-virus interactions, viral replication, and translation. In particular, NS1 is known to interact with the host protein, staufen1 (Stau1). This interaction is important for efficient viral replication. However, the underlying molecular mechanism by which NS1 influences the viral life cycle remains obscure. Here, we show using immunoprecipitation and artificial tethering that the N-terminus of NS1, NS1(1-73), interacts with Stau1, blocks the Stau1-Upf1 interaction, and consequently inhibits the efficiency of Stau1-mediated mRNA decay (SMD), but not nonsense-mediatedmRNA decay (NMD). The regulation of SMD efficiency by NS1 may contribute to building a more favorable cellular environment for viral replication. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Carboxy-terminal cleavage of the human foamy virus Gag precursor molecule is an essential step in the viral life cycle.

PubMed Central

Enssle, J; Fischer, N; Moebes, A; Mauer, B; Smola, U; Rethwilm, A

1997-01-01

Foamy viruses (FVs) express the Gag protein as a precursor with a molecular mass of 74 kDa (pr74) from which a 70-kDa protein (p70) is cleaved by the viral protease. To gain a better understanding of FV Gag protein processing and function, we have generated and analyzed mutants in the C-terminal gag region of an infectious molecular clone. Our results show that p70 is an N-terminal cleavage product of pr74. However, we were unable to identify a p4 molecule. A virus mutant expressing p70 only was found to be replication competent, albeit at very low titers compared to those of wild-type virus. A strong tendency to synthesize and cleave a pr74 molecule was deduced from the occurrence of revertants upon transfection of this mutant. Substitution of the p6gag domain of human immunodeficiency virus type 1 for the p4 domain of FV resulted in a stable chimeric virus which replicated to titers 10 times lower than those of wild-type virus. FV Gag protein was found to be phosphorylated at serine residues. Mutagenesis of serines conserved in the p4 domain had no influence on viral replication in cell culture. The p70/p74 Gag cleavage was found to be required for viral infectivity, since mutagenesis of the putative cleavage site led to replication-incompetent virus. Interestingly, the cleavage site mutants were defective in the intracellular cDNA synthesis of virion DNA, which indicates that correct FV particle formation and the generation of virion DNA are functionally linked. PMID:9311808

A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo.

PubMed

Watanabe, Masahiro; Hashimoto, Koichi; Abe, Yusaku; Kodama, Eiichi N; Nabika, Ryota; Oishi, Shinya; Ohara, Shinichiro; Sato, Masatoki; Kawasaki, Yukihiko; Fujii, Nobutaka; Hosoya, Mitsuaki

2016-01-01

Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model.

A Novel Peptide Derived from the Fusion Protein Heptad Repeat Inhibits Replication of Subacute Sclerosing Panencephalitis Virus In Vitro and In Vivo

PubMed Central

Watanabe, Masahiro; Hashimoto, Koichi; Abe, Yusaku; Kodama, Eiichi N.; Nabika, Ryota; Oishi, Shinya; Ohara, Shinichiro; Sato, Masatoki; Kawasaki, Yukihiko; Fujii, Nobutaka; Hosoya, Mitsuaki

2016-01-01

Subacute sclerosing panencephalitis (SSPE) is a persistent, progressive, and fatal degenerative disease resulting from persistent measles virus (MV) infection of the central nervous system. Most drugs used to treat SSPE have been reported to have limited effects. Therefore, novel therapeutic strategies are urgently required. The SSPE virus, a variant MV strain, differs virologically from wild-type MV strain. One characteristic of the SSPE virus is its defective production of cell-free virus, which leaves cell-to-cell infection as the major mechanism of viral dissemination. The fusion protein plays an essential role in this cell-to-cell spread. It contains two critical heptad repeat regions that form a six-helix bundle in the trimer similar to most viral fusion proteins. In the case of human immunodeficiency virus type-1 (HIV-1), a synthetic peptide derived from the heptad repeat region of the fusion protein enfuvirtide inhibits viral replication and is clinically approved as an anti-HIV-1 agent. The heptad repeat regions of HIV-1 are structurally and functionally similar to those of the MV fusion protein. We therefore designed novel peptides derived from the fusion protein heptad repeat region of the MV and examined their effects on the measles and SSPE virus replication in vitro and in vivo. Some of these synthetic novel peptides demonstrated high antiviral activity against both the measles (Edmonston strain) and SSPE (Yamagata-1 strain) viruses at nanomolar concentrations with no cytotoxicity in vitro. In particular, intracranial administration of one of the synthetic peptides increased the survival rate from 0% to 67% in an SSPE virus-infected nude mouse model. PMID:27612283

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

PubMed

Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

Comparative Proteomic Analysis of Wild-Type and SAP Domain Mutant Foot-and-Mouth Disease Virus-Infected Porcine Cells Identifies the Ubiquitin-Activating Enzyme UBE1 Required for Virus Replication.

PubMed

Zhu, Zixiang; Yang, Fan; Zhang, Keshan; Cao, Weijun; Jin, Ye; Wang, Guoqing; Mao, Ruoqing; Li, Dan; Guo, Jianhong; Liu, Xiangtao; Zheng, Haixue

2015-10-02

Leader protein (L(pro)) of foot-and-mouth disease virus (FMDV) manipulates the activities of several host proteins to promote viral replication and pathogenicity. L(pro) has a conserved protein domain SAP that is suggested to subvert interferon (IFN) production to block antiviral responses. However, apart from blocking IFN production, the roles of the SAP domain during FMDV infection in host cells remain unknown. Therefore, we identified host proteins associated with the SAP domain of L(pro) by a high-throughput quantitative proteomic approach [isobaric tags for relative and absolute quantitation (iTRAQ) in conjunction with liquid chromatography/electrospray ionization tandem mass spectrometry]. Comparison of the differentially regulated proteins in rA/FMDVΔmSAP- versus rA/FMDV-infected SK6 cells revealed 45 down-regulated and 32 up-regulated proteins that were mostly associated with metabolic, ribosome, spliceosome, and ubiquitin-proteasome pathways. The results also imply that the SAP domain has a function similar to SAF-A/B besides its potential protein inhibitor of activated signal transducer and activator of transcription (PIAS) function. One of the identified proteins UBE1 was further analyzed and displayed a novel role for the SAP domain of L(pro). Overexpression of UBE1 enhanced the replication of FMDV, and knockdown of UBE1 decreased FMDV replication. This shows that FMDV manipulates UBE1 for increased viral replication, and the SAP domain was involved in this process.

Picornaviruses and nuclear functions: targeting a cellular compartment distinct from the replication site of a positive-strand RNA virus

PubMed Central

Flather, Dylan; Semler, Bert L.

2015-01-01

The compartmentalization of DNA replication and gene transcription in the nucleus and protein production in the cytoplasm is a defining feature of eukaryotic cells. The nucleus functions to maintain the integrity of the nuclear genome of the cell and to control gene expression based on intracellular and environmental signals received through the cytoplasm. The spatial separation of the major processes that lead to the expression of protein-coding genes establishes the necessity of a transport network to allow biomolecules to translocate between these two regions of the cell. The nucleocytoplasmic transport network is therefore essential for regulating normal cellular functioning. The Picornaviridae virus family is one of many viral families that disrupt the nucleocytoplasmic trafficking of cells to promote viral replication. Picornaviruses contain positive-sense, single-stranded RNA genomes and replicate in the cytoplasm of infected cells. As a result of the limited coding capacity of these viruses, cellular proteins are required by these intracellular parasites for both translation and genomic RNA replication. Being of messenger RNA polarity, a picornavirus genome can immediately be translated upon entering the cell cytoplasm. However, the replication of viral RNA requires the activity of RNA-binding proteins, many of which function in host gene expression, and are consequently localized to the nucleus. As a result, picornaviruses disrupt nucleocytoplasmic trafficking to exploit protein functions normally localized to a different cellular compartment from which they translate their genome to facilitate efficient replication. Furthermore, picornavirus proteins are also known to enter the nucleus of infected cells to limit host-cell transcription and down-regulate innate antiviral responses. The interactions of picornavirus proteins and host-cell nuclei are extensive, required for a productive infection, and are the focus of this review. PMID:26150805

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses

PubMed Central

Johnson, Stephen M.; Eltahla, Auda A.; Aloi, Maria; Aloia, Amanda L.; McDevitt, Christopher A.; Bull, Rowena A.

2017-01-01

ABSTRACT Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3′ untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the Wing, connector, and β-ladder domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies. IMPORTANCE Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as “vesicle packets” (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools. PMID:28956770

A VP26-mNeonGreen Capsid Fusion HSV-2 Mutant Reactivates from Viral Latency in the Guinea Pig Genital Model with Normal Kinetics

PubMed Central

Pieknik, Julianna R.; Tang, Shuang

2018-01-01

Fluorescent herpes simplex viruses (HSV) are invaluable tools for localizing virus in cells, permitting visualization of capsid trafficking and enhancing neuroanatomical research. Fluorescent viruses can also be used to study virus kinetics and reactivation in vivo. Such studies would be facilitated by fluorescent herpes simplex virus recombinants that exhibit wild-type kinetics of replication and reactivation and that are genetically stable. We engineered an HSV-2 strain expressing the fluorescent mNeonGreen protein as a fusion with the VP26 capsid protein. This virus has normal replication and in vivo recurrence phenotypes, providing an essential improved tool for further study of HSV-2 infection. PMID:29738431

Identification of novel host factors via conserved domain search: Cns1 cochaperone is a novel restriction factor of tombusvirus replication in yeast.

PubMed

Lin, Jing-Yi; Nagy, Peter D

2013-12-01

A large number of host-encoded proteins affect the replication of plus-stranded RNA viruses by acting as susceptibility factors. Many other cellular proteins are known to function as restriction factors of viral infections. Previous studies with tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the inhibitory function of TPR (tetratricopeptide repeat) domain-containing cyclophilins, which are members of the large family of host prolyl isomerases, in TBSV replication. In this paper, we tested additional TPR-containing yeast proteins in a cell-free TBSV replication assay and identified the Cns1p cochaperone for heat shock protein 70 (Hsp70) and Hsp90 chaperones as a strong inhibitor of TBSV replication. Cns1p interacted with the viral replication proteins and inhibited the assembly of the viral replicase complex and viral RNA synthesis in vitro. Overexpression of Cns1p inhibited TBSV replication in yeast. The use of a temperature-sensitive (TS) mutant of Cns1p in yeast revealed that at a semipermissive temperature, TS Cns1p could not inhibit TBSV replication. Interestingly, Cns1p and the TPR-containing Cpr7p cyclophilin have similar inhibitory functions during TBSV replication, although some of the details of their viral restriction mechanisms are different. Our observations indicate that TPR-containing cellular proteins could act as virus restriction factors.

Influenza A Virus Encoding Secreted Gaussia Luciferase as Useful Tool to Analyze Viral Replication and Its Inhibition by Antiviral Compounds and Cellular Proteins

PubMed Central

Palanisamy, Navaneethan; Goedecke, Ulrike; Jäger, Nils; Pöhlmann, Stefan; Winkler, Michael

2014-01-01

Reporter genes inserted into viral genomes enable the easy and rapid quantification of virus replication, which is instrumental to efficient in vitro screening of antiviral compounds or in vivo analysis of viral spread and pathogenesis. Based on a published design, we have generated several replication competent influenza A viruses carrying either fluorescent proteins or Gaussia luciferase. Reporter activity could be readily quantified in infected cultures, but the virus encoding Gaussia luciferase was more stable than viruses bearing fluorescent proteins and was therefore analyzed in detail. Quantification of Gaussia luciferase activity in the supernatants of infected culture allowed the convenient and highly sensitive detection of viral spread, and enzymatic activity correlated with the number of infectious particles released from infected cells. Furthermore, the Gaussia luciferase encoding virus allowed the sensitive quantification of the antiviral activity of the neuraminidase inhibitor (NAI) zanamivir and the host cell interferon-inducible transmembrane (IFITM) proteins 1–3, which are known to inhibit influenza virus entry. Finally, the virus was used to demonstrate that influenza A virus infection is sensitive to a modulator of endosomal cholesterol, in keeping with the concept that IFITMs inhibit viral entry by altering cholesterol levels in the endosomal membrane. In sum, we report the characterization of a novel influenza A reporter virus, which allows fast and sensitive detection of viral spread and its inhibition, and we show that influenza A virus entry is sensitive to alterations of endosomal cholesterol levels. PMID:24842154

Investigations of Pro- and Anti-Apoptotic Factors Affecting African Swine Fever Virus Replication and Pathogenesis.

PubMed

Dixon, Linda K; Sánchez-Cordón, Pedro J; Galindo, Inmaculada; Alonso, Covadonga

2017-08-25

African swine fever virus (ASFV) is a large DNA virus that replicates predominantly in the cell cytoplasm and is the only member of the Asfarviridae family. The virus causes an acute haemorrhagic fever, African swine fever (ASF), in domestic pigs and wild boar resulting in the death of most infected animals. Apoptosis is induced at an early stage during virus entry or uncoating. However, ASFV encodes anti-apoptotic proteins which facilitate production of progeny virions. These anti-apoptotic proteins include A179L, a Bcl-2 family member; A224L, an inhibitor of apoptosis proteins (IAP) family member; EP153R a C-type lectin; and DP71L. The latter acts by inhibiting activation of the stress activated pro-apoptotic pathways pro-apoptotic pathways. The mechanisms by which these proteins act is summarised. ASF disease is characterised by massive apoptosis of uninfected lymphocytes which reduces the effectiveness of the immune response, contributing to virus pathogenesis. Mechanisms by which this apoptosis is induced are discussed.

Investigations of Pro- and Anti-Apoptotic Factors Affecting African Swine Fever Virus Replication and Pathogenesis

PubMed Central

Dixon, Linda K.; Sánchez-Cordón, Pedro J.; Galindo, Inmaculada

2017-01-01

African swine fever virus (ASFV) is a large DNA virus that replicates predominantly in the cell cytoplasm and is the only member of the Asfarviridae family. The virus causes an acute haemorrhagic fever, African swine fever (ASF), in domestic pigs and wild boar resulting in the death of most infected animals. Apoptosis is induced at an early stage during virus entry or uncoating. However, ASFV encodes anti-apoptotic proteins which facilitate production of progeny virions. These anti-apoptotic proteins include A179L, a Bcl-2 family member; A224L, an inhibitor of apoptosis proteins (IAP) family member; EP153R a C-type lectin; and DP71L. The latter acts by inhibiting activation of the stress activated pro-apoptotic pathways pro-apoptotic pathways. The mechanisms by which these proteins act is summarised. ASF disease is characterised by massive apoptosis of uninfected lymphocytes which reduces the effectiveness of the immune response, contributing to virus pathogenesis. Mechanisms by which this apoptosis is induced are discussed. PMID:28841179

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.

DNA Replication Arrest and DNA Damage Responses Induced by Alkylating Minor Groove Binders

DTIC Science & Technology

2001-05-01

We are interested in the molecular mechanisms involved in DNA replication arrest by the S phase DNA damage checkpoints. Using in vitro simian virus...40 DNA replication assays, we have found three factors that directly contribute to DNA damage-induced DNA replication arrest: Replication Protein A...trans-acting inhibitors. RPA is the major eukaryotic single-stranded DNA binding protein required for DNA replication , repair and recombination. Upon DNA

Moderate restriction of macrophage-tropic human immunodeficiency virus type 1 by SAMHD1 in monocyte-derived macrophages.

PubMed

Taya, Kahoru; Nakayama, Emi E; Shioda, Tatsuo

2014-01-01

Macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains are able to grow to high titers in human monocyte-derived macrophages. However, it was recently reported that cellular protein SAMHD1 restricts HIV-1 replication in human cells of the myeloid lineage, including monocyte-derived macrophages. Here we show that degradation of SAMHD1 in monocyte-derived macrophages was associated with moderately enhanced growth of the macrophage-tropic HIV-1 strain. SAMHD1 degradation was induced by treating target macrophages with vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 2 (HIV-2) particles containing viral protein X. For undifferentiated monocytes, HIV-2 particle treatment allowed undifferentiated monocytes to be fully permissive for productive infection by the macrophage-tropic HIV-1 strain. In contrast, untreated monocytes were totally resistant to HIV-1 replication. These results indicated that SAMHD1 moderately restricts even a macrophage-tropic HIV-1 strain in monocyte-derived macrophages, whereas the protein potently restricts HIV-1 replication in undifferentiated monocytes.

Defining Hsp70 Subnetworks in Dengue Virus Replication Reveals Key Vulnerability in Flavivirus Infection.

PubMed

Taguwa, Shuhei; Maringer, Kevin; Li, Xiaokai; Bernal-Rubio, Dabeiba; Rauch, Jennifer N; Gestwicki, Jason E; Andino, Raul; Fernandez-Sesma, Ana; Frydman, Judith

2015-11-19

Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here, we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication, and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals. Copyright © 2015 Elsevier Inc. All rights reserved.

Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies

PubMed Central

Baker, Steven F.; Perez, Daniel R.; Martínez-Sobrido, Luis

2016-01-01

Influenza A and B viruses (IAV and IBV, respectively) cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1) was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer’s spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection. PMID:26809059

Replication-Competent Influenza A and B Viruses Expressing a Fluorescent Dynamic Timer Protein for In Vitro and In Vivo Studies.

PubMed

Breen, Michael; Nogales, Aitor; Baker, Steven F; Perez, Daniel R; Martínez-Sobrido, Luis

2016-01-01

Influenza A and B viruses (IAV and IBV, respectively) cause annual seasonal human respiratory disease epidemics. In addition, IAVs have been implicated in occasional pandemics with inordinate health and economic consequences. Studying influenza viruses in vitro or in vivo requires the use of laborious secondary methodologies to identify infected cells. To circumvent this requirement, replication-competent infectious influenza viruses expressing an easily traceable fluorescent reporter protein can be used. Timer is a fluorescent protein that undergoes a time-dependent color emission conversion from green to red. The rate of spectral change is independent of Timer protein concentration and can be used to chronologically measure the duration of its expression. Here, we describe the generation of replication-competent IAV and IBV where the viral non-structural protein 1 (NS1) was fused to the fluorescent dynamic Timer protein. Timer-expressing IAV and IBV displayed similar plaque phenotypes and growth kinetics to wild-type viruses in tissue culture. Within infected cells, Timer's spectral shift can be used to measure the rate and cell-to-cell spread of infection using fluorescent microscopy, plate readers, or flow cytometry. The progression of Timer-expressing IAV infection was also evaluated in a mouse model, demonstrating the feasibility to characterize IAV cell-to-cell infections in vivo. By providing the ability to chronologically track viral spread, Timer-expressing influenza viruses are an excellent option to evaluate the in vitro and in vivo dynamics of viral infection.

Molecular Basis of Latency in Pathogenic Human Viruses

NASA Astrophysics Data System (ADS)

Garcia-Blanco, Mariano A.; Cullen, Bryan R.

1991-11-01

Several human viruses are able to latently infect specific target cell populations in vivo. Analysis of the replication cycles of herpes simplex virus, Epstein-Barr virus, and human immunodeficiency virus suggests that the latent infections established by these human pathogens primarily result from a lack of host factors critical for the expression of viral early gene products. The subsequent activation of specific cellular transcription factors in response to extracellular stimuli can induce the expression of these viral regulatory proteins and lead to a burst of lytic viral replication. Latency in these eukaryotic viruses therefore contrasts with latency in bacteriophage, which is maintained primarily by the expression of virally encoded repressors of lytic replication.

Heat shock protein 72 expression allows permissive replication of oncolytic adenovirus dl1520 (ONYX-015) in rat glioblastoma cells

PubMed Central

Madara, Jonathan; Krewet, James A; Shah, Maulik

2005-01-01

In this study we have made novel observations with regards to potentiation of the tumoricidal activity of the oncolytic adenovirus, dl1520 (ONYX-015) in rat glioblastoma cell lines expressing heat shock protein 72 (HSP72) due to permissive virus replication. ONYX-015 is a conditionally replicating adenovirus that is deleted for the E1B 55 kDA gene product whose normal function is to interact with cell-cycle regulatory proteins to permit virus replication. However, many murine and rodent cell lines are not permissive for adenovirus replication. Previously, it has been reported that the heat shock response is necessary for adenovirus replication and that induction of heat shock proteins is mediated by E1 region gene products. Therefore, we hypothesized that HSP72 expression may allow for permissive replication of ONYX-015 in previously non-permissive cells. Rat glioma cell lines 9L and RT2 were transfected with a plasmids expressing HSP72 or GFP. After infection with ONYX-015, no tumoricidal activity is observed in GFP expressing cell lines despite adequate transduction. In contrast, HSP72 transfected cells show cytopathic effects by 72 hours and greater than 75% loss of viability by 96 hours. Burst assays show active virus replication in the HSP72 expressing cell lines. Therefore, 9L-HSP72 and RT2-HSP72 are ideal models to evaluate the efficacy of ONYX-015 in an immunocompetent rat model. Our study has implications for creating rodent tumor models for pre-clinical studies with E1 region deleted conditionally replicating adenovirus. PMID:15762988

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.

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

You, Jae-Hwan; Reed, Mark L.; Hiscox, Julian A.

The severe acute respiratory syndrome-coronavirus nucleocapsid (N) protein is involved in virus replication and modulation of cell processes. In this latter respect control may in part be achieved through the sub-cellular localisation of the protein. N protein predominately localises in the cytoplasm (the site of virus replication and assembly) but also in the nucleus/nucleolus. Using a combination of live-cell and confocal microscopy coupled to mutagenesis we identified a cryptic nucleolar localisation signal in the central part of the N protein. In addition, based on structural comparison to the avian coronavirus N protein, a nuclear export signal was identified in themore » C-terminal region of the protein.« less

Construction of green fluorescent protein-tagged recombinant iridovirus to assess viral replication.

PubMed

Huang, Youhua; Huang, Xiaohong; Cai, Jia; Ye, Fuzhou; Guan, Liya; Liu, Hong; Qin, Qiwei

2011-09-01

Green fluorescent protein-tagged recombinant virus has been successfully applied to observing the infective dynamics and evaluating viral replication. Here, we identified soft-shelled turtle iridovirus (STIV) ORF55 as an envelope protein (VP55), and developed a recombinant STIV expressing an enhanced green fluorescent protein (EGFP) fused to VP55 (EGFP-STIV). Recombinant EGFP-STIV shared similar single-step growth curves and ultrastructural morphology with wild type STIV (wt-STIV). The green fluorescence distribution during EGFP-STIV infection was consistent with the intracellular distribution of VP55 which was mostly co-localized with virus assembly sites. Furthermore, EGFP-STIV could be used to evaluate viral replication conveniently under drug treatment, and the result showed that STIV replication was significantly inhibited after the addition of antioxidant pyrrolidine dithiocarbamate (PDTC). Thus, the EGFP-tagged recombinant iridovirus will not only be useful for further investigations on the viral replicative dynamics, but also provide an alternative simple strategy to screen for antiviral substances. Copyright © 2011 Elsevier B.V. All rights reserved.

Coat Protein Regulation by CK2, CPIP, HSP70, and CHIP Is Required for Potato Virus A Replication and Coat Protein Accumulation

PubMed Central

Lõhmus, Andres; Hafrén, Anders

2016-01-01

ABSTRACT We demonstrate here that both coat protein (CP) phosphorylation by protein kinase CK2 and a chaperone system formed by two heat shock proteins, CP-interacting protein (CPIP) and heat shock protein 70 (HSP70), are essential for potato virus A (PVA; genus Potyvirus) replication and that all these host proteins have the capacity to contribute to the level of PVA CP accumulation. An E3 ubiquitin ligase called carboxyl terminus Hsc70-interacting protein (CHIP), which may participate in the CPIP-HSP70-mediated CP degradation, is also needed for robust PVA gene expression. Residue Thr243 within the CK2 consensus sequence of PVA CP was found to be essential for viral replication and to regulate CP protein stability. Substitution of Thr243 either with a phosphorylation-mimicking Asp (CPADA) or with a phosphorylation-deficient Ala (CPAAA) residue in CP expressed from viral RNA limited PVA gene expression to the level of nonreplicating PVA. We found that both the CPAAA mutant and CK2 silencing inhibited, whereas CPADA mutant and overexpression of CK2 increased, PVA translation. From our previous studies, we know that phosphorylation reduces the RNA binding capacity of PVA CP and an excess of CP fully blocks viral RNA translation. Together, these findings suggest that binding by nonphosphorylated PVA CP represses viral RNA translation, involving further CP phosphorylation and CPIP-HSP70 chaperone activities as prerequisites for PVA replication. We propose that this mechanism contributes to shifting potyvirus RNA from translation to replication. IMPORTANCE Host protein kinase CK2, two host chaperones, CPIP and HSP70, and viral coat protein (CP) phosphorylation at Thr243 are needed for potato virus A (PVA) replication. Our results show that nonphosphorylated CP blocks viral translation, likely via binding to viral RNA. We propose that this translational block is needed to allow time and space for the formation of potyviral replication complex around the 3′ end of viral RNA. Progression into replication involves CP regulation by both CK2 phosphorylation and chaperones CPIP and HSP70. PMID:27852853

Replication of plant RNA virus genomes in a cell-free extract of evacuolated plant protoplasts

PubMed Central

Komoda, Keisuke; Naito, Satoshi; Ishikawa, Masayuki

2004-01-01

The replication of eukaryotic positive-strand RNA virus genomes occurs through a complex process involving multiple viral and host proteins and intracellular membranes. Here we report a cell-free system that reproduces this process in vitro. This system uses a membrane-containing extract of uninfected plant protoplasts from which the vacuoles had been removed by Percoll gradient centrifugation. We demonstrate that the system supported translation, negative-strand RNA synthesis, genomic RNA replication, and subgenomic RNA transcription of tomato mosaic virus and two other plant positive-strand RNA viruses. The RNA synthesis, which depended on translation of the genomic RNA, produced virus-related RNA species similar to those that are generated in vivo. This system will aid in the elucidation of the mechanisms of genome replication in these viruses. PMID:14769932

[Bioinformatics analysis of mosquito densovirus nostructure protein NS1].

PubMed

Dong, Yun-qiao; Ma, Wen-li; Gu, Jin-bao; Zheng, Wen-ling

2009-12-01

To analyze and predict the structure and function of mosquito densovirus (MDV) nostructual protein1 (NS1). Using different bioinformatics software, the EXPASY pmtparam tool, ClustalX1.83, Bioedit, MEGA3.1, ScanProsite, and Motifscan, respectively to comparatively analyze and predict the physic-chemical parameters, homology, evolutionary relation, secondary structure and main functional motifs of NS1. MDV NS1 protein was a unstable hydrophilic protein and the amino acid sequence was highly conserved which had a relatively closer evolutionary distance with infectious hypodermal and hematopoietic necrosis virus (IHHNV). MDV NS1 has a specific domain of superfamily 3 helicase of small DNA viruses. This domain contains the NTP-binding region with a metal ion-dependent ATPase activity. A virus replication roller rolling-circle replication(RCR) initiation domain was found near the N terminal of this protein. This protien has the biological function of single stranded incision enzyme. The bioinformatics prediction results suggest that MDV NS1 protein plays a key role in viral replication, packaging, and the other stages of viral life.

Ultrastructure of the replication sites of positive-strand RNA viruses

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

Harak, Christian; Lohmann, Volker, E-mail: volker_lohmann@med.uni-heidelberg.de

2015-05-15

Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. Inmore » this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication. - Highlights: • Positive strand RNA viruses induce massive membrane alterations. • Despite the great diversity, replication complexes share many similarities. • Host factors play a pivotal role in replication complex biogenesis. • Use of the same host factors by several viruses hints to similar functions.« less

The Bean Pod Mottle Virus RNA2-Encoded 58-Kilodalton Protein P58 Is Required in cis for RNA2 Accumulation

PubMed Central

Lin, Junyan; Guo, Jiangbo; Finer, John; Dorrance, Anne E.; Redinbaugh, Margaret G.

2014-01-01

ABSTRACT Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58-kDa protein (P58) encoded by RNA2 has not been resolved. P58 and the movement protein (MP) of BPMV are two largely identical proteins differing only at their N termini, with P58 extending MP upstream by 102 amino acid residues. In this report, we unveil a unique role for P58. We show that BPMV RNA2 accumulation in infected cells was abolished when the start codon of P58 was eliminated. The role of P58 does not require the region shared by MP, as RNA2 accumulation in individual cells remained robust even when most of the MP coding sequence was removed. Importantly, the function of P58 required the P58 protein, rather than its coding RNA, as compensatory mutants could be isolated that restored RNA2 accumulation by acquiring new start codons upstream of the original one. Most strikingly, loss of P58 function could not be complemented by P58 provided in trans, suggesting that P58 functions in cis to selectively promote the accumulation of RNA2 copies that encode a functional P58 protein. Finally, we found that all RNA1-encoded proteins are cis-acting relative to RNA1. Together, our results suggest that P58 probably functions by recruiting the RNA1-encoded polyprotein to RNA2 to enable RNA2 reproduction. IMPORTANCE Bean pod mottle virus (BPMV) is one of the most important pathogens of the crop plant soybean, yet its replication mechanism is not well understood, hindering the development of knowledge-based control measures. The current study examined the replication strategy of BPMV RNA2, one of the two genomic RNA segments of this virus, and established an essential role for P58, one of the RNA2-encoded proteins, in the process of RNA2 replication. Our study demonstrates for the first time that P58 functions preferentially with the very RNA from which it is translated, thus greatly advancing our understanding of the replication mechanisms of this and related viruses. Furthermore, this study is important because it provides a potential target for BPMV-specific control, and hence could help to mitigate soybean production losses caused by this virus. PMID:24390330

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface.

PubMed

Knodel, Markus M; Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; Targett-Adams, Paul; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-08

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

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

Ibrahim, Amr; Present address: Genomics Facility, Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza 12619; Hutchens, Heather M.

2012-11-25

To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed withmore » P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.« less

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface

PubMed Central

Nägel, Arne; Reiter, Sebastian; Vogel, Andreas; McLauchlan, John; Herrmann, Eva; Wittum, Gabriel

2018-01-01

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles. PMID:29316722

Proteolysis of mature HIV-1 p6 Gag protein by the insulin-degrading enzyme (IDE) regulates virus replication in an Env-dependent manner.

PubMed

Hahn, Friedrich; Schmalen, Adrian; Setz, Christian; Friedrich, Melanie; Schlößer, Stefan; Kölle, Julia; Spranger, Robert; Rauch, Pia; Fraedrich, Kirsten; Reif, Tatjana; Karius-Fischer, Julia; Balasubramanyam, Ashok; Henklein, Petra; Fossen, Torgils; Schubert, Ulrich

2017-01-01

There is a significantly higher risk for type II diabetes in HIV-1 carriers, albeit the molecular mechanism for this HIV-related pathology remains enigmatic. The 52 amino acid HIV-1 p6 Gag protein is synthesized as the C-terminal part of the Gag polyprotein Pr55. In this context, p6 promotes virus release by its two late (L-) domains, and facilitates the incorporation of the viral accessory protein Vpr. However, the function of p6 in its mature form, after proteolytic release from Gag, has not been investigated yet. We found that the mature p6 represents the first known viral substrate of the ubiquitously expressed cytosolic metalloendopeptidase insulin-degrading enzyme (IDE). IDE is sufficient and required for degradation of p6, and p6 is approximately 100-fold more efficiently degraded by IDE than its eponymous substrate insulin. This observation appears to be specific for HIV-1, as p6 proteins from HIV-2 and simian immunodeficiency virus, as well as the 51 amino acid p9 from equine infectious anaemia virus were insensitive to IDE degradation. The amount of virus-associated p6, as well as the efficiency of release and maturation of progeny viruses does not depend on the presence of IDE in the host cells, as it was shown by CRISPR/Cas9 edited IDE KO cells. However, HIV-1 mutants harboring IDE-insensitive p6 variants exhibit reduced virus replication capacity, a phenomenon that seems to depend on the presence of an X4-tropic Env. Furthermore, competing for IDE by exogenous insulin or inhibiting IDE by the highly specific inhibitor 6bK, also reduced virus replication. This effect could be specifically attributed to IDE since replication of HIV-1 variants coding for an IDE-insensitive p6 were inert towards IDE-inhibition. Our cumulative data support a model in which removal of p6 during viral entry is important for virus replication, at least in the case of X4 tropic HIV-1.

Endoplasmic Reticulum Stress Induced Synthesis of a Novel Viral Factor Mediates Efficient Replication of Genotype-1 Hepatitis E Virus.

PubMed

Nair, Vidya P; Anang, Saumya; Subramani, Chandru; Madhvi, Abhilasha; Bakshi, Karishma; Srivastava, Akriti; Shalimar; Nayak, Baibaswata; Ranjith Kumar, C T; Surjit, Milan

2016-04-01

Hepatitis E virus (HEV) causes acute hepatitis in many parts of the world including Asia, Africa and Latin America. Though self-limiting in normal individuals, it results in ~30% mortality in infected pregnant women. It has also been reported to cause acute and chronic hepatitis in organ transplant patients. Of the seven viral genotypes, genotype-1 virus infects humans and is a major public health concern in South Asian countries. Sporadic cases of genotype-3 and 4 infection in human and animals such as pigs, deer, mongeese have been reported primarily from industrialized countries. Genotype-5, 6 and 7 viruses are known to infect animals such as wild boar and camel, respectively. Genotype-3 and 4 viruses have been successfully propagated in the laboratory in mammalian cell culture. However, genotype-1 virus replicates poorly in mammalian cell culture and no other efficient model exists to study its life cycle. Here, we report that endoplasmic reticulum (ER) stress promotes genotype-1 HEV replication by inducing cap-independent, internal initiation mediated translation of a novel viral protein (named ORF4). Importantly, ORF4 expression and stimulatory effect of ER stress inducers on viral replication is specific to genotype-1. ORF4 protein sequence is mostly conserved among genotype-1 HEV isolates and ORF4 specific antibodies were detected in genotype-1 HEV patient serum. ORF4 interacted with multiple viral and host proteins and assembled a protein complex consisting of viral helicase, RNA dependent RNA polymerase (RdRp), X, host eEF1α1 (eukaryotic elongation factor 1 isoform-1) and tubulinβ. In association with eEF1α1, ORF4 stimulated viral RdRp activity. Furthermore, human hepatoma cells that stably express ORF4 or engineered proteasome resistant ORF4 mutant genome permitted enhanced viral replication. These findings reveal a positive role of ER stress in promoting genotype-1 HEV replication and pave the way towards development of an efficient model of the virus.

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

Zhao, Baoyu; Yi, Guanghui; Du, Fenglei

The recent outbreak of Zika virus (ZIKV) has infected over 1 million people in over 30 countries. ZIKV replicates its RNA genome using virally encoded replication proteins. Nonstructural protein 5 (NS5) contains a methyltransferase for RNA capping and a polymerase for viral RNA synthesis. Here we report the crystal structures of full-length NS5 and its polymerase domain at 3.0 Å resolution. The NS5 structure has striking similarities to the NS5 protein of the related Japanese encephalitis virus. The methyltransferase contains in-line pockets for substrate binding and the active site. Key residues in the polymerase are located in similar positions tomore » those of the initiation complex for the hepatitis C virus polymerase. The polymerase conformation is affected by the methyltransferase, which enables a more efficiently elongation of RNA synthesis in vitro. Altogether, our results will contribute to future studies on ZIKV infection and the development of inhibitors of ZIKV replication.« less

The Cellular Autophagy Pathway Modulates Human T-Cell Leukemia Virus Type 1 Replication

PubMed Central

Tang, Sai-Wen; Chen, Chia-Yen; Klase, Zachary; Zane, Linda

2013-01-01

Autophagy, a general homeostatic process for degradation of cytosolic proteins or organelles, has been reported to modulate the replication of many viruses. The role of autophagy in human T-cell leukemia virus type 1 (HTLV-1) replication has, however, been uncharacterized. Here, we report that HTLV-1 infection increases the accumulation of autophagosomes and that this accumulation increases HTLV-1 production. We found that the HTLV-1 Tax protein increases cellular autophagosome accumulation by acting to block the fusion of autophagosomes to lysosomes, preventing the degradation of the former by the latter. Interestingly, the inhibition of cellular autophagosome-lysosome fusion using bafilomycin A increased the stability of the Tax protein, suggesting that cellular degradation of Tax occurs in part through autophagy. Our current findings indicate that by interrupting the cell's autophagic process, Tax exerts a positive feedback on its own stability. PMID:23175371

Pharmacological cdk inhibitor R-Roscovitine suppresses JC virus proliferation

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

Orba, Yasuko; Laboratory of Molecular and Cellular Pathology, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, 060-8638, Sapporo; Research Fellow of the Japan Society for the Promotion of Science

2008-01-05

The human Polyomavirus JC virus (JCV) utilizes cellular proteins for viral replication and transcription in the host cell nucleus. These cellular proteins represent potential targets for antiviral drugs against the JCV. In this study, we examined the antiviral effects of the pharmacological cyclin-dependent kinase (cdk) inhibitor R-Roscovitine, which has been shown to have antiviral activity against other viruses. We found that Roscovitine significantly inhibited the viral production and cytopathic effects of the JCV in a JCV-infected cell line. Roscovitine attenuated the transcriptional activity of JCV late genes, but not early genes, and also prevented viral replication via inhibiting phosphorylation ofmore » the viral early protein, large T antigen. These data suggest that the JCV requires cdks to transcribe late genes and to replicate its own DNA. That Roscovitine exhibited antiviral activity in JCV-infected cells suggests that Roscovitine might have therapeutic utility in the treatment of progressive multifocal leukoencephalopathy (PML)« less

Inhibition of Avian Influenza A Virus Replication in Human Cells by Host Restriction Factor TUFM Is Correlated with Autophagy.

PubMed

Kuo, Shu-Ming; Chen, Chi-Jene; Chang, Shih-Cheng; Liu, Tzu-Jou; Chen, Yi-Hsiang; Huang, Sheng-Yu; Shih, Shin-Ru

2017-06-13

Avian influenza A viruses generally do not replicate efficiently in human cells, but substitution of glutamic acid (Glu, E) for lysine (Lys, K) at residue 627 of avian influenza virus polymerase basic protein 2 (PB2) can serve to overcome host restriction and facilitate human infectivity. Although PB2 residue 627 is regarded as a species-specific signature of influenza A viruses, host restriction factors associated with PB2 627 E have yet to be fully investigated. We conducted immunoprecipitation, followed by differential proteomic analysis, to identify proteins associating with PB2 627 K (human signature) and PB2 627 E (avian signature) of influenza A/WSN/1933(H1N1) virus, and the results indicated that Tu elongation factor, mitochondrial (TUFM), had a higher binding affinity for PB2 627 E than PB2 627 K in transfected human cells. Stronger binding of TUFM to avian-signature PB2 590 G/ 591 Q and PB2 627 E in the 2009 swine-origin pandemic H1N1 and 2013 avian-origin H7N9 influenza A viruses was similarly observed. Viruses carrying avian-signature PB2 627 E demonstrated increased replication in TUFM-deficient cells, but viral replication decreased in cells overexpressing TUFM. Interestingly, the presence of TUFM specifically inhibited the replication of PB2 627 E viruses, but not PB2 627 K viruses. In addition, enhanced levels of interaction between TUFM and PB2 627 E were noted in the mitochondrial fraction of infected cells. Furthermore, TUFM-dependent autophagy was reduced in TUFM-deficient cells infected with PB2 627 E virus; however, autophagy remained consistent in PB2 627 K virus-infected cells. The results suggest that TUFM acts as a host restriction factor that impedes avian-signature influenza A virus replication in human cells in a manner that correlates with autophagy. IMPORTANCE An understanding of the mechanisms that influenza A viruses utilize to shift host tropism and the identification of host restriction factors that can limit infection are both critical to the prevention and control of emerging viruses that cross species barriers to target new hosts. Using a proteomic approach, we revealed a novel role for TUFM as a host restriction factor that exerts an inhibitory effect on avian-signature PB2 627 E influenza virus propagation in human cells. We further found that increased TUFM-dependent autophagy correlates with the inhibitory effect on avian-signature influenza virus replication and may serve as a key intrinsic mechanism to restrict avian influenza virus infection in humans. These findings provide new insight regarding the TUFM mitochondrial protein and may have important implications for the development of novel antiviral strategies. Copyright © 2017 Kuo et al.

Importin α5 negatively regulates importin β1-mediated nuclear import of Newcastle disease virus matrix protein and viral replication and pathogenicity in chicken fibroblasts.

PubMed

Duan, Zhiqiang; Xu, Haixu; Ji, Xinqin; Zhao, Jiafu; Xu, Houqiang; Hu, Yan; Deng, Shanshan; Hu, Shunlin; Liu, Xiufan

2018-12-31

The matrix (M) protein of Newcastle disease virus (NDV) is demonstrated to localize in the nucleus via intrinsic nuclear localization signal (NLS), but cellular proteins involved in the nuclear import of NDV M protein and the role of M's nuclear localization in the replication and pathogenicity of NDV remain unclear. In this study, importin β1 was screened to interact with NDV M protein by yeast two-hybrid screening. This interaction was subsequently confirmed by co-immunoprecipitation and pull-down assays. In vitro binding studies indicated that the NLS region of M protein and the amino acids 336-433 of importin β1 that belonged to the RanGTP binding region were important for binding. Importantly, a recombinant virus with M/NLS mutation resulted in a pathotype change of NDV and attenuated viral replication and pathogenicity in chicken fibroblasts and SPF chickens. In agreement with the binding data, nuclear import of NDV M protein in digitonin-permeabilized HeLa cells required both importin β1 and RanGTP. Interestingly, importin α5 was verified to interact with M protein through binding importin β1. However, importin β1 or importin α5 depletion by siRNA resulted in different results, which showed the obviously cytoplasmic or nuclear accumulation of M protein and the remarkably decreased or increased replication ability and pathogenicity of NDV in chicken fibroblasts, respectively. Our findings therefore demonstrate for the first time the nuclear import mechanism of NDV M protein and the negative regulation role of importin α5 in importin β1-mediated nuclear import of M protein and the replication and pathogenicity of a paramyxovirus.

Influenza A Virus M2 Ion Channel Activity Is Essential for Efficient Replication in Tissue Culture

PubMed Central

Takeda, Makoto; Pekosz, Andrew; Shuck, Kevin; Pinto, Lawrence H.; Lamb, Robert A.

2002-01-01

The amantadine-sensitive ion channel activity of influenza A virus M2 protein was discovered through understanding the two steps in the virus life cycle that are inhibited by the antiviral drug amantadine: virus uncoating in endosomes and M2 protein-mediated equilibration of the intralumenal pH of the trans Golgi network. Recently it was reported that influenza virus can undergo multiple cycles of replication without M2 ion channel activity (T. Watanabe, S. Watanabe, H. Ito, H. Kida, and Y. Kawaoka, J. Virol. 75:5656–5662, 2001). An M2 protein containing a deletion in the transmembrane (TM) domain (M2-del29–31) has no detectable ion channel activity, yet a mutant virus was obtained containing this deletion. Watanabe and colleagues reported that the M2-del29–31 virus replicated as efficiently as wild-type (wt) virus. We have investigated the effect of amantadine on the growth of four influenza viruses: A/WSN/33; N31S-M2WSN, a mutant in which an asparagine residue at position 31 in the M2 TM domain was replaced with a serine residue; MUd/WSN, which possesses seven RNA segments from WSN plus the RNA segment 7 derived from A/Udorn/72; and A/Udorn/72. N31S-M2WSN was amantadine sensitive, whereas A/WSN/33 was amantadine resistant, indicating that the M2 residue N31 is the sole determinant of resistance of A/WSN/33 to amantadine. The growth of influenza viruses inhibited by amantadine was compared to the growth of an M2-del29–31 virus. We found that the M2-del29–31 virus was debilitated in growth to an extent similar to that of influenza virus grown in the presence of amantadine. Furthermore, in a test of biological fitness, it was found that wt virus almost completely outgrew M2-del29–31 virus in 4 days after cocultivation of a 100:1 ratio of M2-del29–31 virus to wt virus, respectively. We conclude that the M2 ion channel protein, which is conserved in all known strains of influenza virus, evolved its function because it contributes to the efficient replication of the virus in a single cycle. PMID:11773413

Virus reactivation: a panoramic view in human infections

PubMed Central

Traylen, Christopher M; Patel, Hersh R; Fondaw, Wylder; Mahatme, Sheran; Williams, John F; Walker, Lia R; Dyson, Ossie F; Arce, Sergio; Akula, Shaw M

2011-01-01

Viruses are obligate intracellular parasites, relying to a major extent on the host cell for replication. An active replication of the viral genome results in a lytic infection characterized by the release of new progeny virus particles, often upon the lysis of the host cell. Another mode of virus infection is the latent phase, where the virus is ‘quiescent’ (a state in which the virus is not replicating). A combination of these stages, where virus replication involves stages of both silent and productive infection without rapidly killing or even producing excessive damage to the host cells, falls under the umbrella of a persistent infection. Reactivation is the process by which a latent virus switches to a lytic phase of replication. Reactivation may be provoked by a combination of external and/or internal cellular stimuli. Understanding this mechanism is essential in developing future therapeutic agents against viral infection and subsequent disease. This article examines the published literature and current knowledge regarding the viral and cellular proteins that may play a role in viral reactivation. The focus of the article is on those viruses known to cause latent infections, which include herpes simplex virus, varicella zoster virus, Epstein–Barr virus, human cytomegalovirus, human herpesvirus 6, human herpesvirus 7, Kaposi’s sarcoma-associated herpesvirus, JC virus, BK virus, parvovirus and adenovirus. PMID:21799704

Biological and Structural Characterization of a Host-Adapting Amino Acid in Influenza Virus

PubMed Central

Yamada, Shinya; Hatta, Masato; Staker, Bart L.; Watanabe, Shinji; Imai, Masaki; Shinya, Kyoko; Sakai-Tagawa, Yuko; Ito, Mutsumi; Ozawa, Makoto; Watanabe, Tokiko; Sakabe, Saori; Li, Chengjun; Kim, Jin Hyun; Myler, Peter J.; Phan, Isabelle; Raymond, Amy; Smith, Eric; Stacy, Robin; Nidom, Chairul A.; Lank, Simon M.; Wiseman, Roger W.; Bimber, Benjamin N.; O'Connor, David H.; Neumann, Gabriele; Stewart, Lance J.; Kawaoka, Yoshihiro

2010-01-01

Two amino acids (lysine at position 627 or asparagine at position 701) in the polymerase subunit PB2 protein are considered critical for the adaptation of avian influenza A viruses to mammals. However, the recently emerged pandemic H1N1 viruses lack these amino acids. Here, we report that a basic amino acid at position 591 of PB2 can compensate for the lack of lysine at position 627 and confers efficient viral replication to pandemic H1N1 viruses in mammals. Moreover, a basic amino acid at position 591 of PB2 substantially increased the lethality of an avian H5N1 virus in mice. We also present the X-ray crystallographic structure of the C-terminus of a pandemic H1N1 virus PB2 protein. Arginine at position 591 fills the cleft found in H5N1 PB2 proteins in this area, resulting in differences in surface shape and charge for H1N1 PB2 proteins. These differences may affect the protein's interaction with viral and/or cellular factors, and hence its ability to support virus replication in mammals. PMID:20700447

Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

PubMed

Hand, Erin S; Haller, Sherry L; Peng, Chen; Rothenburg, Stefan; Hersperger, Adam R

2015-01-01

As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV.

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.

The Bean pod mottle virus RNA2-encoded 58-kilodalton protein P58 is required in cis for RNA2 accumulation

USDA-ARS?s Scientific Manuscript database

Bean pod mottle virus (BPMV) is a bipartite, positive sense (+) RNA plant virus in the Secoviridae family. Its RNA1 encodes proteins required for genome replication, whereas RNA2 primarily encodes proteins needed for virion assembly and cell-to-cell movement. However, the function of a 58 kilo-dalto...

Suppression of Poxvirus Replication by Resveratrol.

PubMed

Cao, Shuai; Realegeno, Susan; Pant, Anil; Satheshkumar, Panayampalli S; Yang, Zhilong

2017-01-01

Poxviruses continue to cause serious diseases even after eradication of the historically deadly infectious human disease, smallpox. Poxviruses are currently being developed as vaccine vectors and cancer therapeutic agents. Resveratrol is a natural polyphenol stilbenoid found in plants that has been shown to inhibit or enhance replication of a number of viruses, but the effect of resveratrol on poxvirus replication is unknown. In the present study, we found that resveratrol dramatically suppressed the replication of vaccinia virus (VACV), the prototypic member of poxviruses, in various cell types. Resveratrol also significantly reduced the replication of monkeypox virus, a zoonotic virus that is endemic in Western and Central Africa and causes human mortality. The inhibitory effect of resveratrol on poxviruses is independent of VACV N1 protein, a potential resveratrol binding target. Further experiments demonstrated that resveratrol had little effect on VACV early gene expression, while it suppressed VACV DNA synthesis, and subsequently post-replicative gene expression.

Identification of Poxvirus Genome Uncoating and DNA Replication Factors with Mutually Redundant Roles.

PubMed

Liu, Baoming; Panda, Debasis; Mendez-Rios, Jorge D; Ganesan, Sundar; Wyatt, Linda S; Moss, Bernard

2018-04-01

Genome uncoating is essential for replication of most viruses. For poxviruses, the process is divided into two stages: removal of the envelope, allowing early gene expression, and breaching of the core wall, allowing DNA release, replication, and late gene expression. Subsequent studies showed that the host proteasome and the viral D5 protein, which has an essential role in DNA replication, are required for vaccinia virus (VACV) genome uncoating. In a search for additional VACV uncoating proteins, we noted a report that described a defect in DNA replication and late expression when the gene encoding a 68-kDa ankyrin repeat/F-box protein (68k-ank), associated with the cellular SCF (Skp1, cullin1, F-box-containing complex) ubiquitin ligase complex, was deleted from the attenuated modified vaccinia virus Ankara (MVA). Here we showed that the 68k-ank deletion mutant exhibited diminished genome uncoating, formation of DNA prereplication sites, and degradation of viral cores as well as an additional, independent defect in DNA synthesis. Deletion of the 68k-ank homolog of VACV strain WR, however, was without effect, suggesting the existence of compensating genes. By inserting VACV genes into an MVA 68k-ank deletion mutant, we discovered that M2, a member of the poxvirus immune evasion (PIE) domain superfamily and a regulator of NF-κB, and C5, a member of the BTB/Kelch superfamily associated with cullin-3-based ligase complexes, independently rescued the 68k-ank deletion phenotype. Thus, poxvirus uncoating and DNA replication are intertwined processes involving at least three viral proteins with mutually redundant functions in addition to D5. IMPORTANCE Poxviruses comprise a family of large DNA viruses that infect vertebrates and invertebrates and cause diseases of medical and zoological importance. Poxviruses, unlike most other DNA viruses, replicate in the cytoplasm, and their large genomes usually encode 200 or more proteins with diverse functions. About 90 genes may be essential for chordopoxvirus replication based either on their conservation or individual gene deletion studies. However, this number may underestimate the true number of essential functions because of redundancy. Here we show that any one of three seemingly unrelated and individually nonessential proteins is required for the incompletely understood processes of genome uncoating and DNA replication, an example of synthetic lethality. Thus, poxviruses appear to have a complex genetic interaction network that has not been fully appreciated and which will require multifactor deletion screens to assess. Copyright © 2018 American Society for Microbiology.

Role of the Adenovirus DNA-Binding Protein in In Vitro Adeno-Associated Virus DNA Replication

PubMed Central

Ward, Peter; Dean, Frank B.; O’Donnell, Michael E.; Berns, Kenneth I.

1998-01-01

A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences. PMID:9420241

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

Dinh, Phat X.; Panda, Debasis; Das, Phani B.

Using a recombinant vesicular stomatitis virus encoding eGFP fused in-frame with an essential viral replication protein, the phosphoprotein P, we show that during passage in culture, the virus mutates the nucleotide C289 within eGFP of the fusion protein PeGFP to A or T, resulting in R97S/C amino acid substitution and loss of fluorescence. The resultant non-fluorescent virus exhibits increased fitness and growth advantage over its fluorescent counterpart. The growth advantage of the non-fluorescent virus appears to be due to increased transcription and replication activities of the PeGFP protein carrying the R97S/C substitution. Further, our results show that the R97S/C mutationmore » occurs prior to accumulation of mutations that can result in loss of expression of the gene inserted at the G-L gene junction. These results suggest that fitness gain is more important for the recombinant virus than elimination of expression of the heterologous gene.« less

Tumor Necrosis Factor Receptor-Associated Factor 5 Interacts with the NS3 Protein and Promotes Classical Swine Fever Virus Replication.

PubMed

Lv, Huifang; Dong, Wang; Guo, Kangkang; Jin, Mingxing; Li, Xiaomeng; Li, Cunfa; Zhang, Yanming

2018-06-05

Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious and high-mortality viral disease, causing huge economic losses in the swine industry worldwide. CSFV non-structural protein 3 (NS3), a multifunctional protein, plays crucial roles in viral replication. However, how NS3 exactly exerts these functions is currently unknown. Here, we identified tumor necrosis factor receptor-associated factor 5 (TRAF5) as a novel binding partner of the NS3 protein via yeast two-hybrid, co-immunoprecipitation and glutathione S -transferase pull-down assays. Furthermore, we observed that TRAF5 promoted CSFV replication in porcine alveolar macrophages (PAMs). Additionally, CSFV infection or NS3 expression upregulated TRAF5 expression, implying that CSFV may exploit TRAF5 via NS3 for better growth. Moreover, CSFV infection and TRAF5 expression activated p38 mitogen activated protein kinase (MAPK) activity, and inhibition of p38 MAPK activation by the SB203580 inhibitor suppressed CSFV replication. Notably, TRAF5 overexpression did not promote CSFV replication following inhibition of p38 MAPK activation. Our findings reveal that TRAF5 promotes CSFV replication via p38 MAPK activation. This work provides a novel insight into the role of TRAF5 in CSFV replication capacity.

Influenza B virus M2 protein can functionally replace its influenza A virus counterpart in promoting virus replication

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

Wanitchang, Asawin; Wongthida, Phonphimon; Jongkae

The M2 protein (AM2 and BM2) of influenza A and B viruses function as a proton channel essential for viral replication. They also carry a cytoplasmic tail whose functions are not fully delineated. It is currently unknown whether these proteins could be replaced functionally in a viral context. Here, we generated single-cycle influenza A viruses (scIAV-ΔHA) carrying various M2-2A-mCherry constructs in the segment 4 (HA) and evaluated their growth in complementing cells. Intriguingly, the scIAV-ΔHA carrying AM2 and that bearing BM2 grew comparably well in MDCK-HA cells. Furthermore, while the virus carrying chimeric B-AM2 in which the BM2 transmembrane fusedmore » with the AM2 cytoplasmic tail produced robust infection, the one bearing the AM2 transmembrane fused with the BM2 cytoplasmic tail (A-BM2) exhibited severely impaired growth. Altogether, we demonstrate that AM2 and BM2 are functionally interchangeable and underscore the role of compatibility between transmembrane and cytoplasmic tail of the M2 protein. -- Highlights: •Flu A M2 protein (AM2) can be functionally replaced by that of Flu B (BM2). •Both AM2 and BM2 with extended cytoplasmic tail are functional. •Compatibility between the ion channel and the cytoplasmic tail is critical for M2 function. •M2 with higher ion channel activity may augment influenza virus replication.« less

Bioinformatics and functional analyses of coronavirus nonstructural proteins involved in the formation of replicative organelles.

PubMed

Neuman, Benjamin W

2016-11-01

Replication of eukaryotic positive-stranded RNA viruses is usually linked to the presence of membrane-associated replicative organelles. The purpose of this review is to discuss the function of proteins responsible for formation of the coronavirus replicative organelle. This will be done by identifying domains that are conserved across the order Nidovirales, and by summarizing what is known about function and structure at the level of protein domains. Copyright © 2016 Elsevier B.V. All rights reserved.

Rewiring of cellular membrane homeostasis by picornaviruses.

PubMed

Belov, George A; Sztul, Elizabeth

2014-09-01

Viruses are obligatory intracellular parasites and utilize host elements to support key viral processes, including penetration of the plasma membrane, initiation of infection, replication, and suppression of the host's antiviral defenses. In this review, we focus on picornaviruses, a family of positive-strand RNA viruses, and discuss the mechanisms by which these viruses hijack the cellular machinery to form and operate membranous replication complexes. Studies aimed at revealing factors required for the establishment of viral replication structures identified several cellular-membrane-remodeling proteins and led to the development of models in which the virus used a preexisting cellular-membrane-shaping pathway "as is" for generating its replication organelles. However, as more data accumulate, this view is being increasingly questioned, and it is becoming clearer that viruses may utilize cellular factors in ways that are distinct from the normal functions of these proteins in uninfected cells. In addition, the proteincentric view is being supplemented by important new studies showing a previously unappreciated deep remodeling of lipid homeostasis, including extreme changes to phospholipid biosynthesis and cholesterol trafficking. The data on viral modifications of lipid biosynthetic pathways are still rudimentary, but it appears once again that the viruses may rewire existing pathways to generate novel functions. Despite remarkable progress, our understanding of how a handful of viral proteins can completely overrun the multilayered, complex mechanisms that control the membrane organization of a eukaryotic cell remains very limited. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Reverse Genetic Analysis of Ourmiaviruses Reveals the Nucleolar Localization of the Coat Protein in Nicotiana benthamiana and Unusual Requirements for Virion Formation ▿ † ‡

PubMed Central

Crivelli, Giulia; Ciuffo, Marina; Genre, Andrea; Masenga, Vera; Turina, Massimo

2011-01-01

Ourmia melon virus (OuMV) is the type member of the genus Ourmiavirus. These viruses have a trisegmented genome, each part of which encodes a single protein. Ourmiaviruses share a distant similarity with other plant viruses only in their movement proteins (MP), whereas their RNA-dependent RNA polymerase (RdRP) shares features only with fungal viruses of the family Narnaviridae. Thus, ourmiaviruses are in a unique phylogenetic position among existing plant viruses. Here, we developed an agroinoculation system to launch infection in Nicotiana benthamiana plants. Using different combinations of the three segments, we demonstrated that RNA1 is necessary and sufficient for cis-acting replication in the agroinfiltrated area. RNA2 and RNA3, encoding the putative movement protein and the coat protein (CP), respectively, are both necessary for successful systemic infection of N. benthamiana. The CP is dispensable for long-distance transport of the virus through vascular tissues, but its absence prevents efficient systemic infection at the exit sites. Virion formation occurred only when the CP was translated from replication-derived RNA3. Transient expression of a green fluorescent protein-MP (GFP-MP) fusion via agroinfiltration showed that the MP is present in cytoplasmic connections across plant cell walls; in protoplasts the GFP-MP fusion stimulates the formation of tubular protrusions. Expression through agroinfiltration of a GFP-CP fusion displays most of the fluorescence inside the nucleus and within the nucleolus in particular. Nuclear localization of the CP was also confirmed through Western blot analysis of purified nuclei. The significance of several unusual properties of OuMV for replication, virion assembly, and movement is discussed in relation to other positive-strand RNA viruses. PMID:21411534

The critical protein interactions and structures that elicit growth deregulation in cancer and viral replication

PubMed Central

Ou, Horng D.; May, Andrew P.

2010-01-01

One of the greatest challenges in biomedicine is to define the critical targets and network interactions that are subverted to elicit growth deregulation in human cells. Understanding and developing rational treatments for cancer requires a definition of the key molecular targets and how they interact to elicit the complex growth deregulation phenotype. Viral proteins provide discerning and powerful probes to understand both how cells work and how they can be manipulated using a minimal number of components. The small DNA viruses have evolved to target inherent weaknesses in cellular protein interaction networks to hijack the cellular DNA and protein replication machinery. In the battle to escape the inevitability of senescence and programmed cell death, cancers have converged on similar mechanisms, through the acquisition and selection of somatic mutations that drive unchecked cellular replication in tumors. Understanding the dynamic mechanisms through which a minimal number of viral proteins promote host cells to undergo unscheduled and pathological replication is a powerful strategy to identify critical targets that are also disrupted in cancer. Viruses can therefore be used as tools to probe the system-wide protein-protein interactions and structures that drive growth deregulation in human cells. Ultimately this can provide a path for developing system context-dependent therapeutics. This review will describe ongoing experimental approaches using viruses to study pathways deregulated in cancer, with a particular focus on viral cellular protein-protein interactions and structures. PMID:21061422

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

PubMed Central

Neufeldt, Christopher J.; Joyce, Michael A.; Van Buuren, Nicholas; Levin, Aviad; Kirkegaard, Karla; Gale Jr., Michael; Tyrrell, D. Lorne J.; Wozniak, Richard W.

2016-01-01

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication. PMID:26863439

Identification of Cellular Proteins Required for Replication of Human Immunodeficiency Virus Type 1

PubMed Central

Dziuba, Natallia; Ferguson, Monique R.; O'Brien, William A.; Sanchez, Anthony; Prussia, Andrew J.; McDonald, Natalie J.; Friedrich, Brian M.; Li, Guangyu; Shaw, Michael W.; Sheng, Jinsong; Hodge, Thomas W.; Rubin, Donald H.

2012-01-01

Abstract Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation. PMID:22404213

Identification of cellular proteins required for replication of human immunodeficiency virus type 1.

PubMed

Dziuba, Natallia; Ferguson, Monique R; O'Brien, William A; Sanchez, Anthony; Prussia, Andrew J; McDonald, Natalie J; Friedrich, Brian M; Li, Guangyu; Shaw, Michael W; Sheng, Jinsong; Hodge, Thomas W; Rubin, Donald H; Murray, James L

2012-10-01

Cellular proteins are essential for human immunodeficiency virus type 1 (HIV-1) replication and may serve as viable new targets for treating infection. Using gene trap insertional mutagenesis, a high-throughput approach based on random inactivation of cellular genes, candidate genes were found that limit virus replication when mutated. Disrupted genes (N=87) conferring resistance to lytic infection with several viruses were queried for an affect on HIV-1 replication by utilizing small interfering RNA (siRNA) screens in TZM-bl cells. Several genes regulating diverse pathways were found to be required for HIV-1 replication, including DHX8, DNAJA1, GTF2E1, GTF2E2, HAP1, KALRN, UBA3, UBE2E3, and VMP1. Candidate genes were independently tested in primary human macrophages, toxicity assays, and/or Tat-dependent β-galactosidase reporter assays. Bioinformatics analyses indicated that several host factors present in this study participate in canonical pathways and functional processes implicated in prior genome-wide studies. However, the genes presented in this study did not share identity with those found previously. Novel antiviral targets identified in this study should open new avenues for mechanistic investigation.

Viperin Restricts Zika Virus and Tick-Borne Encephalitis Virus Replication by Targeting NS3 for Proteasomal Degradation.

PubMed

Panayiotou, Christakis; Lindqvist, Richard; Kurhade, Chaitanya; Vonderstein, Kirstin; Pasto, Jenny; Edlund, Karin; Upadhyay, Arunkumar S; Överby, Anna K

2018-04-01

Flaviviruses are arthropod-borne viruses that constitute a major global health problem, with millions of human infections annually. Their pathogenesis ranges from mild illness to severe manifestations such as hemorrhagic fever and fatal encephalitis. Type I interferons (IFNs) are induced in response to viral infection and stimulate the expression of interferon-stimulated genes (ISGs), including that encoding viperin (virus-inhibitory protein, endoplasmic reticulum associated, IFN inducible), which shows antiviral activity against a broad spectrum of viruses, including several flaviviruses. Here we describe a novel antiviral mechanism employed by viperin against two prominent flaviviruses, tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin was found to interact and colocalize with the structural proteins premembrane (prM) and envelope (E) of TBEV, as well as with nonstructural (NS) proteins NS2A, NS2B, and NS3. Interestingly, viperin expression reduced the NS3 protein level, and the stability of the other interacting viral proteins, but only in the presence of NS3. We also found that although viperin interacted with NS3 of mosquito-borne flaviviruses (ZIKV, Japanese encephalitis virus, and yellow fever virus), only ZIKV was sensitive to the antiviral effect of viperin. This sensitivity correlated with viperin's ability to induce proteasome-dependent degradation of NS3. ZIKV and TBEV replication was rescued completely when NS3 was overexpressed, suggesting that the viral NS3 is the specific target of viperin. In summary, we present here a novel antiviral mechanism of viperin that is selective for specific viruses in the genus Flavivirus , affording the possible availability of new drug targets that can be used for therapeutic intervention. IMPORTANCE Flaviviruses are a group of enveloped RNA viruses that cause severe diseases in humans and animals worldwide, but no antiviral treatment is yet available. Viperin, a host protein produced in response to infection, effectively restricts the replication of several flaviviruses, but the exact molecular mechanisms have not been elucidated. Here we have identified a novel mechanism employed by viperin to inhibit the replication of two flaviviruses: tick-borne encephalitis virus (TBEV) and Zika virus (ZIKV). Viperin induced selective degradation via the proteasome of TBEV and ZIKV nonstructural 3 (NS3) protein, which is involved in several steps of the viral life cycle. Furthermore, viperin also reduced the stability of several other viral proteins in a NS3-dependent manner, suggesting a central role of NS3 in viperin's antiflavivirus activity. Taking the results together, our work shows important similarities and differences among the members of the genus Flavivirus and could lead to the possibility of therapeutic intervention. Copyright © 2018 American Society for Microbiology.

Proteomic analysis of Frankliniella occidentalis and differentially expressed proteins in response to tomato spotted wilt virus infection.

PubMed

Badillo-Vargas, I E; Rotenberg, D; Schneweis, D J; Hiromasa, Y; Tomich, J M; Whitfield, A E

2012-08-01

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction.

Proteomic Analysis of Frankliniella occidentalis and Differentially Expressed Proteins in Response to Tomato Spotted Wilt Virus Infection

PubMed Central

Badillo-Vargas, I. E.; Rotenberg, D.; Schneweis, D. J.; Hiromasa, Y.; Tomich, J. M.

2012-01-01

Tomato spotted wilt virus (TSWV) is transmitted by Frankliniella occidentalis in a persistent propagative manner. Despite the extensive replication of TSWV in midgut and salivary glands, there is little to no pathogenic effect on F. occidentalis. We hypothesize that the first-instar larva (L1) of F. occidentalis mounts a response to TSWV that protects it from pathogenic effects caused by virus infection and replication in various insect tissues. A partial thrips transcriptome was generated using 454-Titanium sequencing of cDNA generated from F. occidentalis exposed to TSWV. Using these sequences, the L1 thrips proteome that resolved on a two-dimensional gel was characterized. Forty-seven percent of the resolved protein spots were identified using the thrips transcriptome. Real-time quantitative reverse transcriptase PCR (RT-PCR) analysis of virus titer in L1 thrips revealed a significant increase in the normalized abundance of TSWV nucleocapsid RNA from 2 to 21 h after a 3-h acquisition access period on virus-infected plant tissue, indicative of infection and accumulation of virus. We compared the proteomes of infected and noninfected L1s to identify proteins that display differential abundances in response to virus. Using four biological replicates, 26 spots containing 37 proteins were significantly altered in response to TSWV. Gene ontology assignments for 32 of these proteins revealed biological roles associated with the infection cycle of other plant- and animal-infecting viruses and antiviral defense responses. Our findings support the hypothesis that L1 thrips display a complex reaction to TSWV infection and provide new insights toward unraveling the molecular basis of this interaction. PMID:22696645

The Bombyx mori nucleopolyhedrovirus Bm111 affects virulence but not virus replication.

PubMed

Han, Yingying; Xia, Hengchuan; Tang, Qi; Lü, Peng; Ma, Shangshang; Yang, Yanhua; Shao, Dandan; Ma, Quanbing; Chen, Keping

2014-07-01

The Bm111 of Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a small polypeptide (70 amino acids) of which the function remains unknown. To characterize its function, multiple sequence alignments were performed, and the predicted protein was found to share amazingly high (98 %) sequence identity with the Bombyx mandarina nucleopolyhedrovirus ORF110 (Boma110) but negligible with proteins of other insect viruses, indicating the close relationship between these two NPVs with silkworm larvae. The transcription of Bm111 was detected as early as 3 hpi in BmNPV-infected BmN cells, suggesting it is an early gene. To investigate the role of Bm111 in baculovirus life cycle, a Bm111-knockout virus was constructed by bacmid recombination in Escherichia coli. The results showed that knockout of the Bm111 did not affect the replication of virus DNA, but significantly extended the death time of infected silkworm larvae compared to the wild-type or rescued viruses. We also successfully expressed the recombinant protein Bm111 in E. coli to provide sufficient material for subsequent studies. Taken together, our data indicate that Bm111 only affects the virulence of BmNPV, but not its replication.

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.

pelo Is Required for High Efficiency Viral Replication

PubMed Central

Wu, Xiurong; He, Wan-Ting; Tian, Shuye; Meng, Dan; Li, Yuanyue; Chen, Wanze; Li, Lisheng; Tian, Lili; Zhong, Chuan-Qi; Han, Felicia; Chen, Jianming; Han, Jiahuai

2014-01-01

Viruses hijack host factors for their high speed protein synthesis, but information about these factors is largely unknown. In searching for genes that are involved in viral replication, we carried out a forward genetic screen for Drosophila mutants that are more resistant or sensitive to Drosophila C virus (DCV) infection-caused death, and found a virus-resistant line in which the expression of pelo gene was deficient. Our mechanistic studies excluded the viral resistance of pelo deficient flies resulting from the known Drosophila anti-viral pathways, and revealed that pelo deficiency limits the high level synthesis of the DCV capsid proteins but has no or very little effect on the expression of some other viral proteins, bulk cellular proteins, and transfected exogenous genes. The restriction of replication of other types of viruses in pelo deficient flies was also observed, suggesting pelo is required for high level production of capsids of all kinds of viruses. We show that both pelo deficiency and high level DCV protein synthesis increase aberrant 80S ribosomes, and propose that the preferential requirement of pelo for high level synthesis of viral capsids is at least partly due to the role of pelo in dissociation of stalled 80S ribosomes and clearance of aberrant viral RNA and proteins. Our data demonstrated that pelo is a host factor that is required for high efficiency translation of viral capsids and targeting pelo could be a strategy for general inhibition of viral infection. PMID:24722736

Poliovirus Replication Requires the N-terminus but not the Catalytic Sec7 Domain of ArfGEF GBF1

PubMed Central

Belov, George A.; Kovtunovych, Gennadiy; Jackson, Catherine L.; Ehrenfeld, Ellie

2010-01-01

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the Arf GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to BFA, an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. PMID:20497182

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro

PubMed Central

Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3′ untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication. PMID:15141033

The hepatitis C virus Core protein is a potent nucleic acid chaperone that directs dimerization of the viral (+) strand RNA in vitro.

PubMed

Cristofari, Gaël; Ivanyi-Nagy, Roland; Gabus, Caroline; Boulant, Steeve; Lavergne, Jean-Pierre; Penin, François; Darlix, Jean-Luc

2004-01-01

The hepatitis C virus (HCV) is an important human pathogen causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV is an enveloped virus with a positive-sense, single-stranded RNA genome encoding a single polyprotein that is processed to generate viral proteins. Several hundred molecules of the structural Core protein are thought to coat the genome in the viral particle, as do nucleocapsid (NC) protein molecules in Retroviruses, another class of enveloped viruses containing a positive-sense RNA genome. Retroviral NC proteins also possess nucleic acid chaperone properties that play critical roles in the structural remodelling of the genome during retrovirus replication. This analogy between HCV Core and retroviral NC proteins prompted us to investigate the putative nucleic acid chaperoning properties of the HCV Core protein. Here we report that Core protein chaperones the annealing of complementary DNA and RNA sequences and the formation of the most stable duplex by strand exchange. These results show that the HCV Core is a nucleic acid chaperone similar to retroviral NC proteins. We also find that the Core protein directs dimerization of HCV (+) RNA 3' untranslated region which is promoted by a conserved palindromic sequence possibly involved at several stages of virus replication.

Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.

PubMed

Eyre, Nicholas S; Aloia, Amanda L; Joyce, Michael A; Chulanetra, Monrat; Tyrrell, D Lorne; Beard, Michael R

2017-07-01

The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of differentially regulated maize proteins conditioning Sugarcane mosaic virus systemic infection.

PubMed

Chen, Hui; Cao, Yanyong; Li, Yiqing; Xia, Zihao; Xie, Jipeng; Carr, John P; Wu, Boming; Fan, Zaifeng; Zhou, Tao

2017-08-01

Sugarcane mosaic virus (SCMV) is the most important cause of maize dwarf mosaic disease. To identify maize genes responsive to SCMV infection and that may be involved in pathogenesis, a comparative proteomic analysis was performed using the first and second systemically infected leaves (termed 1 SL and 2 SL, respectively). Seventy-one differentially expressed proteins were identified in 1 SL and 2 SL upon SCMV infection. Among them, eight proteins showed the same changing patterns in both 1 SL and 2 SL. Functional annotations of regulated proteins and measurement of photosynthetic activity revealed that photosynthesis was more inhibited and defensive gene expression more pronounced in 1 SL than in 2 SL. Knockdown of regulated proteins in both 1 SL and 2 SL by a brome mosaic virus-based gene silencing vector in maize indicated that protein disulfide isomerase-like and phosphoglycerate kinase were required for optimal SCMV replication. By contrast, knockdown of polyamine oxidase (ZmPAO) significantly increased SCMV accumulation, implying that ZmPAO activity might contribute to resistance or tolerance. The results suggest that combining comparative proteomic analyses of different tissues and virus-induced gene silencing is an efficient way to identify host proteins supporting virus replication or enhancing resistance to virus infection. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Escape from R-peptide deletion in a {gamma}-retrovirus

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

Schneider, Irene C.; Eckhardt, Manon; Brynza, Julia

2011-09-30

The R peptide in the cytoplasmic tail (C-tail) of {gamma}-retroviral envelope proteins (Env) prevents membrane fusion before budding. To analyse its role in the formation of replication competent, infectious particles, we developed chimeric murine leukaemia viruses (MLV) with unmodified or R-peptide deleted Env proteins of the gibbon ape leukaemia virus (GaLV). While titres of these viruses were unaffected, R-peptide deficiency led to strongly impaired spreading. Most remarkably, we isolated an escape mutant which had restored an open reading frame for a C-terminal extension of the truncated C-tail. A reconstituted virus encoding this escape C-tail replicated in cell culture. In contrastmore » to R-peptide deficient Env, particle incorporation of the escape Env was effective due to an enhanced protein expression and restored intracellular co-localisation with Gag proteins. Our data demonstrate that the R peptide not only regulates membrane fusion but also mediates efficient Env protein particle incorporation in {gamma}-retrovirus infected cells.« less

Roles for the Cytoplasmic Tails of the Fusion and Hemagglutinin-Neuraminidase Proteins in Budding of the Paramyxovirus Simian Virus 5

PubMed Central

Waning, David L.; Schmitt, Anthony P.; Leser, George P.; Lamb, Robert A.

2002-01-01

The efficient release of many enveloped viruses from cells involves the coalescence of viral components at sites of budding on the plasma membrane of infected cells. This coalescence is believed to require interactions between the cytoplasmic tails of surface glycoproteins and the matrix (M) protein. For the paramyxovirus simian virus 5 (SV5), the cytoplasmic tail of the hemagglutinin-neuraminidase (HN) protein has been shown previously to be important for normal virus budding. To investigate a role for the cytoplasmic tail of the fusion (F) protein in virus assembly and budding, we generated a series of F cytoplasmic tail-truncated recombinant viruses. Analysis of these viruses in tissue culture indicated that the cytoplasmic tail of the F protein was dispensable for normal virus replication and budding. To investigate further the requirements for assembly and budding of SV5, we generated two double-mutant recombinant viruses that lack 8 amino acids of the predicted 17-amino-acid HN protein cytoplasmic tail in combination with truncation of either 10 or 18 amino acids from the predicted 20-amino-acid F protein cytoplasmic tail. Both of the double mutant recombinant viruses displayed a replication defect in tissue culture and a budding defect, the extent of which was dependant on the length of the remaining F cytoplasmic tail. Taken together, this work and our earlier data on virus-like particle formation (A. P. Schmitt, G. P. Leser, D. L. Waning, and R. A. Lamb, J. Virol. 76:3953-3964, 2002) suggest a redundant role for the cytoplasmic tails of the HN and F proteins in virus assembly and budding. PMID:12186912

Phosphorylation of paramyxovirus phosphoprotein and its role in viral gene expression.

PubMed

Fuentes, Sandra M; Sun, Dengyun; Schmitt, Anthony P; He, Biao

2010-01-01

Paramyxoviruses include many important human and animal pathogens such as measles virus, mumps virus, human parainfluenza viruses, and respiratory syncytial virus, as well as emerging viruses such as Nipah virus and Hendra virus. The paramyxovirus RNA-dependent RNA polymerase consists of the phosphoprotein (P) and the large protein. Both of these proteins are essential for viral RNA synthesis. The P protein is phosphorylated at multiple sites, probably by more than one host kinase. While it is thought that the phosphorylation of P is important for its role in viral RNA synthesis, the precise role of P protein phosphorylation remains an enigma. For instance, it was demonstrated that the putative CKII phosphorylation sites of the P protein of respiratory syncytial virus play a role in viral RNA synthesis using a minigenome replicon system; however, mutating these putative CKII phosphorylation sites within a viral genome had no effect on viral RNA synthesis, leading to the hypothesis that P protein phosphorylation, at least by CKII, does not play a role in viral RNA synthesis. Recently, it has been reported that the phosphorylation state of the P protein of parainfluenza virus 5, a prototypical paramyxovirus, correlates with the ability of P protein to synthesize viral RNA, indicating that P protein phosphorylation does in fact play a role in viral RNA synthesis. Furthermore, host kinases PLK1, as well as AKT1 have been found to play critical roles in paramyxovirus RNA synthesis through regulation of P protein phosphorylation status. Beyond furthering our understanding of paramyxovirus RNA replication, these recent discoveries may also result in a new paradigm in treating infections caused by these viruses, as host kinases that regulate paramyxovirus replication are investigated as potential targets of therapeutic intervention.

The pH of activation of the hemagglutinin protein regulates H5N1 influenza virus replication and pathogenesis in mice.

PubMed

Zaraket, Hassan; Bridges, Olga A; Russell, Charles J

2013-05-01

After receptor binding and internalization during influenza virus entry, the hemagglutinin (HA) protein is triggered by low pH to undergo irreversible conformational changes that mediate membrane fusion. To investigate how mutations that alter the activation pH of the HA protein influence the fitness of an avian H5N1 influenza virus in a mammalian model, we infected C57BL/6J or DBA/2J mice and compared the replication and virulence of recombinant A/chicken/Vietnam/C58/04 (H5N1) HA-Y231H mutant, wild-type, and HA-H241Q and HA-K582I mutant viruses that have HA activation pH values of 6.3, 5.9, 5.6, and 5.4, respectively. The HA-Y231H mutant virus was highly susceptible to acid inactivation in vitro and was attenuated for growth and virulence in mice, suggesting that an H5N1 HA protein triggered at pH 6.3 is too unstable for the virus to remain fit. Wild-type and HA-H241Q viruses were similar in pathogenicity and grew to similar levels in mice, ducks, and cell cultures derived from both avian and mammalian tissues, suggesting that H5N1 HA proteins triggered at pH values in the range of 5.9 to 5.6 broadly support replication. The HA-K582I mutant virus had greater growth and virulence in DBA/2J mice than the wild type did, although the mutant virus was highly attenuated in ducks. The data suggest that adaptation of avian H5N1 influenza virus for infection in mammals is supported by a decrease in the HA activation pH to 5.4. Identification of the HA activation pH as a host-specific infectivity factor is expected to aid in the surveillance and risk assessment of currently circulating H5N1 influenza viruses.

Heterogeneous Nuclear Ribonucleoprotein K Supports Vesicular Stomatitis Virus Replication by Regulating Cell Survival and Cellular Gene Expression

PubMed Central

Dinh, Phat X.; Das, Anshuman; Franco, Rodrigo

2013-01-01

The heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a member of the family of hnRNPs and was recently shown in a genome-wide small interfering RNA (siRNA) screen to support vesicular stomatitis virus (VSV) growth. To decipher the role of hnRNP K in VSV infection, we conducted studies which suggest that the protein is required for VSV spreading. Virus binding to cells, entry, and nucleocapsid uncoating steps were not adversely affected in the absence of hnRNP K, whereas viral genome transcription and replication were reduced slightly. These results indicate that hnRNP K is likely involved in virus assembly and/or release from infected cells. Further studies showed that hnRNP K suppresses apoptosis of virus-infected cells, resulting in increased cell survival during VSV infection. The increased survival of the infected cells was found to be due to the suppression of proapoptotic proteins such as Bcl-XS and Bik in a cell-type-dependent manner. Additionally, depletion of hnRNP K resulted in not only significantly increased levels of T-cell-restricted intracellular antigen 1 (TIA1) but also switching of the expression of the two isoforms of the protein (TIA1a and TIA1b), both of which inhibited VSV replication. hnRNP K was also found to support expression of several cellular proteins known to be required for VSV infection. Overall, our studies demonstrate hnRNP K to be a multifunctional protein that supports VSV infection via its role(s) in suppressing apoptosis of infected cells, inhibiting the expression of antiviral proteins, and maintaining the expression of proteins required for the virus. PMID:23843646

Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses

PubMed Central

Fuchs, Jonas; Hölzer, Martin; Schilling, Mirjam; Patzina, Corinna; Schoen, Andreas; Zimmer, Gert; Marz, Manja; Müller, Marcel A.

2017-01-01

ABSTRACT Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned Mx1 cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat Mx1 genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat Mx1 genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts. IMPORTANCE Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity against Ebola virus and bat influenza A-like virus, and we describe here their phylogenetic relationship, revealing patterns of positive selection that suggest a coevolution with viral pathogens. By understanding the molecular mechanisms of the innate resistance of bats against viral diseases, we might gain important insights into how to prevent and fight human zoonotic infections caused by bat-borne viruses. PMID:28490593

Evolution and Antiviral Specificities of Interferon-Induced Mx Proteins of Bats against Ebola, Influenza, and Other RNA Viruses.

PubMed

Fuchs, Jonas; Hölzer, Martin; Schilling, Mirjam; Patzina, Corinna; Schoen, Andreas; Hoenen, Thomas; Zimmer, Gert; Marz, Manja; Weber, Friedemann; Müller, Marcel A; Kochs, Georg

2017-08-01

Bats serve as a reservoir for various, often zoonotic viruses, including significant human pathogens such as Ebola and influenza viruses. However, for unknown reasons, viral infections rarely cause clinical symptoms in bats. A tight control of viral replication by the host innate immune defense might contribute to this phenomenon. Transcriptomic studies revealed the presence of the interferon-induced antiviral myxovirus resistance (Mx) proteins in bats, but detailed functional aspects have not been assessed. To provide evidence that bat Mx proteins might act as key factors to control viral replication we cloned Mx1 cDNAs from three bat families, Pteropodidae, Phyllostomidae, and Vespertilionidae. Phylogenetically these bat Mx1 genes cluster closely with their human ortholog MxA. Using transfected cell cultures, minireplicon systems, virus-like particles, and virus infections, we determined the antiviral potential of the bat Mx1 proteins. Bat Mx1 significantly reduced the polymerase activity of viruses circulating in bats, including Ebola and influenza A-like viruses. The related Thogoto virus, however, which is not known to infect bats, was not inhibited by bat Mx1. Further, we provide evidence for positive selection in bat Mx1 genes that might explain species-specific antiviral activities of these proteins. Together, our data suggest a role for Mx1 in controlling these viruses in their bat hosts. IMPORTANCE Bats are a natural reservoir for various viruses that rarely cause clinical symptoms in bats but are dangerous zoonotic pathogens, like Ebola or rabies virus. It has been hypothesized that the interferon system might play a key role in controlling viral replication in bats. We speculate that the interferon-induced Mx proteins might be key antiviral factors of bats and have coevolved with bat-borne viruses. This study evaluated for the first time a large set of bat Mx1 proteins spanning three major bat families for their antiviral potential, including activity against Ebola virus and bat influenza A-like virus, and we describe here their phylogenetic relationship, revealing patterns of positive selection that suggest a coevolution with viral pathogens. By understanding the molecular mechanisms of the innate resistance of bats against viral diseases, we might gain important insights into how to prevent and fight human zoonotic infections caused by bat-borne viruses. Copyright © 2017 American Society for Microbiology.

A Novel Expression Cassette of Lyssavirus Shows that the Distantly Related Mokola Virus Can Rescue a Defective Rabies Virus Genome

PubMed Central

Le Mercier, Philippe; Jacob, Yves; Tanner, Kyle; Tordo, Noël

2002-01-01

By comparing three expression vectors for the rabies virus (Rv) minigenome, we show that the characteristic of the Rv RNA is important for efficient rescue despite its not being crucial for replication. Moreover, we show that the coexpression of the viral proteins from helper Rv and Mokola virus could rescue the Rv minigenome while Rv-related European bat lyssavirus 1 could not, suggesting that the signals controlling transcription and replication are conserved in the distantly related Rv and Mokola virus. PMID:11799201

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

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.

Hepatitis B virus molecular biology and pathogenesis.

PubMed

Lamontagne, R Jason; Bagga, Sumedha; Bouchard, Michael J

2016-01-01

As obligate intracellular parasites, viruses need a host cell to provide a milieu favorable to viral replication. Consequently, viruses often adopt mechanisms to subvert host cellular signaling processes. While beneficial for the viral replication cycle, virus-induced deregulation of host cellular signaling processes can be detrimental to host cell physiology and can lead to virus-associated pathogenesis, including, for oncogenic viruses, cell transformation and cancer progression. Included among these oncogenic viruses is the hepatitis B virus (HBV). Despite the availability of an HBV vaccine, 350-500 million people worldwide are chronically infected with HBV, and a significant number of these chronically infected individuals will develop hepatocellular carcinoma (HCC). Epidemiological studies indicate that chronic infection with HBV is the leading risk factor for the development of HCC. Globally, HCC is the second highest cause of cancer-associated deaths, underscoring the need for understanding mechanisms that regulate HBV replication and the development of HBV-associated HCC. HBV is the prototype member of the Hepadnaviridae family; members of this family of viruses have a narrow host range and predominately infect hepatocytes in their respective hosts. The extremely small and compact hepadnaviral genome, the unique arrangement of open reading frames, and a replication strategy utilizing reverse transcription of an RNA intermediate to generate the DNA genome are distinguishing features of the Hepadnaviridae . In this review, we provide a comprehensive description of HBV biology, summarize the model systems used for studying HBV infections, and highlight potential mechanisms that link a chronic HBV-infection to the development of HCC. For example, the HBV X protein (HBx), a key regulatory HBV protein that is important for HBV replication, is thought to play a cofactor role in the development of HBV-induced HCC, and we highlight the functions of HBx that may contribute to the development of HBV-associated HCC.

Secretome Screening Reveals Fibroblast Growth Factors as Novel Inhibitors of Viral Replication.

PubMed

van Asten, Saskia D; Raaben, Matthijs; Nota, Benjamin; Spaapen, Robbert M

2018-06-13

Cellular antiviral programs can efficiently inhibit viral infection. These programs are often initiated through signaling cascades induced by secreted proteins such as type I interferons, IL-6 or TNF-α. Here, we generated an arrayed library of 756 human secreted proteins to perform a secretome screen focused on the discovery of novel modulators of viral entry and/or replication. The individual secreted proteins were tested for their capacity to inhibit infection by two replication-competent recombinant vesicular stomatitis viruses (VSV) with distinct glycoproteins utilizing different entry pathways. Fibroblast growth factor 16 (FGF16) was identified and confirmed as the most prominent novel inhibitor of both VSVs and therefore of viral replication and not entry. Importantly, an antiviral interferon signature was completely absent in FGF16 treated cells. Nevertheless, the antiviral effect of FGF16 is broad as it was evident on multiple cell types and also on infection of Coxsackievirus. In addition, other members of the FGF family also inhibited viral infection. Thus, our unbiased secretome screen revealed a novel protein family capable of inducing a cellular antiviral state. This previously unappreciated role of the FGF family may have implications for the development of new antivirals and the efficacy of oncolytic virus therapy. Importance Viruses infect human cells in order to replicate, while human cells aim to resist infection. Several cellular antiviral programs have therefore evolved to resist infection. Knowledge of these programs is essential for the design of antiviral therapeutics in the future. The induction of antiviral programs is often initiated by secreted proteins such as interferons. We hypothesized that other secreted proteins may also promote resistance to viral infection. Thus we tested 756 human secreted proteins for their capacity to inhibit two pseudotypes of vesicular stomatitis virus (VSV). In this first secretome screen on viral infection we identified fibroblast growth factor 16 (FGF16) as a novel antiviral against multiple VSV pseudotypes as well as Coxsackievirus. Subsequent testing of other FGF family members revealed that FGF signaling generally inhibits viral infection. This finding may lead to the development of new antivirals and may also be applicable to enhance oncolytic virus therapy. Copyright © 2018 American Society for Microbiology.

Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge

PubMed Central

Goatley, Lynnette C.; Jabbar, Tamara; Sanchez-Cordon, Pedro J.; Netherton, Christopher L.; Chapman, David A. G.; Dixon, Linda K.

2017-01-01

ABSTRACT Many of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus in vivo. All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼106 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization. IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines. PMID:28978700

Deletion of the African Swine Fever Virus Gene DP148R Does Not Reduce Virus Replication in Culture but Reduces Virus Virulence in Pigs and Induces High Levels of Protection against Challenge.

PubMed

Reis, Ana L; Goatley, Lynnette C; Jabbar, Tamara; Sanchez-Cordon, Pedro J; Netherton, Christopher L; Chapman, David A G; Dixon, Linda K

2017-12-15

Many of the approximately 165 proteins encoded by the African swine fever virus (ASFV) genome do not have significant similarity to known proteins and have not been studied experimentally. One such protein is DP148R. We showed that the DP148R gene is transcribed at early times postinfection. Deletion of this gene did not reduce virus replication in macrophages, showing that it is not essential for replication in these cells. However, deletion of this gene from a virulent isolate, Benin 97/1, producing the BeninΔDP148R virus, dramatically reduced the virulence of the virus in vivo All pigs infected with the BeninΔDP148R virus survived infection, showing only transient mild clinical signs soon after immunization. Following challenge with the parental virulent virus, all pigs immunized by the intramuscular route (11/11) and all except one immunized by the intranasal route (5/6) survived. Mild or no clinical signs were observed after challenge. As expected, control nonimmune pigs developed signs of acute African swine fever (ASF). The virus genome and infectious virus were observed soon after immunization, coincident with the onset of clinical signs (∼10 6 genome copies or 50% tissue culture infective doses/ml). The levels of the virus genome declined over an extended period up to 60 days postimmunization. In contrast, infectious virus was no longer detectable by days 30 to 35. Gamma interferon (IFN-γ) was detected in serum between days 4 and 7 postimmunization, and IFN-γ-producing cells were detected in all pigs analyzed following stimulation of immune lymphocytes with whole virus. ASFV-specific antibodies were first detected from day 10 postimmunization. IMPORTANCE African swine fever (ASF) is endemic in Africa, parts of the Trans Caucasus, the Russian Federation, and several European countries. The lack of a vaccine hinders control. Many of the ASF virus genes lack similarity to known genes and have not been characterized. We have shown that one of these, DP148R, is transcribed early during virus replication in cells and can be deleted from the virus genome without reducing virus replication. The virus with the gene deletion, BeninΔDP148R, caused mild clinical signs in pigs and induced high levels of protection against challenge with the parental virulent virus. Therefore, deletion of this gene can provide a target for the rational development of vaccines. Copyright © 2017 Reis et al.

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.

Suppression of Zika Virus Infection and Replication in Endothelial Cells and Astrocytes by PKA Inhibitor PKI 14-22.

PubMed

Cheng, Fan; Ramos da Silva, Suzane; Huang, I-Chueh; Jung, Jae U; Gao, Shou-Jiang

2018-02-15

The recent outbreak of Zika virus (ZIKV), a reemerging flavivirus, and its associated neurological disorders, such as Guillain-Barré (GB) syndrome and microcephaly, have generated an urgent need to develop effective ZIKV vaccines and therapeutic agents. Here, we used human endothelial cells and astrocytes, both of which represent key cell types for ZIKV infection, to identify potential inhibitors of ZIKV replication. Because several pathways, including the AMP-activated protein kinase (AMPK), protein kinase A (PKA), and mitogen-activated protein kinase (MAPK) signaling pathways, have been reported to play important roles in flavivirus replication, we tested inhibitors and agonists of these pathways for their effects on ZIKV replication. We identified the PKA inhibitor PKI 14-22 (PKI) to be a potent inhibitor of ZIKV replication. PKI effectively suppressed the replication of ZIKV from both the African and Asian/American lineages with a high efficiency and minimal cytotoxicity. While ZIKV infection does not induce PKA activation, endogenous PKA activity is essential for supporting ZIKV replication. Interestingly, in addition to PKA, PKI also inhibited another unknown target(s) to block ZIKV replication. PKI inhibited ZIKV replication at the postentry stage by preferentially affecting negative-sense RNA synthesis as well as viral protein translation. Together, these results have identified a potential inhibitor of ZIKV replication which could be further explored for future therapeutic application. IMPORTANCE There is an urgent need to develop effective vaccines and therapeutic agents against Zika virus (ZIKV) infection, a reemerging flavivirus associated with neurological disorders, including Guillain-Barré (GB) syndrome and microcephaly. By screening for inhibitors of several cellular pathways, we have identified the PKA inhibitor PKI 14-22 (PKI) to be a potent inhibitor of ZIKV replication. We show that PKI effectively suppresses the replication of all ZIKV strains tested with minimal cytotoxicity to human endothelial cells and astrocytes, two key cell types for ZIKV infection. Furthermore, we show that PKI inhibits ZIKV negative-sense RNA synthesis and viral protein translation. This study has identified a potent inhibitor of ZIKV infection which could be further explored for future therapeutic application. Copyright © 2018 American Society for Microbiology.

Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library.

PubMed

Hou, Peili; Zhao, Guimin; He, Chengqiang; Wang, Hongmei; He, Hongbin

2018-01-04

The bovine ephemeral fever virus (BEFV) glycoprotein neutralization site 1 (also referred as G 1 protein), is a critical protein responsible for virus infectivity and eliciting immune-protection, however, binding peptides of BEFV G 1 protein are still unclear. Thus, the aim of the present study was to screen specific polypeptides, which bind BEFV G 1 protein with high-affinity and inhibit BEFV replication. The purified BEFV G 1 was coated and then reacted with the M13-based Ph.D.-7 phage random display library. The peptides for target binding were automated sequenced after four rounds of enrichment biopanning. The amino acid sequences of polypeptide displayed on positive clones were deduced and the affinity of positive polypeptides with BEFV G 1 was assayed by ELISA. Then the roles of specific G 1 -binding peptides in the context of BEFV infection were analyzed. The results showed that 27 specific peptide ligands displaying 11 different amino acid sequences were obtained, and the T18 and T25 clone had a higher affinity to G 1 protein than the other clones. Then their antiviral roles of two phage clones (T25 and T18) showed that both phage polypeptide T25 and T18 exerted inhibition on BEFV replication compared to control group. Moreover, synthetic peptide based on T18 (HSIRYDF) and T25 (YSLRSDY) alone or combined use on BEFV replication showed that the synthetic peptides could effectively inhibit the formation of cytopathic plaque and significantly inhibit BEFV RNA replication in a dose-dependent manner. Two antiviral peptide ligands binding to bovine ephemeral fever virus G 1 protein from phage display peptide library were identified, which may provide a potential research tool for diagnostic reagents and novel antiviral agents.

Subgenomic Reporter RNA System for Detection of Alphavirus Infection in Mosquitoes

PubMed Central

Steel, J. Jordan; Franz, Alexander W. E.; Sanchez-Vargas, Irma; Olson, Ken E.; Geiss, Brian J.

2013-01-01

Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes. PMID:24367703

Levels of the E2 interacting protein TopBP1 modulate papillomavirus maintenance stage replication

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

Kanginakudru, Sriramana, E-mail: skangina@iu.edu; DeSmet, Marsha, E-mail: mdesmet@iupui.edu; Thomas, Yanique, E-mail: ysthomas@umail.iu.edu

2015-04-15

The evolutionarily conserved DNA topoisomerase II beta-binding protein 1 (TopBP1) functions in DNA replication, DNA damage response, and cell survival. We analyzed the role of TopBP1 in human and bovine papillomavirus genome replication. Consistent with prior reports, TopBP1 co-localized in discrete nuclear foci and was in complex with papillomavirus E2 protein. Similar to E2, TopBP1 is recruited to the region of the viral origin of replication during G1/S and early S phase. TopBP1 knockdown increased, while over-expression decreased transient virus replication, without affecting cell cycle. Similarly, using cell lines harboring HPV-16 or HPV-31 genome, TopBP1 knockdown increased while over-expression reducedmore » viral copy number relative to genomic DNA. We propose a model in which TopBP1 serves dual roles in viral replication: it is essential for initiation of replication yet it restricts viral copy number. - Highlights: • Protein interaction study confirmed In-situ interaction between TopBP1 and E2. • TopBP1 present at papillomavirus ori in G1/S and early S phase of cell cycle. • TopBP1 knockdown increased, over-expression reduced virus replication. • TopBP1 protein level change did not influence cell survival or cell cycle. • TopBP1 displaced from papillomavirus ori after initiation of replication.« less

Monkey Viperin Restricts Porcine Reproductive and Respiratory Syndrome Virus Replication.

PubMed

Fang, Jianyu; Wang, Haiyan; Bai, Juan; Zhang, Qiaoya; Li, Yufeng; Liu, Fei; Jiang, Ping

2016-01-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is an important pathogen which causes huge economic damage globally in the swine industry. Current vaccination strategies provide only limited protection against PRRSV infection. Viperin is an interferon (IFN) stimulated protein that inhibits some virus infections via IFN-dependent or IFN-independent pathways. However, the role of viperin in PRRSV infection is not well understood. In this study, we cloned the full-length monkey viperin (mViperin) complementary DNA (cDNA) from IFN-α-treated African green monkey Marc-145 cells. It was found that the mViperin is up-regulated following PRRSV infection in Marc-145 cells along with elevated IRF-1 gene levels. IFN-α induced mViperin expression in a dose- and time-dependent manner and strongly inhibits PRRSV replication in Marc-145 cells. Overexpression of mViperin suppresses PRRSV replication by blocking the early steps of PRRSV entry and genome replication and translation but not inhibiting assembly and release. And mViperin co-localized with PRRSV GP5 and N protein, but only interacted with N protein in distinct cytoplasmic loci. Furthermore, it was found that the 13-16 amino acids of mViperin were essential for inhibiting PRRSV replication, by disrupting the distribution of mViperin protein from the granular distribution to a homogeneous distribution in the cytoplasm. These results could be helpful in the future development of novel antiviral therapies against PRRSV infection.

Inhibition of Poliovirus-Induced Cleavage of Cellular Protein PCBP2 Reduces the Levels of Viral RNA Replication

PubMed Central

Chase, Amanda J.; Daijogo, Sarah

2014-01-01

ABSTRACT Due to their small genome size, picornaviruses must utilize host proteins to mediate cap-independent translation and viral RNA replication. The host RNA-binding protein poly(rC) binding protein 2 (PCBP2) is involved in both processes in poliovirus infected cells. It has been shown that the viral proteinase 3CD cleaves PCBP2 and contributes to viral translation inhibition. However, cleaved PCBP2 remains active in viral RNA replication. This would suggest that both cleaved and intact forms of PCBP2 have a role in the viral RNA replication cycle. The picornavirus genome must act as a template for both translation and RNA replication. However, a template that is actively being translated cannot function as a template for RNA replication, suggesting that there is a switch in template usage from translation to RNA replication. We demonstrate that the cleavage of PCBP2 by the poliovirus 3CD proteinase is a necessary step for efficient viral RNA replication and, as such, may be important for mediating a switch in template usage from translation to RNA replication. IMPORTANCE Poliovirus, like all positive-strand RNA viruses that replicate in the cytoplasm of eukaryotic cells, uses its genomic RNA as a template for both viral protein synthesis and RNA replication. Given that these processes cannot occur simultaneously on the same template, poliovirus has evolved a mechanism(s) to facilitate the switch from using templates for translation to using them for RNA synthesis. This study explores one possible scenario for how the virus alters the functions of a host cell RNA binding protein to mediate, in part, this important transition. PMID:24371074

Molecular biology of human herpesvirus 8: novel functions and virus-host interactions implicated in viral pathogenesis and replication.

PubMed

Cousins, Emily; Nicholas, John

2014-01-01

Human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), is the second identified human gammaherpesvirus. Like its relative Epstein-Barr virus, HHV-8 is linked to B-cell tumors, specifically primary effusion lymphoma and multicentric Castleman's disease, in addition to endothelial-derived KS. HHV-8 is unusual in its possession of a plethora of "accessory" genes and encoded proteins in addition to the core, conserved herpesvirus and gammaherpesvirus genes that are necessary for basic biological functions of these viruses. The HHV-8 accessory proteins specify not only activities deducible from their cellular protein homologies but also novel, unsuspected activities that have revealed new mechanisms of virus-host interaction that serve virus replication or latency and may contribute to the development and progression of virus-associated neoplasia. These proteins include viral interleukin-6 (vIL-6), viral chemokines (vCCLs), viral G protein-coupled receptor (vGPCR), viral interferon regulatory factors (vIRFs), and viral antiapoptotic proteins homologous to FLICE (FADD-like IL-1β converting enzyme)-inhibitory protein (FLIP) and survivin. Other HHV-8 proteins, such as signaling membrane receptors encoded by open reading frames K1 and K15, also interact with host mechanisms in unique ways and have been implicated in viral pathogenesis. Additionally, a set of micro-RNAs encoded by HHV-8 appear to modulate expression of multiple host proteins to provide conditions conducive to virus persistence within the host and could also contribute to HHV-8-induced neoplasia. Here, we review the molecular biology underlying these novel virus-host interactions and their potential roles in both virus biology and virus-associated disease.

Human Cytomegalovirus Strategies to Maintain and Promote mRNA Translation

PubMed Central

Vincent, Heather A.; Ziehr, Benjamin; Moorman, Nathaniel J.

2016-01-01

mRNA translation requires the ordered assembly of translation initiation factors and ribosomal subunits on a transcript. Host signaling pathways regulate each step in this process to match levels of protein synthesis to environmental cues. In response to infection, cells activate multiple defenses that limit viral protein synthesis, which viruses must counteract to successfully replicate. Human cytomegalovirus (HCMV) inhibits host defenses that limit viral protein expression and manipulates host signaling pathways to promote the expression of both host and viral proteins necessary for virus replication. Here we review key regulatory steps in mRNA translation, and the strategies used by HCMV to maintain protein synthesis in infected cells. PMID:27089357

Autophagic machinery activated by dengue virus enhances virus replication

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

Lee, Y.-R.; Lei, H.-Y.; Department of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan, Taiwan

2008-05-10

Autophagy is a cellular response against stresses which include the infection of viruses and bacteria. We unravel that Dengue virus-2 (DV2) can trigger autophagic process in various infected cell lines demonstrated by GFP-LC3 dot formation and increased LC3-II formation. Autophagosome formation was also observed under the transmission electron microscope. DV2-induced autophagy further enhances the titers of extracellular and intracellular viruses indicating that autophagy can promote viral replication in the infected cells. Moreover, our data show that ATG5 protein is required to execute DV2-induced autophagy. All together, we are the first to demonstrate that DV can activate autophagic machinery that ismore » favorable for viral replication.« less

Replication-Competent Influenza A Viruses Expressing Reporter Genes.

PubMed

Breen, Michael; Nogales, Aitor; Baker, Steven F; Martínez-Sobrido, Luis

2016-06-23

Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo.

Replication-Competent Influenza A Viruses Expressing Reporter Genes

PubMed Central

Breen, Michael; Nogales, Aitor; Baker, Steven F.; Martínez-Sobrido, Luis

2016-01-01

Influenza A viruses (IAV) cause annual seasonal human respiratory disease epidemics. In addition, IAV have been implicated in occasional pandemics with inordinate health and economic consequences. Studying IAV, in vitro or in vivo, requires the use of laborious secondary methodologies to identify virus-infected cells. To circumvent this requirement, replication-competent IAV expressing an easily traceable reporter protein can be used. Here we discuss the development and applications of recombinant replication-competent IAV harboring diverse fluorescent or bioluminescent reporter genes in different locations of the viral genome. These viruses have been employed for in vitro and in vivo studies, such as the screening of neutralizing antibodies or antiviral compounds, the identification of host factors involved in viral replication, cell tropism, the development of vaccines, or the assessment of viral infection dynamics. In summary, reporter-expressing, replicating-competent IAV represent a powerful tool for the study of IAV both in vitro and in vivo. PMID:27347991

Gene Rearrangement Attenuates Expression and Lethality of a Nonsegmented Negative Strand RNA Virus

NASA Astrophysics Data System (ADS)

Williams Wertz, Gail; Perepelitsa, Victoria P.; Ball, L. Andrew

1998-03-01

The nonsegmented negative strand RNA viruses comprise hundreds of human, animal, insect, and plant pathogens. Gene expression of these viruses is controlled by the highly conserved order of genes relative to the single transcriptional promoter. We utilized this regulatory mechanism to alter gene expression levels of vesicular stomatitis virus by rearranging the gene order. This report documents that gene expression levels and the viral phenotype can be manipulated in a predictable manner. Translocation of the promoter-proximal nucleocapsid protein gene N, whose product is required stoichiometrically for genome replication, to successive positions down the genome reduced N mRNA and protein expression in a stepwise manner. The reduction in N gene expression resulted in a stepwise decrease in genomic RNA replication. Translocation of the N gene also attenuated the viruses to increasing extents for replication in cultured cells and for lethality in mice, without compromising their ability to elicit protective immunity. Because monopartite negative strand RNA viruses have not been reported to undergo homologous recombination, gene rearrangement should be irreversible and may provide a rational strategy for developing stably attenuated live vaccines against this type of virus.

Affinity purification combined with mass spectrometry to identify herpes simplex virus protein-protein interactions.

PubMed

Meckes, David G

2014-01-01

The identification and characterization of herpes simplex virus protein interaction complexes are fundamental to understanding the molecular mechanisms governing the replication and pathogenesis of the virus. Recent advances in affinity-based methods, mass spectrometry configurations, and bioinformatics tools have greatly increased the quantity and quality of protein-protein interaction datasets. In this chapter, detailed and reliable methods that can easily be implemented are presented for the identification of protein-protein interactions using cryogenic cell lysis, affinity purification, trypsin digestion, and mass spectrometry.

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

Identification and characterization of the host protein DNAJC14 as a broadly active flavivirus replication modulator.

PubMed

Yi, Zhigang; Sperzel, Lindsey; Nürnberger, Cindy; Bredenbeek, Peter J; Lubick, Kirk J; Best, Sonja M; Stoyanov, Cristina T; Law, Lok Man J; Yuan, Zhenghong; Rice, Charles M; MacDonald, Margaret R

2011-01-13

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule.

Characterization of the catalytic center of the Ebola virus L polymerase.

PubMed

Schmidt, Marie Luisa; Hoenen, Thomas

2017-10-01

Ebola virus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing antivirals. Targeting the ribonucleoprotein complex (RNP) proteins, which facilitate genome replication and transcription, and particularly the polymerase L, is a promising antiviral approach since these processes are essential for the virus life cycle. However, until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp) of L, which is one of the most promising molecular targets, has never been experimentally characterized. Using multiple sequence alignments with other negative sense single-stranded RNA viruses we identified the putative catalytic center of the EBOV RdRp. An L protein with mutations in this center was then generated and characterized using various life cycle modelling systems. These systems are based on minigenomes, i.e. miniature versions of the viral genome, in which the viral genes are exchanged against a reporter gene. When such minigenomes are coexpressed with RNP proteins in mammalian cells, the RNP proteins recognize them as authentic templates for replication and transcription, resulting in reporter activity reflecting these processes. Replication-competent minigenome systems indicated that our L catalytic domain mutant was impaired in genome replication and/or transcription, and by using replication-deficient minigenome systems, as well as a novel RT-qPCR-based genome replication assay, we showed that it indeed no longer supported either of these processes. However, it still showed similar expression to wild-type L, and retained its ability to be incorporated into inclusion bodies, which are the sites of EBOV genome replication. We have experimentally defined the catalytic center of the EBOV RdRp, and thus a promising antiviral target regulating an essential aspect of the EBOV life cycle.

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication.

PubMed

Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R; Li, Melody M H; Rice, Charles M; MacDonald, Margaret R

2016-01-06

DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

PubMed Central

Bozzacco, Leonia; Yi, Zhigang; Andreo, Ursula; Conklin, Claire R.; Li, Melody M. H.; Rice, Charles M.

2016-01-01

ABSTRACT DNAJC14, a heat shock protein 40 (Hsp40) cochaperone, assists with Hsp70-mediated protein folding. Overexpressed DNAJC14 is targeted to sites of yellow fever virus (YFV) replication complex (RC) formation, where it interacts with viral nonstructural (NS) proteins and inhibits viral RNA replication. How RCs are assembled and the roles of chaperones in this coordinated process are largely unknown. We hypothesized that chaperones are diverted from their normal cellular protein quality control function to play similar roles during viral infection. Here, we show that DNAJC14 overexpression affects YFV polyprotein processing and alters RC assembly. We monitored YFV NS2A-5 polyprotein processing by the viral NS2B-3 protease in DNAJC14-overexpressing cells. Notably, DNAJC14 mutants that did not inhibit YFV replication had minimal effects on polyprotein processing, while overexpressed wild-type DNAJC14 affected the NS3/4A and NS4A/2K cleavage sites, resulting in altered NS3-to-NS3-4A ratios. This suggests that DNAJC14's folding activity normally modulates NS3/4A/2K cleavage events to liberate appropriate levels of NS3 and NS4A and promote RC formation. We introduced amino acid substitutions at the NS3/4A site to alter the levels of the NS3 and NS4A products and examined their effects on YFV replication. Residues with reduced cleavage efficiency did not support viral RNA replication, and only revertant viruses with a restored wild-type arginine or lysine residue at the NS3/4A site were obtained. We conclude that DNAJC14 inhibition of RC formation upon DNAJC14 overexpression is likely due to chaperone dysregulation and that YFV probably utilizes DNAJC14's cochaperone function to modulate processing at the NS3/4A site as a mechanism ensuring virus replication. IMPORTANCE Flaviviruses are single-stranded RNA viruses that cause a wide range of illnesses. Upon host cell entry, the viral genome is translated on endoplasmic reticulum (ER) membranes to produce a single polyprotein, which is cleaved by host and viral proteases to generate viral proteins required for genome replication and virion production. Several studies suggest a role for molecular chaperones during these processes. While the details of chaperone roles have been elusive, in this report we show that overexpression of the ER-resident cochaperone DNAJC14 affects YFV polyprotein processing at the NS3/4A site. This work reveals that DNAJC14 modulation of NS3/4A site processing is an important mechanism to ensure virus replication. Our work highlights the importance of finely regulating flavivirus polyprotein processing. In addition, it suggests future studies to address similarities and/or differences among flaviviruses and to interrogate the precise mechanisms employed for polyprotein processing, a critical step that can ultimately be targeted for novel drug development. PMID:26739057

Modified Newcastle disease virus vectors expressing the H5 hemagglutinin induce enhanced protection against highly pathogenic H5N1 avian influenza virus in chickens

PubMed Central

Kim, Shin-Hee; Paldurai, Anandan; Xiao, Sa; Collins, Peter L.; Samal, Siba K.

2016-01-01

Naturally-occurring attenuated strains of Newcastle disease virus (NDV) are being developed as vaccine vectors for use in poultry and humans. However, some NDV strains, such as Beaudette C (BC), may retain too much virulence in poultry for safe use, and more highly attenuated strains may be suboptimally immunogenic. We therefore modified the BC strain by changing the multibasic cleavage site sequence of the F protein to the dibasic sequence of avirulent strain LaSota. Additionally, the BC, F, and HN proteins were modified in several ways to enhance virus replication. These modified BC-derived vectors and the LaSota strain were engineered to express the hemagglutin (HA) protein of H5N1 highly pathogenic influenza virus (HPAIV). In general, the modified BC-based vectors expressing HA replicated better than LaSota/HA, and expressed higher levels of HA protein. Pathogenicity tests indicated that all the modified viruses were highly attenuated in chickens. Based on in vitro characterization, two of the modified BC vectors were chosen for evaluation in chickens as vaccine vectors against H5N1 HPAIV A/Vietnam/1203/04. Immunization of chickens with rNDV vector vaccines followed by challenge with HPAIV demonstrated high levels of protection against clinical disease and mortality. However, only those chickens immunized with modified BC/HA in which residues 271–330 from the F protein had been replaced with the corresponding sequence from the NDV AKO strain conferred complete protection against challenge virus shedding. Our findings suggest that this modified rNDV can be used safely as a vaccine vector with enhanced replication, expression, and protective efficacy in avian species, and potentially in humans. PMID:24968158

Role of host protein Ebp1 in influenza virus growth: intracellular localization of Ebp1 in virus-infected and uninfected cells.

PubMed

Honda, Ayae

2008-01-20

The cellular protein Ebp1 was identified to interact with PB1 protein of influenza virus RNA polymerase, and inhibit both RNA synthesis in vitro and influenza virus replication in vivo [Honda, A., Okamoto, T., Ishihama, A., 2007. Host factor Ebp1: selective inhibitor of influenza virus transcriptase. Genes Cells 12, 133-142]. The intracellular localization of Ebp1 that is involved in cell proliferation control was analyzed by direct immunostaining of cells before and after influenza virus infection. Ebp1 was found to localize in the nuclear membrane of uninfected cells, and to form nuclear aggregates with viral P proteins in virus-infected cells.

LaSota fusion (F) cleavage motif-mediated fusion activity is affected by other regions of the F protein from different genotype Newcastle disease virus in a chimeric virus: implication for virulence attenuation.

PubMed

Kim, Shin-Hee; Xiao, Sa; Collins, Peter L; Samal, Siba K

2016-06-01

The cleavage site sequence of the fusion (F) protein contributes to a wide range of virulence of Newcastle disease virus (NDV). In this study, we identified other important amino acid sequences of the F protein that affect cleavage and modulation of fusion. We generated chimeric Beaudette C (BC) viruses containing the cleavage site sequence of avirulent strain LaSota (Las-Fc) together with various regions of the F protein of another virulent strain AKO. We found that the F1 subunit is important for cleavage inhibition. Further dissection of the F1 subunit showed that replacement of four amino acids in the BC/Las-Fc protein with their AKO counterparts (T341S, M384I, T385A and I386L) resulted in an increase in fusion and replication in vitro. In contrast, the mutation N403D greatly reduced cleavage and viral replication, and affected protein conformation. These findings will be useful in developing improved live NDV vaccines and vaccine vectors.

Two Proline-Rich Nuclear Localization Signals in the Amino- and Carboxyl-Terminal Regions of the Borna Disease Virus Phosphoprotein

PubMed Central

Shoya, Yuko; Kobayashi, Takeshi; Koda, Toshiaki; Ikuta, Kazuyoshi; Kakinuma, Mitsuaki; Kishi, Masahiko

1998-01-01

Borna disease virus (BDV) uses a unique strategy of replication and transcription which takes place in the nucleus, unlike other known, nonsegmented, negative-stranded RNA viruses of animal origin. In this process, viral constituents necessary for replication must be transported to the nucleus from the cytoplasm. We report here the evidence that BDV P protein, which may play an important role in viral replication and transcription, is transported into the nucleus in the absence of other viral constituents. This transportation is accomplished by its own nuclear localization signals (NLSs), which are present in both N-terminal (29PRPRKIPR36) and C-terminal (181PPRIYPQLPSAPT193) regions of the protein. These two NLSs can function independently and both have several Pro residues as key amino acids. PMID:9811710

Influenza A virus-induced degradation of eukaryotic translation initiation factor 4B contributes to viral replication by suppressing IFITM3 protein expression.

PubMed

Wang, Song; Chi, Xiaojuan; Wei, Haitao; Chen, Yuhai; Chen, Zhilong; Huang, Shile; Chen, Ji-Long

2014-08-01

Although alteration in host cellular translation machinery occurs in virus-infected cells, the role of such alteration and the precise pathogenic processes are not well understood. Influenza A virus (IAV) infection shuts off host cell gene expression at transcriptional and translational levels. Here, we found that the protein level of eukaryotic translation initiation factor 4B (eIF4B), an integral component of the translation initiation apparatus, was dramatically reduced in A549 cells as well as in the lung, spleen, and thymus of mice infected with IAV. The decrease in eIF4B level was attributed to lysosomal degradation of eIF4B, which was induced by viral NS1 protein. Silencing eIF4B expression in A549 cells significantly promoted IAV replication, and conversely, overexpression of eIF4B markedly inhibited the viral replication. Importantly, we observed that eIF4B knockdown transgenic mice were more susceptible to IAV infection, exhibiting faster weight loss, shorter survival time, and more-severe organ damage. Furthermore, we demonstrated that eIF4B regulated the expression of interferon-induced transmembrane protein 3 (IFITM3), a critical protein involved in immune defense against a variety of RNA viruses, including influenza virus. Taken together, our findings reveal that eIF4B plays an important role in host defense against IAV infection at least by regulating the expression of IFITM3, which restricts viral entry and thereby blocks early stages of viral production. These data also indicate that influenza virus has evolved a strategy to overcome host innate immunity by downregulating eIF4B protein. Influenza A virus (IAV) infection stimulates the host innate immune system, in part, by inducing interferons (IFNs). Secreted IFNs activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, leading to elevated transcription of a large group of IFN-stimulated genes that have antiviral function. To circumvent the host innate immune response, influenza virus has evolved multiple strategies for suppressing the production of IFNs. Here, we show that IAV infection induces lysosomal degradation of eIF4B protein; and eIF4B inhibits IAV replication by upregulating expression of interferon-induced transmembrane protein 3 (IFITM3), a key protein that protects the host from virus infection. Our finding illustrates a critical role of eIF4B in the host innate immune response and provides novel insights into the complex mechanisms by which influenza virus interacts with its host. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs.

PubMed

Tilston-Lunel, Natasha L; Acrani, Gustavo Olszanski; Randall, Richard E; Elliott, Richard M

2015-12-23

Oropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955, and since then, it has been known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV, however, remains one of the most poorly understood emerging viral zoonoses. Here we describe the successful recovery of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding region. Characterization of the recombinant viruses carried out in vitro demonstrated that the NSs protein of OROV is an interferon (IFN) antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-α when cells were pretreated; however, the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-α, in contrast to the family prototype BUNV. We found that OROV lacking the NSm protein displayed characteristics similar to those of the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in the mammalian and mosquito cell lines that were tested. Oropouche virus (OROV) is a public health threat in Central and South America, where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus, and with the current rates of urban expansion, more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV, we have successfully rescued the virus along with mutants. We have established that the C terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell culture. The tools described in this paper are important in terms of understanding this important yet neglected human pathogen. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Generation of Recombinant Oropouche Viruses Lacking the Nonstructural Protein NSm or NSs

PubMed Central

Randall, Richard E.; Elliott, Richard M.

2015-01-01

ABSTRACT Oropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955, and since then, it has been known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV, however, remains one of the most poorly understood emerging viral zoonoses. Here we describe the successful recovery of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding region. Characterization of the recombinant viruses carried out in vitro demonstrated that the NSs protein of OROV is an interferon (IFN) antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-α when cells were pretreated; however, the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-α, in contrast to the family prototype BUNV. We found that OROV lacking the NSm protein displayed characteristics similar to those of the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in the mammalian and mosquito cell lines that were tested. IMPORTANCE Oropouche virus (OROV) is a public health threat in Central and South America, where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus, and with the current rates of urban expansion, more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV, we have successfully rescued the virus along with mutants. We have established that the C terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell culture. The tools described in this paper are important in terms of understanding this important yet neglected human pathogen. PMID:26699638

Production of pseudoinfectious yellow fever virus with a two-component genome.

PubMed

Shustov, Alexandr V; Mason, Peter W; Frolov, Ilya

2007-11-01

Application of genetically modified, deficient-in-replication flaviviruses that are incapable of developing productive, spreading infection is a promising means of designing safe and effective vaccines. Here we describe a two-component genome yellow fever virus (YFV) replication system in which each of the genomes encodes complete sets of nonstructural proteins that form the replication complex but expresses either only capsid or prM/E instead of the entire structural polyprotein. Upon delivery to the same cell, these genomes produce together all of the viral structural proteins, and cells release a combination of virions with both types of genomes packaged into separate particles. In tissue culture, this modified YFV can be further passaged at an escalating scale by using a high multiplicity of infection (MOI). However, at a low MOI, only one of the genomes is delivered into the cells, and infection cannot spread. The replicating prM/E-encoding genome produces extracellular E protein in the form of secreted subviral particles that are known to be an effective immunogen. The presented strategy of developing viruses defective in replication might be applied to other flaviviruses, and these two-component genome viruses can be useful for diagnostic or vaccine applications, including the delivery and expression of heterologous genes. In addition, the achieved separation of the capsid-coding sequence and the cyclization signal in the YFV genome provides a new means for studying the mechanism of the flavivirus packaging process.

Neuraminidase inhibiting antibody responses in pigs differ between influenza A virus N2 lineages and by vaccine type

USDA-ARS?s Scientific Manuscript database

The neuraminidase (NA) protein of influenza A viruses (IAV) has important functional roles in the viral replication cycle. Antibodies specific to NA can reduce viral replication and limit disease severity, but are not routinely measured. We analyzed NA inhibiting (NI) antibody titers in serum and re...

Five unique open reading frames of infectious laryngotracheitis virus are expressed during infection but are dispensable for virus replication in cell culture.

PubMed

Veits, Jutta; Mettenleiter, Thomas C; Fuchs, Walter

2003-06-01

The chicken alphaherpesvirus infectious laryngotracheitis virus (ILTV) exhibits several unique genetic features including an internal inversion of a conserved part of the unique long genome region. At one end, this inversion is preceded by a cluster of five open reading frames (ORFs) of 335-411 codons, designated ORF A to ORF E, that are not present in any other known herpesvirus genome. In this report we analysed expression of these genes and identified the corresponding viral RNA and protein products. Northern blot analyses showed 3'-coterminal transcripts of ORFs A and B, and monocistronic mRNAs of ORFs C and D. ORF E is part of a 3'-coterminal transcription unit that includes the conserved glycoprotein H and thymidine kinase genes. Monospecific antisera obtained after immunization of rabbits with bacterial fusion proteins allowed detection of the protein products of ORF A (40 kDa), ORF B (34 kDa), ORF C (38 and 30 kDa), ORF D (41 kDa) and ORF E (44 kDa) in ILTV-infected cells. For functional analyses, five virus recombinants possessing deletions within the individual ORFs and concomitant insertions of a reporter gene cassette encoding green fluorescent protein were generated. All virus mutants were replication competent in cell culture, but exhibited reduced virus titres or plaque sizes when compared to wild-type ILTV. These findings indicate that the ILTV-specific ORF A to ORF E genes might be important for virus replication in the natural host organism.

Heat Shock Protein 70 Modulates Influenza A Virus Polymerase Activity*

PubMed Central

Manzoor, Rashid; Kuroda, Kazumichi; Yoshida, Reiko; Tsuda, Yoshimi; Fujikura, Daisuke; Miyamoto, Hiroko; Kajihara, Masahiro; Kida, Hiroshi; Takada, Ayato

2014-01-01

The role of heat shock protein 70 (Hsp70) in virus replication has been discussed for many viruses. The known suppressive role of Hsp70 in influenza virus replication is based on studies conducted in cells with various Hsp70 expression levels. In this study, we determined the role of Hsp70 in influenza virus replication in HeLa and HEK293T cells, which express Hsp70 constitutively. Co-immunoprecipitation and immunofluorescence studies revealed that Hsp70 interacted with PB2 or PB1 monomers and PB2/PB1 heterodimer but not with the PB1/PA heterodimer or PB2/PB1/PA heterotrimer and translocated into the nucleus with PB2 monomers or PB2/PB1 heterodimers. Knocking down Hsp70 resulted in reduced virus transcription and replication activities. Reporter gene assay, immunofluorescence assay, and Western blot analysis of nuclear and cytoplasmic fractions from infected cells demonstrated that the increase in viral polymerase activity during the heat shock phase was accompanied with an increase in Hsp70 and viral polymerases levels in the nuclei, where influenza virus replication takes place, whereas a reduction in viral polymerase activity was accompanied with an increase in cytoplasmic relocation of Hsp70 along with viral polymerases. Moreover, significantly higher levels of viral genomic RNA (vRNA) were observed during the heat shock phase than during the recovery phase. Overall, for the first time, these findings suggest that Hsp70 may act as a chaperone for influenza virus polymerase, and the modulatory effect of Hsp70 appears to be a sequel of shuttling of Hsp70 between nuclear and cytoplasmic compartments. PMID:24474693

Biochemical Activities of Minute Virus of Mice Nonstructural Protein NS1 Are Modulated In Vitro by the Phosphorylation State of the Polypeptide

PubMed Central

Nüesch, Jürg P. F.; Corbau, Romuald; Tattersall, Peter; Rommelaere, Jean

1998-01-01

NS1, the 83-kDa major nonstructural protein of minute virus of mice (MVM), is a multifunctional nuclear phosphoprotein which is required in a variety of steps during progeny virus production, early as well as late during infection. NS1 is the initiator protein for viral DNA replication. It binds specifically to target DNA motifs; has site-specific single-strand nickase, intrinsic ATPase, and helicase activities; trans regulates viral and cellular promoters; and exerts cytotoxic stress on the host cell. To investigate whether these multiple activities of NS1 depend on posttranslational modifications, in particular phosphorylation, we expressed His-tagged NS1 in HeLa cells by using recombinant vaccinia viruses, dephosphorylated it at serine and threonine residues with calf intestine alkaline phosphatase, and compared the biochemical activities of the purified un(der)phosphorylated (NS1O) and the native (NS1P) polypeptides. Biochemical analyses of replicative functions of NS1O revealed a severe reduction of intrinsic helicase activity and, to a minor extent, of ATPase and nickase activities, whereas its affinity for the target DNA sequence [ACCA]2–3 was enhanced compared to that of NS1P. In the presence of endogenous protein kinases found in replication extracts, NS1O showed all functions necessary for resolution and replication of the 3′ dimer bridge, indicating reactivation of NS1O by rephosphorylation. Partial reactivation of the helicase activity was found as well when NS1O was incubated with protein kinase C. PMID:9733839

Suppression of HTLV-1 replication by Tax-mediated rerouting of the p13 viral protein to nuclear speckles

PubMed Central

Andresen, Vibeke; Pise-Masison, Cynthia A.; Sinha-Datta, Uma; Bellon, Marcia; Valeri, Valerio; Washington Parks, Robyn; Cecchinato, Valentina; Fukumoto, Risaku; Nicot, Christophe

2011-01-01

Disease development in human T-cell leukemia virus type 1 (HTLV-1)–infected individuals is positively correlated with the level of integrated viral DNA in T cells. HTLV-1 replication is positively regulated by Tax and Rex and negatively regulated by the p30 and HBZ proteins. In the present study, we demonstrate that HTLV-1 encodes another negative regulator of virus expression, the p13 protein. Expressed separately, p13 localizes to the mitochondria, whereas in the presence of Tax, part of it is ubiquitinated, stabilized, and rerouted to the nuclear speckles. The p13 protein directly binds Tax, decreases Tax binding to the CBP/p300 transcriptional coactivator, and, by reducing Tax transcriptional activity, suppresses viral expression. Because Tax stabilizes its own repressor, these findings suggest that HTLV-1 has evolved a complex mechanism to control its own replication. Further, these results highlight the importance of studying the function of the HTLV-1 viral proteins, not only in isolation, but also in the context of full viral replication. PMID:21677314

A host YB-1 ribonucleoprotein complex is hijacked by hepatitis C virus for the control of NS3-dependent particle production.

PubMed

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin; Lamarre, Daniel

2013-11-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.

A Host YB-1 Ribonucleoprotein Complex Is Hijacked by Hepatitis C Virus for the Control of NS3-Dependent Particle Production

PubMed Central

Chatel-Chaix, Laurent; Germain, Marie-Anne; Motorina, Alena; Bonneil, Éric; Thibault, Pierre; Baril, Martin

2013-01-01

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production. PMID:23986595

Vesicular Egress of Non-Enveloped Lytic Parvoviruses Depends on Gelsolin Functioning

PubMed Central

Bär, Séverine; Daeffler, Laurent; Rommelaere, Jean; Nüesch, Jürg P. F.

2008-01-01

The autonomous parvovirus Minute Virus of Mice (MVM) induces specific changes in the cytoskeleton filaments of infected permissive cells, causing in particular the degradation of actin fibers and the generation of “actin patches.” This is attributed to a virus-induced imbalance between the polymerization factor N-WASP (Wiscott-Aldrich syndrome protein) and gelsolin, a multifunctional protein cleaving actin filaments. Here, the focus is on the involvement of gelsolin in parvovirus propagation and virus-induced actin processing. Gelsolin activity was knocked-down, and consequences thereof were determined for virus replication and egress and for actin network integrity. Though not required for virus replication or progeny particle assembly, gelsolin was found to control MVM (and related H1-PV) transport from the nucleus to the cell periphery and release into the culture medium. Gelsolin-dependent actin degradation and progeny virus release were both controlled by (NS1)/CKIIα, a recently identified complex between a cellular protein kinase and a MVM non-structural protein. Furthermore, the export of newly synthesized virions through the cytoplasm appeared to be mediated by (virus-modified) lysomal/late endosomal vesicles. By showing that MVM release, like entry, is guided by the cytoskeleton and mediated by vesicles, these results challenge the current view that egress of non-enveloped lytic viruses is a passive process. PMID:18704167

Novel host restriction factors implicated in HIV-1 replication.

PubMed

Ghimire, Dibya; Rai, Madhu; Gaur, Ritu

2018-04-01

Human immunodeficiency virus-1 (HIV-1) is known to interact with multiple host cellular proteins during its replication in the target cell. While many of these host cellular proteins facilitate viral replication, a number of them are reported to inhibit HIV-1 replication at various stages of its life cycle. These host cellular proteins, which are known as restriction factors, constitute an integral part of the host's first line of defence against the viral pathogen. Since the discovery of apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G) as an HIV-1 restriction factor, several human proteins have been identified that exhibit anti-HIV-1 restriction. While each restriction factor employs a distinct mechanism of inhibition, the HIV-1 virus has equally evolved complex counter strategies to neutralize their inhibitory effect. APOBEC3G, tetherin, sterile alpha motif and histidine-aspartate domain 1 (SAMHD1), and trim-5α are some of the best known HIV-1 restriction factors that have been studied in great detail. Recently, six novel restriction factors were discovered that exhibit significant antiviral activity: endoplasmic reticulum α1,2-mannosidase I (ERManI), translocator protein (TSPO), guanylate-binding protein 5 (GBP5), serine incorporator (SERINC3/5) and zinc-finger antiviral protein (ZAP). The focus of this review is to discuss the antiviral mechanism of action of these six restriction factors and provide insights into the probable counter-evasion strategies employed by the HIV-1 virus. The recent discovery of new restriction factors substantiates the complex host-pathogen interactions occurring during HIV-1 pathogenesis and makes it imperative that further investigations are conducted to elucidate the molecular basis of HIV-1 replication.

The Short Form of the Zinc Finger Antiviral Protein Inhibits Influenza A Virus Protein Expression and Is Antagonized by the Virus-Encoded NS1.

PubMed

Tang, Qiannan; Wang, Xinlu; Gao, Guangxia

2017-01-15

Zinc finger antiviral protein (ZAP) is a host factor that specifically inhibits the replication of certain viruses. There are two ZAP isoforms arising from alternative splicing, which differ only at the C termini. It was recently reported that the long isoform (ZAPL) promotes proteasomal degradation of influenza A virus (IAV) proteins PA and PB2 through the C-terminal poly(ADP-ribose) polymerase (PARP) domain, which is missing in the short form (ZAPS), and that this antiviral activity is antagonized by the viral protein PB1. Here, we report that ZAP inhibits IAV protein expression in a PARP domain-independent manner. Overexpression of ZAPS inhibited the expression of PA, PB2, and neuraminidase (NA), and downregulation of the endogenous ZAPS enhanced their expression. We show that ZAPS inhibited PB2 protein expression by reducing the encoding viral mRNA levels and repressing its translation. However, downregulation of ZAPS only modestly enhanced the early stage of viral replication. We provide evidence showing that the antiviral activity of ZAPS is antagonized by the viral protein NS1. A recombinant IAV carrying an NS1 mutant that lost the ZAPS-antagonizing activity replicated better in ZAPS-deficient cells. We further provide evidence suggesting that NS1 antagonizes ZAPS by inhibiting its binding to target mRNA. These results uncover a distinct mechanism underlying the interactions between ZAP and IAV. ZAP is a host antiviral factor that has been extensively reported to inhibit the replication of certain viruses by repressing the translation and promoting the degradation of the viral mRNAs. There are two ZAP isoforms, ZAPL and ZAPS. ZAPL was recently reported to promote IAV protein degradation through the PARP domain. Whether ZAPS, which lacks the PARP domain, inhibits IAV and the underlying mechanisms remained to be determined. Here, we show that ZAPS posttranscriptionally inhibits IAV protein expression. This antiviral activity of ZAP is antagonized by the viral protein NS1. The fact that ZAP uses two distinct mechanisms to inhibit IAV infection and that the virus evolved different antagonists suggests an important role of ZAP in the host effort to control IAV infection and the importance of the threat of ZAP to the virus. The results reported here help us to comprehensively understand the interactions between ZAP and IAV. Copyright © 2017 American Society for Microbiology.

Generation of mutant Uukuniemi viruses lacking the nonstructural protein NSs by reverse genetics indicates that NSs is a weak interferon antagonist.

PubMed

Rezelj, Veronica V; Överby, Anna K; Elliott, Richard M

2015-05-01

Uukuniemi virus (UUKV) is a tick-borne member of the Phlebovirus genus (family Bunyaviridae) and has been widely used as a safe laboratory model to study aspects of bunyavirus replication. Recently, a number of new tick-borne phleboviruses have been discovered, some of which, like severe fever with thrombocytopenia syndrome virus and Heartland virus, are highly pathogenic in humans. UUKV could now serve as a useful comparator to understand the molecular basis for the different pathogenicities of these related viruses. We established a reverse-genetics system to recover UUKV entirely from cDNA clones. We generated two recombinant viruses, one in which the nonstructural protein NSs open reading frame was deleted from the S segment and one in which the NSs gene was replaced with green fluorescent protein (GFP), allowing convenient visualization of viral infection. We show that the UUKV NSs protein acts as a weak interferon antagonist in human cells but that it is unable to completely counteract the interferon response, which could serve as an explanation for its inability to cause disease in humans. Uukuniemi virus (UUKV) is a tick-borne phlebovirus that is apathogenic for humans and has been used as a convenient model to investigate aspects of phlebovirus replication. Recently, new tick-borne phleboviruses have emerged, such as severe fever with thrombocytopenia syndrome virus in China and Heartland virus in the United States, that are highly pathogenic, and UUKV will now serve as a comparator to aid in the understanding of the molecular basis for the virulence of these new viruses. To help such investigations, we have developed a reverse-genetics system for UUKV that permits manipulation of the viral genome. We generated viruses lacking the nonstructural protein NSs and show that UUKV NSs is a weak interferon antagonist. In addition, we created a virus that expresses GFP and thus allows convenient monitoring of virus replication. These new tools represent a significant advance in the study of tick-borne phleboviruses. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A Drosophila Toolkit for the Visualization and Quantification of Viral Replication Launched from Transgenic Genomes

PubMed Central

Wernet, Mathias F.; Klovstad, Martha; Clandinin, Thomas R.

2014-01-01

Arthropod RNA viruses pose a serious threat to human health, yet many aspects of their replication cycle remain incompletely understood. Here we describe a versatile Drosophila toolkit of transgenic, self-replicating genomes (‘replicons’) from Sindbis virus that allow rapid visualization and quantification of viral replication in vivo. We generated replicons expressing Luciferase for the quantification of viral replication, serving as useful new tools for large-scale genetic screens for identifying cellular pathways that influence viral replication. We also present a new binary system in which replication-deficient viral genomes can be activated ‘in trans’, through co-expression of an intact replicon contributing an RNA-dependent RNA polymerase. The utility of this toolkit for studying virus biology is demonstrated by the observation of stochastic exclusion between replicons expressing different fluorescent proteins, when co-expressed under control of the same cellular promoter. This process is analogous to ‘superinfection exclusion’ between virus particles in cell culture, a process that is incompletely understood. We show that viral polymerases strongly prefer to replicate the genome that encoded them, and that almost invariably only a single virus genome is stochastically chosen for replication in each cell. Our in vivo system now makes this process amenable to detailed genetic dissection. Thus, this toolkit allows the cell-type specific, quantitative study of viral replication in a genetic model organism, opening new avenues for molecular, genetic and pharmacological dissection of virus biology and tool development. PMID:25386852

Cellular Chaperonin CCTγ Contributes to Rabies Virus Replication during Infection

PubMed Central

Zhang, Jinyang; Wu, Xiaopeng; Zan, Jie; Wu, Yongping; Ye, Chengjin; Ruan, Xizhen

2013-01-01

Rabies, as the oldest known infectious disease, remains a serious threat to public health worldwide. The eukaryotic cytosolic chaperonin TRiC/CCT complex facilitates the folding of proteins through ATP hydrolysis. Here, we investigated the expression, cellular localization, and function of neuronal CCTγ during neurotropic rabies virus (RABV) infection using mouse N2a cells as a model. Following RABV infection, 24 altered proteins were identified by using two-dimensional electrophoresis and mass spectrometry, including 20 upregulated proteins and 4 downregulated proteins. In mouse N2a cells infected with RABV or cotransfected with RABV genes encoding nucleoprotein (N) and phosphoprotein (P), confocal microscopy demonstrated that upregulated cellular CCTγ was colocalized with viral proteins N and P, which formed a hollow cricoid inclusion within the region around the nucleus. These inclusions, which correspond to Negri bodies (NBs), did not form in mouse N2a cells only expressing the viral protein N or P. Knockdown of CCTγ by lentivirus-mediated RNA interference led to significant inhibition of RABV replication. These results demonstrate that the complex consisting of viral proteins N and P recruits CCTγ to NBs and identify the chaperonin CCTγ as a host factor that facilitates intracellular RABV replication. This work illustrates how viruses can utilize cellular chaperonins and compartmentalization for their own benefit. PMID:23637400

Rab1A is required for assembly of classical swine fever virus particle.

PubMed

Lin, Jihui; Wang, Chengbao; Liang, Wulong; Zhang, Jing; Zhang, Longxiang; Lv, Huifang; Dong, Wang; Zhang, Yanming

2018-01-15

Rab1A belongs to the small Rab GTPase family and is involved in the lifecycle of numerous viruses. Here, knockdown of Rab1A inhibited CSFV growth. Further study revealed that Rab1A depletion decreased intracellular and extracellular CSFV titers, but did not affect intracellular virus genome copies and E2 protein expression within a virus lifecycle, which suggested that Rab1A is required for CSFV particle assembly rather than for genome replication or virion release. This was proofed by blocking the spread of virus using neutralizing antibodies, through which the negative effects of Rab1A knockdown on multi-cycle replication of CSFV were eliminated. Moreover, co-immunoprecipitation and confocal microscopy assays showed that Rab1A bound to CSFV NS5A protein, indicating that Rab1A and viral NS5A proteins may work cooperatively during CSFV particle assembly. In conclusion, this study demonstrated for the first time that Rab1A is required for CSFV particle assembly and binds to viral particle assembly-related NS5A protein. Copyright © 2017 Elsevier Inc. All rights reserved.

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.

In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.

PubMed

Erban, Tomas; Harant, Karel; Hubalek, Martin; Vitamvas, Pavel; Kamler, Martin; Poltronieri, Palmiro; Tyl, Jan; Markovic, Martin; Titera, Dalibor

2015-09-11

We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed.

In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite

PubMed Central

Erban, Tomas; Harant, Karel; Hubalek, Martin; Vitamvas, Pavel; Kamler, Martin; Poltronieri, Palmiro; Tyl, Jan; Markovic, Martin; Titera, Dalibor

2015-01-01

We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed. PMID:26358842

The RNA synthesis machinery of negative-stranded RNA viruses

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

Ortín, Juan, E-mail: jortin@cnb.csic.es; Martín-Benito, Jaime, E-mail: jmartinb@cnb.csic.es

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins. Here we review our knowledge on these large RNA-synthesis machines, including the structure ofmore » their components, the interactions among them and their enzymatic activities, and we discuss models showing how they perform the virus transcription and replication programmes. - Highlights: • Overall organisation of NSV RNA synthesis machines. • Structure and function of the ribonucleoprotein components: Atomic structure of the RNA polymerase complex. • Commonalities and differences between segmented- and non-segmented NSVs. • Transcription versus replication programmes.« less

Recombination Promoted by DNA Viruses: Phage λ to Herpes Simplex Virus

PubMed Central

Weller, Sandra K.; Sawitzke, James A.

2015-01-01

The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies. PMID:25002096

The ATR Signaling Pathway Is Disabled during Infection with the Parvovirus Minute Virus of Mice

PubMed Central

Adeyemi, Richard O.

2014-01-01

ABSTRACT The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. IMPORTANCE Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. PMID:24965470

The ATR signaling pathway is disabled during infection with the parvovirus minute virus of mice.

PubMed

Adeyemi, Richard O; Pintel, David J

2014-09-01

The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. Upon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Uracil DNA glycosylase BKRF3 contributes to Epstein-Barr virus DNA replication through physical interactions with proteins in viral DNA replication complex.

PubMed

Su, Mei-Tzu; Liu, I-Hua; Wu, Chia-Wei; Chang, Shu-Ming; Tsai, Ching-Hwa; Yang, Pei-Wen; Chuang, Yu-Chia; Lee, Chung-Pei; Chen, Mei-Ru

2014-08-01

Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmuno-precipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A Novel Self-Replicating Chimeric Lentivirus-Like Particle

PubMed Central

Young, Kelly R.; Madden, Victoria J.; Johnson, Philip R.; Johnston, Robert E.

2012-01-01

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4+ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation. PMID:22013035

A novel self-replicating chimeric lentivirus-like particle.

PubMed

Jurgens, Christy K; Young, Kelly R; Madden, Victoria J; Johnson, Philip R; Johnston, Robert E

2012-01-01

Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

Non-Structural Proteins of Arthropod-Borne Bunyaviruses: Roles and Functions

PubMed Central

Eifan, Saleh; Schnettler, Esther; Dietrich, Isabelle; Kohl, Alain; Blomström, Anne-Lie

2013-01-01

Viruses within the Bunyaviridae family are tri-segmented, negative-stranded RNA viruses. The family includes several emerging and re-emerging viruses of humans, animals and plants, such as Rift Valley fever virus, Crimean-Congo hemorrhagic fever virus, La Crosse virus, Schmallenberg virus and tomato spotted wilt virus. Many bunyaviruses are arthropod-borne, so-called arboviruses. Depending on the genus, bunyaviruses encode, in addition to the RNA-dependent RNA polymerase and the different structural proteins, one or several non-structural proteins. These non-structural proteins are not always essential for virus growth and replication but can play an important role in viral pathogenesis through their interaction with the host innate immune system. In this review, we will summarize current knowledge and understanding of insect-borne bunyavirus non-structural protein function(s) in vertebrate, plant and arthropod. PMID:24100888

Valosin-containing protein (VCP/p97) plays a role in the replication of West Nile virus.

PubMed

Phongphaew, Wallaya; Kobayashi, Shintaro; Sasaki, Michihito; Carr, Michael; Hall, William W; Orba, Yasuko; Sawa, Hirofumi

2017-01-15

Valosin-containing protein (VCP) is classified as a member of the type II AAA + ATPase protein family. VCP functions in several cellular processes, including protein degradation, membrane fusion, vesicular trafficking and disassembly of stress granules. Moreover, VCP is considered to play a role in the replication of several viruses, albeit through different mechanisms. In the present study, we have investigated the role of VCP in West Nile virus (WNV) infection. Endogenous VCP expression was inhibited using either VCP inhibitors or by siRNA knockdown. It could be shown that the inhibition of endogenous VCP expression significantly inhibited WNV infection. The entry assay revealed that silencing of endogenous VCP caused a significant reduction in the expression levels of WNV-RNA compared to control siRNA-treated cells. This indicates that VCP may play a role in early steps either the binding or entry steps of the WNV life cycle. Using WNV virus like particles and WNV-DNA-based replicon, it could be demonstrated that perturbation of VCP expression decreased levels of newly synthesized WNV genomic RNA. These findings suggest that VCP is involved in early steps and during genome replication of the WNV life cycle. Copyright © 2016 Elsevier B.V. All rights reserved.

Poliovirus replication requires the N-terminus but not the catalytic Sec7 domain of ArfGEF GBF1.

PubMed

Belov, George A; Kovtunovych, Gennadiy; Jackson, Catherine L; Ehrenfeld, Ellie

2010-10-01

Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the ADP-ribosylation factor (Arf) GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to brefeldin A (BFA), an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism. © Published 2010. This article is a US Government work and is in the public domain in the USA.

Mother-to-Child HIV Transmission Bottleneck Selects for Consensus Virus with Lower Gag-Protease-Driven Replication Capacity

PubMed Central

Naidoo, Vanessa L.; Mann, Jaclyn K.; Noble, Christie; Adland, Emily; Carlson, Jonathan M.; Thomas, Jake; Brumme, Chanson J.; Thobakgale-Tshabalala, Christina F.; Brumme, Zabrina L.; Goulder, Philip J. R.

2017-01-01

ABSTRACT In the large majority of cases, HIV infection is established by a single variant, and understanding the characteristics of successfully transmitted variants is relevant to prevention strategies. Few studies have investigated the viral determinants of mother-to-child transmission. To determine the impact of Gag-protease-driven viral replication capacity on mother-to-child transmission, the replication capacities of 148 recombinant viruses encoding plasma-derived Gag-protease from 53 nontransmitter mothers, 48 transmitter mothers, and 47 infected infants were assayed in an HIV-1-inducible green fluorescent protein reporter cell line. All study participants were infected with HIV-1 subtype C. There was no significant difference in replication capacities between the nontransmitter (n = 53) and transmitter (n = 44) mothers (P = 0.48). Infant-derived Gag-protease NL4-3 recombinant viruses (n = 41) were found to have a significantly lower Gag-protease-driven replication capacity than that of viruses derived from the mothers (P < 0.0001 by a paired t test). High percent similarities to consensus subtype C Gag, p17, p24, and protease sequences were also found in the infants (n = 28) in comparison to their mothers (P = 0.07, P = 0.002, P = 0.03, and P = 0.02, respectively, as determined by a paired t test). These data suggest that of the viral quasispecies found in mothers, the HIV mother-to-child transmission bottleneck favors the transmission of consensus-like viruses with lower viral replication capacities. IMPORTANCE Understanding the characteristics of successfully transmitted HIV variants has important implications for preventative interventions. Little is known about the viral determinants of HIV mother-to-child transmission (MTCT). We addressed the role of viral replication capacity driven by Gag, a major structural protein that is a significant determinant of overall viral replicative ability and an important target of the host immune response, in the MTCT bottleneck. This study advances our understanding of the genetic bottleneck in MTCT by revealing that viruses transmitted to infants have a lower replicative ability as well as a higher similarity to the population consensus (in this case HIV subtype C) than those of their mothers. Furthermore, the observation that “consensus-like” virus sequences correspond to lower in vitro replication abilities yet appear to be preferentially transmitted suggests that viral characteristics favoring transmission are decoupled from those that enhance replicative capacity. PMID:28637761

Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

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

Imura, Yoshiyuki, E-mail: imura@brs.nihon-u.ac.jp; Molho, Melissa; Chuang, Chingkai

Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement bothmore » defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly. - Highlights: • Tombusvirus p33 replication protein interacts with cellular RAD6/Ubc2 E2 enzymes. • Deletion of RAD6 reduces tombusvirus replication in yeast. • Silencing of UBC2 in plants inhibits tombusvirus replication. • Mono- and bi-ubiquitination of p33 replication protein in yeast and in vitro. • Rad6p promotes the recruitment of cellular ESCRT proteins into the tombusvirus replicase.« less

Measles virus induces persistent infection by autoregulation of viral replication.

PubMed

Doi, Tomomitsu; Kwon, Hyun-Jeong; Honda, Tomoyuki; Sato, Hiroki; Yoneda, Misako; Kai, Chieko

2016-11-24

Natural infection with measles virus (MV) establishes lifelong immunity. Persistent infection with MV is likely involved in this phenomenon, as non-replicating protein antigens never induce such long-term immunity. Although MV establishes stable persistent infection in vitro and possibly in vivo, the mechanism by which this occurs is largely unknown. Here, we demonstrate that MV changes the infection mode from lytic to non-lytic and evades the innate immune response to establish persistent infection without viral genome mutation. We found that, in the persistent phase, the viral RNA level declined with the termination of interferon production and cell death. Our analysis of viral protein dynamics shows that during the establishment of persistent infection, the nucleoprotein level was sustained while the phosphoprotein and large protein levels declined. The ectopic expression of nucleoprotein suppressed viral replication, indicating that viral replication is self-regulated by nucleoprotein accumulation during persistent infection. The persistently infected cells were able to produce interferon in response to poly I:C stimulation, suggesting that MV does not interfere with host interferon responses in persistent infection. Our results may provide mechanistic insight into the persistent infection of this cytopathic RNA virus that induces lifelong immunity.

MicroRNA-like viral small RNA from porcine reproductive and respiratory syndrome virus negatively regulates viral replication by targeting the viral nonstructural protein 2.

PubMed

Li, Na; Yan, Yunhuan; Zhang, Angke; Gao, Jiming; Zhang, Chong; Wang, Xue; Hou, Gaopeng; Zhang, Gaiping; Jia, Jinbu; Zhou, En-Min; Xiao, Shuqi

2016-12-13

Many viruses encode microRNAs (miRNAs) that are small non-coding single-stranded RNAs which play critical roles in virus-host interactions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically impactful viruses in the swine industry. The present study sought to determine whether PRRSV encodes miRNAs that could regulate PRRSV replication. Four viral small RNAs (vsRNAs) were mapped to the stem-loop structures in the ORF1a, ORF1b and GP2a regions of the PRRSV genome by bioinformatics prediction and experimental verification. Of these, the structures with the lowest minimum free energy (MFE) values predicted for PRRSV-vsRNA1 corresponded to typical stem-loop, hairpin structures. Inhibition of PRRSV-vsRNA1 function led to significant increases in viral replication. Transfection with PRRSV-vsRNA1 mimics significantly inhibited PRRSV replication in primary porcine alveolar macrophages (PAMs). The time-dependent increase in the abundance of PRRSV-vsRNA1 mirrored the gradual upregulation of PRRSV RNA expression. Knockdown of proteins associated with cellular miRNA biogenesis demonstrated that Drosha and Argonaute (Ago2) are involved in PRRSV-vsRNA1 biogenesis. Moreover, PRRSV-vsRNA1 bound specifically to the nonstructural protein 2 (NSP2)-coding sequence of PRRSV genome RNA. Collectively, the results reveal that PRRSV encodes a functional PRRSV-vsRNA1 which auto-regulates PRRSV replication by directly targeting and suppressing viral NSP2 gene expression. These findings not only provide new insights into the mechanism of the pathogenesis of PRRSV, but also explore a potential avenue for controlling PRRSV infection using viral small RNAs.

Human parainfluenza virus type 3 (HPIV-3); Construction and rescue of an infectious, recombinant virus expressing the enhanced green fluorescent protein (EGFP).

USDA-ARS?s Scientific Manuscript database

The ability to rescue an infectious, recombinant, RNA virus from a cDNA clone, has led to new opportunities for measuring viral replication from a viral expressed reporter gene. In this protocol, the process of inserting enhanced green fluorescent protein (EGFP) gene into the human parainfluenza vi...

Cell-Free and Cell-Based Approaches to Explore the Roles of Host Membranes and Lipids in the Formation of Viral Replication Compartment Induced by Tombusviruses.

PubMed

Nagy, Peter D; Pogany, Judit; Xu, Kai

2016-03-03

Plant positive strand RNA viruses are intracellular infectious agents that take advantage of cellular lipids and membranes to support replication and protect viral RNA from degradation by host antiviral responses. In this review, we discuss how Tomato bushy stunt virus (TBSV) co-opts lipid transfer proteins and modulates lipid metabolism and transport to facilitate the assembly of the membrane-bound viral replicase complexes within intricate replication compartments. Identification and characterization of the proviral roles of specific lipids and proteins involved in lipid metabolism based on results from yeast (Saccharomyces cerevisiae) model host and cell-free approaches are discussed. The review also highlights the advantage of using liposomes with chemically defined composition to identify specific lipids required for TBSV replication. Remarkably, all the known steps in TBSV replication are dependent on cellular lipids and co-opted membranes.

Antiretroviral Agents Effectively Block HIV Replication after Cell-to-Cell Transfer

PubMed Central

Permanyer, Marc; Ballana, Ester; Ruiz, Alba; Badia, Roger; Riveira-Munoz, Eva; Gonzalo, Encarna; Clotet, Bonaventura

2012-01-01

Cell-to-cell transmission of HIV has been proposed as a mechanism contributing to virus escape to the action of antiretrovirals and a mode of HIV persistence during antiretroviral therapy. Here, cocultures of infected HIV-1 cells with primary CD4+ T cells or lymphoid cells were used to evaluate virus transmission and the effect of known antiretrovirals. Transfer of HIV antigen from infected to uninfected cells was resistant to the reverse transcriptase inhibitors (RTIs) zidovudine (AZT) and tenofovir, but was blocked by the attachment inhibitor IgGb12. However, quantitative measurement of viral DNA production demonstrated that all anti-HIV agents blocked virus replication with similar potency to cell-free virus infections. Cell-free and cell-associated infections were equally sensitive to inhibition of viral replication when HIV-1 long terminal repeat (LTR)-driven green fluorescent protein (GFP) expression in target cells was measured. However, detection of GFP by flow cytometry may incorrectly estimate the efficacy of antiretrovirals in cell-associated virus transmission, due to replication-independent Tat-mediated LTR transactivation as a consequence of cell-to-cell events that did not occur in short-term (48-h) cell-free virus infections. In conclusion, common markers of virus replication may not accurately correlate and measure infectivity or drug efficacy in cell-to-cell virus transmission. When accurately quantified, active drugs blocked proviral DNA and virus replication in cell-to-cell transmission, recapitulating the efficacy of antiretrovirals in cell-free virus infections and in vivo. PMID:22696642

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.

Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens

PubMed Central

Meliopoulos, Victoria A.; Andersen, Lauren E.; Birrer, Katherine F.; Simpson, Kaylene J.; Lowenthal, John W.; Bean, Andrew G. D.; Stambas, John; Stewart, Cameron R.; Tompkins, S. Mark; van Beusechem, Victor W.; Fraser, Iain; Mhlanga, Musa; Barichievy, Samantha; Smith, Queta; Leake, Devin; Karpilow, Jon; Buck, Amy; Jona, Ghil; Tripp, Ralph A.

2012-01-01

Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.—Meliopoulos, V. A., Andersen, L. E., Birrer, K. F., Simpson, K. J., Lowenthal, J. W., Bean, A. G. D., Stambas, J., Stewart, C. R., Tompkins, S. M., van Beusechem, V. W., Fraser, I., Mhlanga, M., Barichievy, S., Smith, Q., Leake, D., Karpilow, J., Buck, A., Jona, G., Tripp, R. A. Host gene targets for novel influenza therapies elucidated by high-throughput RNA interference screens. PMID:22247330

Filovirus pathogenesis and immune evasion: insights from Ebola virus and Marburg virus

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

Messaoudi, Ilhem; Amarasinghe, Gaya K.; Basler, Christopher F.

Ebola viruses and Marburg viruses, members of the filovirus family, are zoonotic pathogens that cause severe disease in people, as highlighted by the latest Ebola virus epidemic in West Africa. Filovirus disease is characterized by uncontrolled virus replication and the activation of host responses that contribute to pathogenesis. Underlying these phenomena is the potent suppression of host innate antiviral responses, particularly the type I interferon response, by viral proteins, which allows high levels of viral replication. In this Review, we describe the mechanisms used by filoviruses to block host innate immunity and discuss the links between immune evasion and filovirusmore » pathogenesis.« less

Filovirus pathogenesis and immune evasion: insights from Ebola virus and Marburg virus.

PubMed

Messaoudi, Ilhem; Amarasinghe, Gaya K; Basler, Christopher F

2015-11-01

Ebola viruses and Marburg viruses, members of the filovirus family, are zoonotic pathogens that cause severe disease in people, as highlighted by the latest Ebola virus epidemic in West Africa. Filovirus disease is characterized by uncontrolled virus replication and the activation of host responses that contribute to pathogenesis. Underlying these phenomena is the potent suppression of host innate antiviral responses, particularly the type I interferon response, by viral proteins, which allows high levels of viral replication. In this Review, we describe the mechanisms used by filoviruses to block host innate immunity and discuss the links between immune evasion and filovirus pathogenesis.

Plant rhabdoviruses.

PubMed

Redinbaugh, M G; Hogenhout, S A

2005-01-01

This chapter provides an overview of plant rhabdovirus structure and taxonomy, genome structure, protein function, and insect and plant infection. It is focused on recent research and unique aspects of rhabdovirus biology. Plant rhabdoviruses are transmitted by aphid, leafhopper or planthopper vectors, and the viruses replicate in both their insect and plant hosts. The two plant rhabdovirus genera, Nucleorhabdovirus and Cytorhabdovirus, can be distinguished on the basis of their intracellular site of morphogenesis in plant cells. All plant rhabdoviruses carry analogs of the five core genes: the nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G) and large or polymerase (L). However, compared to vesiculoviruses that are composed of the five core genes, all plant rhabdoviruses encode more than these five genes, at least one of which is inserted between the P and M genes in the rhabdoviral genome. Interestingly, while these extra genes are not similar among plant rhabdoviruses, two encode proteins with similarity to the 30K superfamily of plant virus movement proteins. Analysis of nucleorhabdoviral protein sequences revealed nuclear localization signals for the N, P, M and L proteins, consistent with virus replication and morphogenesis of these viruses in the nucleus. Plant and insect factors that limit virus infection and transmission are discussed.

The small envelope protein of porcine reproductive and respiratory syndrome virus possesses ion channel protein-like properties

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

Lee, Changhee; Yoo, Dongwan

The small envelope (E) protein of porcine reproductive and respiratory syndrome virus (PRRSV) is a hydrophobic 73 amino acid protein encoded in the internal open reading frame (ORF) of the bicistronic mRNA2. As a first step towards understanding the biological role of E protein during PRRSV replication, E gene expression was blocked in a full-length infectious clone by mutating the ATG translational initiation to GTG, such that the full-length mutant genomic clone was unable to synthesize the E protein. DNA transfection of PRRSV-susceptible cells with the E gene knocked-out genomic clone showed the absence of virus infectivity. P129-{delta}E-transfected cells howevermore » produced virion particles in the culture supernatant, and these particles contained viral genomic RNA, demonstrating that the E protein is essential for PRRSV infection but dispensable for virion assembly. Electron microscopy suggests that the P129-{delta}E virions assembled in the absence of E had a similar appearance to the wild-type particles. Strand-specific RT-PCR demonstrated that the E protein-negative, non-infectious P129-{delta}E virus particles were able to enter cells but further steps of replication were interrupted. The entry of PRRSV has been suggested to be via receptor-mediated endocytosis, and lysomotropic basic compounds and known ion-channel blocking agents both inhibited PRRSV replication effectively during the uncoating process. The expression of E protein in Escherichia coli-mediated cell growth arrests and increased the membrane permeability. Cross-linking experiments in cells infected with PRRSV or transfected with E gene showed that the E protein was able to form homo-oligomers. Taken together, our data suggest that the PRRSV E protein is likely an ion-channel protein embedded in the viral envelope and facilitates uncoating of virus and release of the genome in the cytoplasm.« less

Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector.

PubMed

Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C; Miller, W Allen

2016-11-18

Understanding the molecular mechanisms involved in plant virus-vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus , Luteoviridae ) and Pea enation mosaic virus 2 (PEMV2, Umbravirus , Tombusviridae ) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum , and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum . Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits.

Sequences outside that of residues 93-102 of 3A protein can contribute to the ability of foot-and-mouth disease virus (FMDV) to replicate in bovine-derived cells.

PubMed

Ma, Xueqing; Li, Pinghua; Bai, Xingwen; Sun, Pu; Bao, Huifang; Lu, Zengjun; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

2014-10-13

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals. During 2010 and 2011, there was an epidemic of the Mya-98 lineage of the Southeast Asia (SEA) topotype in East Asia, including China. Changes in the FMDV 3A protein have been previously reported to be associated with the inability of FMDV to grow in bovine cells and cause disease in cattle. In this paper, we report the generation of a full-length infectious cDNA clone of FMDV O/SEA/Mya-98 strain O/GZSB/2011 for the first time along with two genetically modified viruses with deletion at positions 93-102 and 133-143 in 3A based on the established infectious clone. All the recombinant viruses grew well and displayed growth properties and plaque phenotypes similar to those of the parental virus in baby hamster kidney (BHK-21) cells, porcine kidney (PK-15) cells, and primary fetal porcine kidney (FPK) cells. While the recombinant viruses rvGZSB and rvSBΔ133-143 exhibited similar growth properties and plaque phenotypes with the parental virus in primary fetal bovine kidney (FBK) cells, the recombinant virus rvSBΔ93-102, containing deletion at positions 93-102 in 3A, grew at a slower rate and had a smaller plaque size phenotype in FBK cells than that of the parental virus. Therefore, the results suggest that the deletion at positions 93-102 of 3A protein does not affect FMDV replication efficiency in BHK-21, PK-15 and FPK cells, but affects virus replication efficiency in FBK cells, although, cannot alone account for the inability to replicate in bovine cells. Copyright © 2014 Elsevier B.V. All rights reserved.

GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement

PubMed Central

Kaido, Masanori; Abe, Kazutomo; Mine, Akira; Hyodo, Kiwamu; Taniguchi, Takako; Taniguchi, Hisaaki; Mise, Kazuyuki; Okuno, Tetsuro

2014-01-01

The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process. PMID:25411849

Quantitative Proteomic Analysis of Mosquito C6/36 Cells Reveals Host Proteins Involved in Zika Virus Infection.

PubMed

Xin, Qi-Lin; Deng, Cheng-Lin; Chen, Xi; Wang, Jun; Wang, Shao-Bo; Wang, Wei; Deng, Fei; Zhang, Bo; Xiao, Gengfu; Zhang, Lei-Ke

2017-06-15

Zika virus (ZIKV) is an emerging arbovirus belonging to the genus Flavivirus of the family Flaviviridae During replication processes, flavivirus manipulates host cell systems to facilitate its replication, while the host cells activate antiviral responses. Identification of host proteins involved in the flavivirus replication process may lead to the discovery of antiviral targets. The mosquitoes Aedes aegypti and Aedes albopictus are epidemiologically important vectors for ZIKV, and effective restrictions of ZIKV replication in mosquitoes will be vital in controlling the spread of virus. In this study, an iTRAQ-based quantitative proteomic analysis of ZIKV-infected Aedes albopictus C6/36 cells was performed to investigate host proteins involved in the ZIKV infection process. A total of 3,544 host proteins were quantified, with 200 being differentially regulated, among which CHCHD2 can be upregulated by ZIKV infection in both mosquito C6/36 and human HeLa cells. Our further study indicated that CHCHD2 can promote ZIKV replication and inhibit beta interferon (IFN-β) production in HeLa cells, suggesting that ZIKV infection may upregulate CHCHD2 to inhibit IFN-I production and thus promote virus replication. Bioinformatics analysis of regulated host proteins highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the 20S proteasome, bortezomib, can inhibit ZIKV infection in vivo Our study illustrated how host cells respond to ZIKV infection and also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. IMPORTANCE ZIKV infection poses great threats to human health, and there is no FDA-approved drug available for the treatment of ZIKV infection. During replication, ZIKV manipulates host cell systems to facilitate its replication, while host cells activate antiviral responses. Identification of host proteins involved in the ZIKV replication process may lead to the discovery of antiviral targets. In this study, the first quantitative proteomic analysis of ZIKV-infected cells was performed to investigate host proteins involved in the ZIKV replication process. Bioinformatics analysis highlighted several ZIKV infection-regulated biological processes. Further study indicated that the ubiquitin proteasome system (UPS) plays roles in the ZIKV entry process and that an FDA-approved inhibitor of the UPS, bortezomib, can inhibit ZIKV infection in vivo Our study not only illustrated how host cells respond to ZIKV infection but also provided a candidate drug for the control of ZIKV infection in mosquitoes and treatment of ZIKV infection in patients. Copyright © 2017 American Society for Microbiology.

Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

PubMed

Khalil, Mohamed I; Sommer, Marvin H; Hay, John; Ruyechan, William T; Arvin, Ann M

2015-07-01

The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication. Copyright © 2015 Elsevier Inc. All rights reserved.

Inhibition of mTORC1 inhibits lytic replication of Epstein-Barr virus in a cell-type specific manner.

PubMed

Adamson, Amy L; Le, Brandi T; Siedenburg, Brian D

2014-06-11

Epstein-Barr virus is a human herpesvirus that infects a majority of the human population. Primary infection of Epstein-Barr virus (EBV) causes the syndrome infectious mononucleosis. This virus is also associated with several cancers, including Burkitt's lymphoma, post-transplant lymphoproliferative disorder and nasopharyngeal carcinoma. As all herpesvirus family members, EBV initially replicates lytically to produce abundant virus particles, then enters a latent state to remain within the host indefinitely. Through a genetic screen in Drosophila, we determined that reduction of Drosophila Tor activity altered EBV immediate-early protein function. To further investigate this finding, we inhibited mTOR in EBV-positive cells and investigated subsequent changes to lytic replication via Western blotting, flow cytometry, and quantitative PCR. The student T-test was used to evaluate significance. mTOR, the human homolog of Drosophila Tor, is an important protein at the center of a major signaling pathway that controls many aspects of cell biology. As the EBV immediate-early genes are responsible for EBV lytic replication, we examined the effect of inhibition of mTORC1 on EBV lytic replication in human EBV-positive cell lines. We determined that treatment of cells with rapamycin, which is an inhibitor of mTORC1 activity, led to a reduction in the ability of B cell lines to undergo lytic replication. In contrast, EBV-positive epithelial cell lines underwent higher levels of lytic replication when treated with rapamycin. Overall, the responses of EBV-positive cell lines vary when treated with mTOR inhibitors, and this may be important when considering such inhibitors as anti-cancer therapeutic agents.

Hepatitis E virus persists in the presence of a type III interferon response.

PubMed

Yin, Xin; Li, Xinlei; Ambardekar, Charuta; Hu, Zhimin; Lhomme, Sébastien; Feng, Zongdi

2017-05-01

The RIG-I-like RNA helicase (RLR)-mediated interferon (IFN) response plays a pivotal role in the hepatic antiviral immunity. The hepatitis A virus (HAV) and the hepatitis C virus (HCV) counter this response by encoding a viral protease that cleaves the mitochondria antiviral signaling protein (MAVS), a common signaling adaptor for RLRs. However, a third hepatotropic RNA virus, the hepatitis E virus (HEV), does not appear to encode a functional protease yet persists in infected cells. We investigated HEV-induced IFN responses in human hepatoma cells and primary human hepatocytes. HEV infection resulted in persistent virus replication despite poor spread. This was companied by a type III IFN response that upregulated multiple IFN-stimulated genes (ISGs), but type I IFNs were barely detected. Blocking type III IFN production or signaling resulted in reduced ISG expression and enhanced HEV replication. Unlike HAV and HCV, HEV did not cleave MAVS; MAVS protein size, mitochondrial localization, and function remained unaltered in HEV-replicating cells. Depletion of MAVS or MDA5, and to a less extent RIG-I, also diminished IFN production and increased HEV replication. Furthermore, persistent activation of the JAK/STAT signaling rendered infected cells refractory to exogenous IFN treatment, and depletion of MAVS or the receptor for type III IFNs restored the IFN responsiveness. Collectively, these results indicate that unlike other hepatotropic RNA viruses, HEV does not target MAVS and its persistence is associated with continuous production of type III IFNs.

Selective replication of oncolytic virus M1 results in a bystander killing effect that is potentiated by Smac mimetics.

PubMed

Cai, Jing; Lin, Yuan; Zhang, Haipeng; Liang, Jiankai; Tan, Yaqian; Cavenee, Webster K; Yan, Guangmei

2017-06-27

Oncolytic virotherapy is a treatment modality that uses native or genetically modified viruses that selectively replicate in and kill tumor cells. Viruses represent a type of pathogen-associated molecular pattern and thereby induce the up-regulation of dozens of cytokines via activating the host innate immune system. Second mitochondria-derived activator of caspases (Smac) mimetic compounds (SMCs), which antagonize the function of inhibitor of apoptosis proteins (IAPs) and induce apoptosis, sensitize tumor cells to multiple cytokines. Therefore, we sought to determine whether SMCs sensitize tumor cells to cytokines induced by the oncolytic M1 virus, thus enhancing a bystander killing effect. Here, we report that SMCs potentiate the oncolytic effect of M1 in vitro, in vivo, and ex vivo. This strengthened oncolytic efficacy resulted from the enhanced bystander killing effect caused by the M1 virus via cytokine induction. Through a microarray analysis and subsequent validation using recombinant cytokines, we identified IL-8, IL-1A, and TRAIL as the key cytokines in the bystander killing effect. Furthermore, SMCs increased the replication of M1, and the accumulation of virus protein induced irreversible endoplasmic reticulum stress- and c-Jun N-terminal kinase-mediated apoptosis. Nevertheless, the combined treatment with M1 and SMCs had little effect on normal and human primary cells. Because SMCs selectively and significantly enhance the bystander killing effect and the replication of oncolytic virus M1 specifically in cancer cells, this combined treatment may represent a promising therapeutic strategy.

Effects of Shield1 on the viral replication of varicella-zoster virus containing FKBP-tagged ORF4 and 48

PubMed Central

Li, Shuying; Liu, Zhanjun; Li, Ji; Liu, Aihua; Zhu, Lihua; Yu, Kui; Zhang, Ke

2018-01-01

The present study aimed to explore the effects of a stabilizing ligand, Shield-1, on the replication of recombinant varicella-zoster virus (VZV) containing FK506 binding protein (FKPB) tags in essential open reading frames (ORF) 4 and 48. A specific galactokinase (galK) selection method was conducted, following the addition of galK labels to VZV ORF4 and 48, using a SW102 VZV bacterial artificial chromosome (BAC) system. Subsequently, recombinant VZV containing FKPB tags in ORF4 and 48 was constructed by counterselection and homologous recombination. Recombinant viral plasmids containing FKPB-tagged VZV ORF4 and 48 were extracted and transfected into human acute retinal pigment epithelial ARPE-19 cells. The results demonstrated that the FKPB-tagged viral protein was rapidly degraded by proteases in recombinant virus-infected ARPE-19 cells. In addition, the recombinant VZVORF4-FKBP-ORF48-FKBP virus could not grow if a synthetic ligand of FKBP, Shield1, was not added to the ARPE-19 cell culture medium; however, the degradation of FKPB-tagged viral protein was prevented if Shield1 was added to the ARPE-19 cell culture medium, thereby allowing viral replication in ARPE-19 cells. These results indicated that Shield1 may regulate replication of recombinant VZVORF4-FKBP-ORF48-FKBP following transfection into human epithelial cells. PMID:29115621

Two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins

PubMed Central

Soler, Nicolas; Marguet, Evelyne; Cortez, Diego; Desnoues, Nicole; Keller, Jenny; van Tilbeurgh, Herman; Sezonov, Guennadi; Forterre, Patrick

2010-01-01

Thermococcales (phylum Euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. Here we describe three new plasmids from Thermococcales that could provide new tools and model systems for genetic and molecular studies in Archaea. The plasmids pTN2 from Thermococcus nautilus sp. 30-1 and pP12-1 from Pyrococcus sp. 12-1 belong to the same family. They have similar size (∼12 kb) and share six genes, including homologues of genes encoded by the virus PAV1 from Pyrococcus abyssi. The plasmid pT26-2 from Thermococcus sp. 26-2 (21.5 kb), that corresponds to another plasmid family, encodes many proteins having homologues in virus-like elements integrated in several genomes of Thermococcales and Methanococcales. Our analyses confirm that viruses and plasmids are evolutionary related and co-evolve with their hosts. Whereas all plasmids previously isolated from Thermococcales replicate by the rolling circle mechanism, the three plasmids described here probably replicate by the theta mechanism. The plasmids pTN2 and pP12-1 encode a putative helicase of the SFI superfamily and a new family of DNA polymerase, whose activity was demonstrated in vitro, whereas pT26-2 encodes a putative new type of helicase. This strengthens the idea that plasmids and viruses are a reservoir of novel protein families involved in DNA replication. PMID:20403814

Protein Phosphatase-1 regulates Rift Valley fever virus replication.

PubMed

Baer, Alan; Shafagati, Nazly; Benedict, Ashwini; Ammosova, Tatiana; Ivanov, Andrey; Hakami, Ramin M; Terasaki, Kaori; Makino, Shinji; Nekhai, Sergei; Kehn-Hall, Kylene

2016-03-01

Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target. Copyright © 2016 Elsevier B.V. All rights reserved.

Various plus unique: Viral protein U as a plurifunctional protein for HIV-1 replication.

PubMed

Soper, Andrew; Juarez-Fernandez, Guillermo; Aso, Hirofumi; Moriwaki, Miyu; Yamada, Eri; Nakano, Yusuke; Koyanagi, Yoshio; Sato, Kei

2017-04-01

Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome, encodes four accessory genes, one of which is viral protein U (Vpu). Recently, the study of Vpu has been of great interest. For instance, various cellular proteins are degraded (e.g. CD4) and down-modulated (e.g. tetherin) by Vpu. Vpu also antagonizes the function of tetherin and inhibits NF-κB. Moreover, Vpu is a viroporin forming ion channels and may represent a promising target for anti-HIV-1 drugs. In this review, we summarize the domains/residues that are responsible for Vpu's functions, describe the current understanding of the role of Vpu in HIV-1-infected cells, and review the effect of Vpu on HIV-1 in replication and pathogenesis. Future investigations that simultaneously assess a combination of Vpu functions are required to clearly delineate the most important functions for viral replication. Impact statement Viral protein U (Vpu) is a unique protein encoded by human immunodeficiency virus type 1 (HIV-1) and related lentiviruses, playing multiple roles in viral replication and pathogenesis. In this review, we briefly summarize the most up-to-date knowledge of HIV-1 Vpu.

The chaperonin CCTα is required for efficient transcription and replication of rabies virus.

PubMed

Zhang, Jinyang; Ye, Chengjin; Ruan, Xizhen; Zan, Jie; Xu, Yunbin; Liao, Min; Zhou, Jiyong

2014-10-01

Negri bodies (NBs) are formed in the cytoplasm of rabies virus (RABV)-infected cells and are accompanied by a number of host factors to NBs, in which replication and transcription occur. Here, it was found that chaperonin containing TCP-1 subunit alpha (CCTα) relocalizes to NBs in RABV-infected cells, and that cotransfection of nucleo- and phospho-proteins of RABV is sufficient to recruit CCTα to the NBs' structure. Inhibition of CCTα expression by specific short hairpin RNA knockdown inhibited the replication and transcription of RABV. Therefore, this study showed that the host factor CCTα is associated with RABV infection and is very likely required for efficient virus transcription and replication. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

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

Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells.

PubMed

Jose, Joyce; Taylor, Aaron B; Kuhn, Richard J

2017-02-14

Sindbis virus (SINV [genus Alphavirus , family Togaviridae ]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels. IMPORTANCE Reemerging mosquito-borne alphaviruses cause serious human epidemics worldwide. Several structural and imaging studies have helped to define the life cycle of alphaviruses in mammalian cells, but the mode of virus replication and assembly in the invertebrate vector and mechanisms producing two disease outcomes in two types of cells are yet to be identified. Using transmission electron microscopy and live-cell imaging with dual fluorescent protein-tagged SINV, we show that while insect and mammalian cells display similarities in entry and exit, they present distinct spatial and temporal organizations in virus replication and assembly. By characterizing acutely and persistently infected cells, we provide new insights into alphavirus replication and assembly in two distinct hosts, resulting in high-titer virus production in mammalian cells and continuous virus production at reduced levels in mosquito cells-presumably a prerequisite for alphavirus maintenance in nature. Copyright © 2017 Jose et al.

CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes

PubMed Central

Arthos, James; Rubbert, Andrea; Rabin, Ronald L.; Cicala, Claudia; Machado, Elizabeth; Wildt, Kathryne; Hanbach, Meredith; Steenbeke, Tavis D.; Swofford, Ruth; Farber, Joshua M.; Fauci, Anthony S.

2000-01-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1β. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1α, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages. PMID:10864653

CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes.

PubMed

Arthos, J; Rubbert, A; Rabin, R L; Cicala, C; Machado, E; Wildt, K; Hanbach, M; Steenbeke, T D; Swofford, R; Farber, J M; Fauci, A S

2000-07-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.

Identification of amino acid residues in infectious hematopoietic necrosis virus (IHNV) NV protein necessary for viral replication and pathogenicity.

PubMed

Wu, Yang; Wang, Li; Guo, Tiantian; Jiang, Yanping; Qiao, Xinyuan; Sun, Li; Liu, Min; Tang, Lijie; Xu, Yigang; Li, Yijing

2018-05-18

Our previous studies demonstrated that the nonstructural NV protein of infectious hematopoietic necrosis virus (IHNV) was essential for efficient viral replication and pathogenicity, and that the amino acid residues 32 EGDL 35 of the NV protein were responsible for nuclear localization, and played important roles in suppressing IFN and inhibiting NF-κB activity. However, little is known about the influence of 32 EGDL 35 on IHNV replication and pathogenicity. In the present study, two recombinant IHNV strains with deletions of NV 32 EGDL 35 were generated and the effect on IHNV replication and pathogenicity was explored. Our results showed that both mutants stably replicated in Chinook salmon embryo cells for 15 consecutive passages, and had similar host-tropism as wild-type (wt) IHNV; however, titers of the mutants were lower than those of wt IHNV in CHSE-214 cells. Infection of rainbow trout showed wt IHNV produced 90% cumulative mortality, while the mutants produced 55% and 60% cumulative mortality, respectively. Histopathological evaluation showed that tissues from the liver, brain, kidney, and heart of fish infected with wt IHNV exhibited pathological changes, but significant lesions were found only in the liver and heart of fish infected with the recombinant viruses. In addition, the recombinant viruses induced higher expression levels of IFN1, Mx-1, and IL-6 compared with those induced by wt IHNV. These results indicated that the 32 EGDL 35 residues were essential for the efficient anti-IFN and NF-κB-inhibiting activity of NV. Our results provide a basis for understanding the roles of 32 EGDL 35 in IHNV replication and pathogenicity, and may prove beneficial in the prevention and control of IHNV infections of fish. Copyright © 2018. Published by Elsevier Ltd.

Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication.

PubMed

Herod, Morgan R; Ferrer-Orta, Cristina; Loundras, Eleni-Anna; Ward, Joseph C; Verdaguer, Nuria; Rowlands, David J; Stonehouse, Nicola J

2016-08-01

The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further. Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., in trans) while others must originate from the template (i.e., in cis). Here, we present an analysis of cis and trans activities of the RNA-dependent RNA polymerase 3D. We demonstrate a novel cis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen. Copyright © 2016 Herod et al.

Both cis and trans Activities of Foot-and-Mouth Disease Virus 3D Polymerase Are Essential for Viral RNA Replication

PubMed Central

Herod, Morgan R.; Ferrer-Orta, Cristina; Loundras, Eleni-Anna; Ward, Joseph C.; Verdaguer, Nuria; Rowlands, David J.

2016-01-01

ABSTRACT The Picornaviridae is a large family of positive-sense RNA viruses that contains numerous human and animal pathogens, including foot-and-mouth disease virus (FMDV). The picornavirus replication complex comprises a coordinated network of protein-protein and protein-RNA interactions involving multiple viral and host-cellular factors. Many of the proteins within the complex possess multiple roles in viral RNA replication, some of which can be provided in trans (i.e., via expression from a separate RNA molecule), while others are required in cis (i.e., expressed from the template RNA molecule). In vitro studies have suggested that multiple copies of the RNA-dependent RNA polymerase (RdRp) 3D are involved in the viral replication complex. However, it is not clear whether all these molecules are catalytically active or what other function(s) they provide. In this study, we aimed to distinguish between catalytically active 3D molecules and those that build a replication complex. We report a novel nonenzymatic cis-acting function of 3D that is essential for viral-genome replication. Using an FMDV replicon in complementation experiments, our data demonstrate that this cis-acting role of 3D is distinct from the catalytic activity, which is predominantly trans acting. Immunofluorescence studies suggest that both cis- and trans-acting 3D molecules localize to the same cellular compartment. However, our genetic and structural data suggest that 3D interacts in cis with RNA stem-loops that are essential for viral RNA replication. This study identifies a previously undescribed aspect of picornavirus replication complex structure-function and an important methodology for probing such interactions further. IMPORTANCE Foot-and-mouth disease virus (FMDV) is an important animal pathogen responsible for foot-and-mouth disease. The disease is endemic in many parts of the world with outbreaks within livestock resulting in major economic losses. Propagation of the viral genome occurs within replication complexes, and understanding this process can facilitate the development of novel therapeutic strategies. Many of the nonstructural proteins involved in replication possess multiple functions in the viral life cycle, some of which can be supplied to the replication complex from a separate genome (i.e., in trans) while others must originate from the template (i.e., in cis). Here, we present an analysis of cis and trans activities of the RNA-dependent RNA polymerase 3D. We demonstrate a novel cis-acting role of 3D in replication. Our data suggest that this role is distinct from its enzymatic functions and requires interaction with the viral genome. Our data further the understanding of genome replication of this important pathogen. PMID:27194768

Active RNA replication of hepatitis C virus downregulates CD81 expression.

PubMed

Ke, Po-Yuan; Chen, Steve S-L

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81.

Active RNA Replication of Hepatitis C Virus Downregulates CD81 Expression

PubMed Central

Ke, Po-Yuan; Chen, Steve S.-L.

2013-01-01

So far how hepatitis C virus (HCV) replication modulates subsequent virus growth and propagation still remains largely unknown. Here we determine the impact of HCV replication status on the consequential virus growth by comparing normal and high levels of HCV RNA expression. We first engineered a full-length, HCV genotype 2a JFH1 genome containing a blasticidin-resistant cassette inserted at amino acid residue of 420 in nonstructural (NS) protein 5A, which allowed selection of human hepatoma Huh7 cells stably-expressing HCV. Short-term establishment of HCV stable cells attained a highly-replicating status, judged by higher expressions of viral RNA and protein as well as higher titer of viral infectivity as opposed to cells harboring the same genome without selection. Interestingly, maintenance of highly-replicating HCV stable cells led to decreased susceptibility to HCV pseudotyped particle (HCVpp) infection and downregulated cell surface level of CD81, a critical HCV entry (co)receptor. The decreased CD81 cell surface expression occurred through reduced total expression and cytoplasmic retention of CD81 within an endoplasmic reticulum -associated compartment. Moreover, productive viral RNA replication in cells harboring a JFH1 subgenomic replicon containing a similar blasticidin resistance gene cassette in NS5A and in cells robustly replicating full-length infectious genome also reduced permissiveness to HCVpp infection through decreasing the surface expression of CD81. The downregulation of CD81 surface level in HCV RNA highly-replicating cells thus interfered with reinfection and led to attenuated viral amplification. These findings together indicate that the HCV RNA replication status plays a crucial determinant in HCV growth by modulating the expression and intracellular localization of CD81. PMID:23349980

ICTV Virus Taxonomy Profile: Virgaviridae

USDA-ARS?s Scientific Manuscript database

The family Virgaviridae is comprised of plant-infecting viruses with rod-shaped particles, single stranded RNA genomes with 3' terminal tRNA-like structures, and replication proteins typical of alphalike viruses. Differences in the number of genome components, genome organization and transmission m...

Elimination of A-type inclusion formation enhances cowpox virus replication in mice: implications for orthopoxvirus evolution.

PubMed

Kastenmayer, Robin J; Maruri-Avidal, Liliana; Americo, Jeffrey L; Earl, Patricia L; Weisberg, Andrea S; Moss, Bernard

2014-03-01

Some orthopoxviruses including cowpox virus embed virus particles in dense bodies, comprised of the A-type inclusion (ATI) protein, which may provide long-term environmental protection. This strategy could be beneficial if the host population is sparse or spread is inefficient or indirect. However, the formation of ATI may be neutral or disadvantageous for orthopoxviruses that rely on direct respiratory spread. Disrupted ATI open reading frames in orthopoxviruses such as variola virus, the agent of smallpox, and monkeypox virus suggests that loss of this feature provided positive selection. To test this hypothesis, we constructed cowpox virus mutants with deletion of the ATI gene or another gene required for embedding virions. The ATI deletion mutant caused greater weight loss and higher replication in the respiratory tract than control viruses, supporting our hypothesis. Deletion of the gene for embedding virions had a lesser effect, possibly due to known additional functions of the encoded protein. Published by Elsevier Inc.

Replication of adeno-associated virus in cells irradiated with UV light at 254 nm

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

Yakobson, B.; Hrynko, T.A.; Peak, M.J.

1989-03-01

Irradiation of simian virus 40 (ori mutant)-transformed Chinese hamster embryo cells (OD4 line) with UV light induced a cellular capacity which supported a full cycle of helper-independent adeno-associated virus replication. Monochromatic UV light at 254 nm was about 1,000-fold more effective than UV light at 313 nm, indicating that cellular nucleic acid is the primary chromophore in the UV-induced process leading to permissiveness for adeno-associated virus replication. The UV irradiation and the infection could be separated for up to 12 h without substantial loss of permissiveness. During this time interval, the induction process was partly sensitive to cycloheximide, suggesting amore » requirement for de novo protein synthesis.« less

Cellular Protein WDR11 Interacts with Specific Herpes Simplex Virus Proteins at the trans-Golgi Network To Promote Virus Replication

PubMed Central

Taylor, Kathryne E.

2015-01-01

ABSTRACT It has recently been proposed that the herpes simplex virus (HSV) protein ICP0 has cytoplasmic roles in blocking antiviral signaling and in promoting viral replication in addition to its well-known proteasome-dependent functions in the nucleus. However, the mechanisms through which it produces these effects remain unclear. While investigating this further, we identified a novel cytoplasmic interaction between ICP0 and the poorly characterized cellular protein WDR11. During an HSV infection, WDR11 undergoes a dramatic change in localization at late times in the viral replication cycle, moving from defined perinuclear structures to a dispersed cytoplasmic distribution. While this relocation was not observed during infection with viruses other than HSV-1 and correlated with efficient HSV-1 replication, the redistribution was found to occur independently of ICP0 expression, instead requiring viral late gene expression. We demonstrate for the first time that WDR11 is localized to the trans-Golgi network (TGN), where it interacts specifically with some, but not all, HSV virion components, in addition to ICP0. Knockdown of WDR11 in cultured human cells resulted in a modest but consistent decrease in yields of both wild-type and ICP0-null viruses, in the supernatant and cell-associated fractions, without affecting viral gene expression. Although further study is required, we propose that WDR11 participates in viral assembly and/or secondary envelopment. IMPORTANCE While the TGN has been proposed to be the major site of HSV-1 secondary envelopment, this process is incompletely understood, and in particular, the role of cellular TGN components in this pathway is unknown. Additionally, little is known about the cellular functions of WDR11, although the disruption of this protein has been implicated in multiple human diseases. Therefore, our finding that WDR11 is a TGN-resident protein that interacts with specific viral proteins to enhance viral yields improves both our understanding of basic cellular biology as well as how this protein is co-opted by HSV. PMID:26178983

Cyclosporin A associated helicase-like protein facilitates the association of hepatitis C virus RNA polymerase with its cellular cyclophilin B.

PubMed

Morohashi, Kengo; Sahara, Hiroeki; Watashi, Koichi; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-04-29

Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology.

Phosphatidic Acid Produced by Phospholipase D Promotes RNA Replication of a Plant RNA Virus

PubMed Central

Hyodo, Kiwamu; Taniguchi, Takako; Manabe, Yuki; Kaido, Masanori; Mise, Kazuyuki; Sugawara, Tatsuya; Taniguchi, Hisaaki; Okuno, Tetsuro

2015-01-01

Eukaryotic positive-strand RNA [(+)RNA] viruses are intracellular obligate parasites replicate using the membrane-bound replicase complexes that contain multiple viral and host components. To replicate, (+)RNA viruses exploit host resources and modify host metabolism and membrane organization. Phospholipase D (PLD) is a phosphatidylcholine- and phosphatidylethanolamine-hydrolyzing enzyme that catalyzes the production of phosphatidic acid (PA), a lipid second messenger that modulates diverse intracellular signaling in various organisms. PA is normally present in small amounts (less than 1% of total phospholipids), but rapidly and transiently accumulates in lipid bilayers in response to different environmental cues such as biotic and abiotic stresses in plants. However, the precise functions of PLD and PA remain unknown. Here, we report the roles of PLD and PA in genomic RNA replication of a plant (+)RNA virus, Red clover necrotic mosaic virus (RCNMV). We found that RCNMV RNA replication complexes formed in Nicotiana benthamiana contained PLDα and PLDβ. Gene-silencing and pharmacological inhibition approaches showed that PLDs and PLDs-derived PA are required for viral RNA replication. Consistent with this, exogenous application of PA enhanced viral RNA replication in plant cells and plant-derived cell-free extracts. We also found that a viral auxiliary replication protein bound to PA in vitro, and that the amount of PA increased in RCNMV-infected plant leaves. Together, our findings suggest that RCNMV hijacks host PA-producing enzymes to replicate. PMID:26020241

Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection.

PubMed

Albecka, Anna; Owen, Danielle J; Ivanova, Lyudmila; Brun, Juliane; Liman, Rukayya; Davies, Laura; Ahmed, M Firoz; Colaco, Susanna; Hollinshead, Michael; Graham, Stephen C; Crump, Colin M

2017-01-15

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells. Copyright © 2017 Albecka et al.

Porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes

USDA-ARS?s Scientific Manuscript database

Membrane modification of host subcellular compartments is critical to the replication of many RNA viruses. Enveloped viruses additionally require the ability to requisition cellular membranes during egress for the development of infectious progeny. Porcine reproductive and respiratory syndrome virus...

Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B

PubMed Central

Kong, Lingbao; Fujimoto, Akira; Nakamura, Mariko; Aoyagi, Haruyo; Matsuda, Mami; Watashi, Koichi; Suzuki, Ryosuke; Arita, Minetaro; Yamagoe, Satoshi; Dohmae, Naoshi; Suzuki, Takehiro; Sakamaki, Yuriko; Ichinose, Shizuko; Suzuki, Tetsuro; Wakita, Takaji

2016-01-01

ABSTRACT It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCE The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors. PMID:26739056

Proteomic Analysis of Virus-Host Interactions in an Infectious Context Using Recombinant Viruses*

PubMed Central

Komarova, Anastassia V.; Combredet, Chantal; Meyniel-Schicklin, Laurène; Chapelle, Manuel; Caignard, Grégory; Camadro, Jean-Michel; Lotteau, Vincent; Vidalain, Pierre-Olivier; Tangy, Frédéric

2011-01-01

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells. PMID:21911578

In vivo dynamics of EBNA1-oriP interaction during latent and lytic replication of Epstein-Barr virus.

PubMed

Daikoku, Tohru; Kudoh, Ayumi; Fujita, Masatoshi; Sugaya, Yutaka; Isomura, Hiroki; Tsurumi, Tatsuya

2004-12-24

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is required for maintenance of the viral genome DNA during the latent phase of EBV replication but continues to be synthesized after the induction of viral productive replication. An EBV genome-wide chromatin immunoprecipitation assay revealed that EBNA1 constantly binds to oriP of the EBV genome during not only latent but also lytic infection. Although the total levels of EBNA1 proved constant throughout the latter, the levels of the oriP-bound form were increased as lytic infection proceeded. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins are clustered. Confocal laser microscopic analyses revealed that whereas EBNA1 was distributed broadly in nuclei as fine punctate dots during the latent phase of infection, the protein became redistributed to the viral replication compartments and localized as distinct spots within and/or nearby the compartments after the induction of lytic replication. Taking these findings into consideration, oriP regions of the EBV genome might be organized by EBNA1 into replication domains that may set up scaffolding for lytic replication and transcription.

Applications of Replicating-Competent Reporter-Expressing Viruses in Diagnostic and Molecular Virology.

PubMed

Li, Yongfeng; Li, Lian-Feng; Yu, Shaoxiong; Wang, Xiao; Zhang, Lingkai; Yu, Jiahui; Xie, Libao; Li, Weike; Ali, Razim; Qiu, Hua-Ji

2016-05-06

Commonly used tests based on wild-type viruses, such as immunostaining, cannot meet the demands for rapid detection of viral replication, high-throughput screening for antivirals, as well as for tracking viral proteins or virus transport in real time. Notably, the development of replicating-competent reporter-expressing viruses (RCREVs) has provided an excellent option to detect directly viral replication without the use of secondary labeling, which represents a significant advance in virology. This article reviews the applications of RCREVs in diagnostic and molecular virology, including rapid neutralization tests, high-throughput screening systems, identification of viral receptors and virus-host interactions, dynamics of viral infections in vitro and in vivo, vaccination approaches and others. However, there remain various challenges associated with RCREVs, including pathogenicity alterations due to the insertion of a reporter gene, instability or loss of the reporter gene expression, or attenuation of reporter signals in vivo. Despite all these limitations, RCREVs have become powerful tools for both basic and applied virology with the development of new technologies for generating RCREVs, the inventions of novel reporters and the better understanding of regulation of viral replication.

Cyclophilin A Interacts with Viral VP4 and Inhibits the Replication of Infectious Bursal Disease Virus.

PubMed

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

2015-01-01

Nonstructural protein VP4, a serine protease of infectious bursal disease virus (IBDV) that catalyzes the hydrolysis of polyprotein pVP2-VP4-VP3 to form the viral proteins VP2, VP4, and VP3, is essential to the replication of IBDV. However, the interacting partners of VP4 in host cells and the effects of the interaction on the IBDV lifecycle remain incompletely elucidated. In this study, using the yeast two-hybrid system, the putative VP4-interacting partner cyclophilin A (CypA) was obtained from a chicken embryo fibroblast (CEF) expression library. CypA was further confirmed to interact with VP4 of IBDV using co-immunoprecipitation (CO-IP), GST pull-down, and confocal microscopy assays. Moreover, we found that the overexpression of CypA suppressed IBDV replication, whereas the knock-down of CypA by small interfering RNAs promoted the replication of IBDV. Taken together, our findings indicate that the host cell protein CypA interacts with viral VP4 and inhibits the replication of IBDV.

Free fatty acids or high-concentration glucose enhances hepatitis A virus replication in association with a reduction in glucose-regulated protein 78 expression.

PubMed

Nwe Win, Nan; Kanda, Tatsuo; Nakamura, Masato; Nakamoto, Shingo; Okamoto, Hiroaki; Yokosuka, Osamu; Shirasawa, Hiroshi

2017-01-29

Although the interaction between host and hepatitis A virus (HAV) factors could lead to severe hepatitis A, the exact mechanism of acute liver failure caused by HAV infection is not yet fully understood. The effects of metabolic diseases such as dyslipidemia or diabetes mellitus on HAV replication are still unknown. Here, we examined the effects of free fatty acids or high-concentration glucose on HAV replication and the effects on mitogen-activated protein kinase signaling pathway-related genes in human hepatocytes. We discovered a novel effect of free fatty acids or high-concentration glucose on HAV replication in association with a reduction in the expression of glucose-regulated protein 78 (GRP78). We also observed that thapsigargin induced GRP78 expression and inhibited HAV replication. These findings may provide a new interpretation of the relationship between metabolic diseases and severity of hepatitis A and suggest a new understanding of the mechanism of severe HAV infection. Copyright © 2016 Elsevier Inc. All rights reserved.

The role of porcine reproductive and respiratory syndrome (PRRS) virus structural and non-structural proteins in virus pathogenesis.

PubMed

Music, Nedzad; Gagnon, Carl A

2010-12-01

Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease affecting the swine industry worldwide. The etiological agent, PRRS virus (PRRSV), possesses a RNA viral genome with nine open reading frames (ORFs). The ORF1a and ORF1b replicase-associated genes encode the polyproteins pp1a and pp1ab, respectively. The pp1a is processed in nine non-structural proteins (nsps): nsp1α, nsp1β, and nsp2 to nsp8. Proteolytic cleavage of pp1ab generates products nsp9 to nsp12. The proteolytic pp1a cleavage products process and cleave pp1a and pp1ab into nsp products. The nsp9 to nsp12 are involved in virus genome transcription and replication. The 3' end of the viral genome encodes four minor and three major structural proteins. The GP(2a), GP₃ and GP₄ (encoded by ORF2a, 3 and 4), are glycosylated membrane associated minor structural proteins. The fourth minor structural protein, the E protein (encoded by ORF2b), is an unglycosylated membrane associated protein. The viral envelope contains two major structural proteins: a glycosylated major envelope protein GP₅ (encoded by ORF5) and an unglycosylated membrane M protein (encoded by ORF6). The third major structural protein is the nucleocapsid N protein (encoded by ORF7). All PRRSV non-structural and structural proteins are essential for virus replication, and PRRSV infectivity is relatively intolerant to subtle changes within the structural proteins. PRRSV virulence is multigenic and resides in both the non-structural and structural viral proteins. This review discusses the molecular characteristics, biological and immunological functions of the PRRSV structural and nsps and their involvement in the virus pathogenesis.

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

DNA Unwinding Functions of Minute Virus of Mice NS1 Protein Are Modulated Specifically by the Lambda Isoform of Protein Kinase C

PubMed Central

Dettwiler, Sabine; Rommelaere, Jean; Nüesch, Jürg P. F.

1999-01-01

The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966–9977, 1998). The present study led to the identification of atypical PKCλ/ι as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKCλ phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKCλ phosphorylation at residue S473. PMID:10438831

Stress Granule-Inducing Eukaryotic Translation Initiation Factor 4A Inhibitors Block Influenza A Virus Replication

PubMed Central

Slaine, Patrick D.; Kleer, Mariel; Smith, Nathan K.; Khaperskyy, Denys A.

2017-01-01

Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5′ untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host protein synthesis machinery can be targeted to block viral replication. PMID:29258238

The roles of membranes and associated cytoskeleton in plant virus replication and cell-to-cell movement.

PubMed

Pitzalis, Nicolas; Heinlein, Manfred

2017-12-18

The infection of plants by viruses depends on cellular mechanisms that support the replication of the viral genomes, and the cell-to-cell and systemic movement of the virus via plasmodesmata (PD) and the connected phloem. While the propagation of some viruses requires the conventional endoplasmic reticulum (ER)-Golgi pathway, others replicate and spread between cells in association with the ER and are independent of this pathway. Using selected viruses as examples, this review re-examines the involvement of membranes and the cytoskeleton during virus infection and proposes potential roles of class VIII myosins and membrane-tethering proteins in controlling viral functions at specific ER subdomains, such as cortical microtubule-associated ER sites, ER-plasma membrane contact sites, and PD. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification.

PubMed

Emara, Mohamed M; Liu, Hsuan; Davis, William G; Brinton, Margo A

2008-11-01

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.

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.

A novel begomovirus isolated from sida contains putative cis- and trans-acting replication specificity determinants that have evolved independently in several geographical lineages.

PubMed

Mauricio-Castillo, J A; Torres-Herrera, S I; Cárdenas-Conejo, Y; Pastor-Palacios, G; Méndez-Lozano, J; Argüello-Astorga, G R

2014-09-01

A novel begomovirus isolated from a Sida rhombifolia plant collected in Sinaloa, Mexico, was characterized. The genomic components of sida mosaic Sinaloa virus (SiMSinV) shared highest sequence identity with DNA-A and DNA-B components of chino del tomate virus (CdTV), suggesting a vertical evolutionary relationship between these viruses. However, recombination analysis indicated that a short segment of SiMSinV DNA-A encompassing the plus-strand replication origin and the 5´-proximal 43 codons of the Rep gene was derived from tomato mottle Taino virus (ToMoTV). Accordingly, the putative cis- and trans-acting replication specificity determinants of SiMSinV were identical to those of ToMoTV but differed from those of CdTV. Modeling of the SiMSinV and CdTV Rep proteins revealed significant differences in the region comprising the small β1/β5 sheet element, where five putative DNA-binding specificity determinants (SPDs) of Rep (i.e., amino acid residues 5, 8, 10, 69 and 71) were previously identified. Computer-assisted searches of public databases led to identification of 33 begomoviruses from three continents encoding proteins with SPDs identical to those of the Rep encoded by SiMSinV. Sequence analysis of the replication origins demonstrated that all 33 begomoviruses harbor potential Rep-binding sites identical to those of SiMSinV. These data support the hypothesis that the Rep β1/β5 sheet region determines specificity of this protein for DNA replication origin sequences.

Antiviral effect of PmRab7 knock-down on inhibition of Laem-Singh virus replication in black tiger shrimp.

PubMed

Ongvarrasopone, Chalermporn; Chomchay, Ekapol; Panyim, Sakol

2010-10-01

PmRab7 is a Penaeus monodon small GTPase protein possibly involved in replication of several shrimp viruses. In this study RNA interference (RNAi) using double-stranded RNA (dsRNA) targeting PmRab7 gene (dsRNA-PmRab7) was employed to silence the expression of PmRab7 to investigate the inhibitory effect on Laem-Singh virus (LSNV) replication. Injection of dsRNA-PmRab7 24h before challenge with the virus resulted in a drastic decrease of PmRab7 mRNA and complete inhibition of LSNV replication at 3 days post-challenge. In a therapeutic mode, shrimp injected with dsRNA-PmRab7 1 day but not at 3 or 5 days post-LSNV challenge resulted in inhibition of LSNV replication. These results pave the way to use dsRNA-PmRab7 to prevent or cure LSNV infection in shrimp. Copyright © 2010 Elsevier B.V. All rights reserved.

Role of Pea Enation Mosaic Virus Coat Protein in the Host Plant and Aphid Vector

PubMed Central

Doumayrou, Juliette; Sheber, Melissa; Bonning, Bryony C.; Miller, W. Allen

2016-01-01

Understanding the molecular mechanisms involved in plant virus–vector interactions is essential for the development of effective control measures for aphid-vectored epidemic plant diseases. The coat proteins (CP) are the main component of the viral capsids, and they are implicated in practically every stage of the viral infection cycle. Pea enation mosaic virus 1 (PEMV1, Enamovirus, Luteoviridae) and Pea enation mosaic virus 2 (PEMV2, Umbravirus, Tombusviridae) are two RNA viruses in an obligate symbiosis causing the pea enation mosaic disease. Sixteen mutant viruses were generated with mutations in different domains of the CP to evaluate the role of specific amino acids in viral replication, virion assembly, long-distance movement in Pisum sativum, and aphid transmission. Twelve mutant viruses were unable to assemble but were able to replicate in inoculated leaves, move long-distance, and express the CP in newly infected leaves. Four mutant viruses produced virions, but three were not transmissible by the pea aphid, Acyrthosiphon pisum. Three-dimensional modeling of the PEMV CP, combined with biological assays for virion assembly and aphid transmission, allowed for a model of the assembly of PEMV coat protein subunits. PMID:27869713

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.

A homolog of the variola virus B22 membrane protein contributes to ectromelia virus pathogenicity in the mouse footpad model.

PubMed

Reynolds, Sara E; Earl, Patricia L; Minai, Mahnaz; Moore, Ian; Moss, Bernard

2017-01-15

Most poxviruses encode a homolog of a ~200,000-kDa membrane protein originally identified in variola virus. We investigated the importance of the ectromelia virus (ECTV) homolog C15 in a natural infection model. In cultured mouse cells, the replication of a mutant virus with stop codons near the N-terminus (ECTV-C15Stop) was indistinguishable from a control virus (ECTV-C15Rev). However, for a range of doses injected into the footpads of BALB/c mice there was less mortality with the mutant. Similar virus loads were present at the site of infection with mutant or control virus whereas there was less ECTV-C15Stop in popliteal and inguinal lymph nodes, spleen and liver indicating decreased virus spread and replication. The latter results were supported by immunohistochemical analyses. Decreased spread was evidently due to immune modulatory activity of C15, rather than to an intrinsic viral function, as the survival of infected mice depended on CD4+ and CD8+ T cells. Published by Elsevier Inc.

Hepatitis B virus molecular biology and pathogenesis

PubMed Central

Lamontagne, R. Jason; Bagga, Sumedha; Bouchard, Michael J.

2016-01-01

As obligate intracellular parasites, viruses need a host cell to provide a milieu favorable to viral replication. Consequently, viruses often adopt mechanisms to subvert host cellular signaling processes. While beneficial for the viral replication cycle, virus-induced deregulation of host cellular signaling processes can be detrimental to host cell physiology and can lead to virus-associated pathogenesis, including, for oncogenic viruses, cell transformation and cancer progression. Included among these oncogenic viruses is the hepatitis B virus (HBV). Despite the availability of an HBV vaccine, 350–500 million people worldwide are chronically infected with HBV, and a significant number of these chronically infected individuals will develop hepatocellular carcinoma (HCC). Epidemiological studies indicate that chronic infection with HBV is the leading risk factor for the development of HCC. Globally, HCC is the second highest cause of cancer-associated deaths, underscoring the need for understanding mechanisms that regulate HBV replication and the development of HBV-associated HCC. HBV is the prototype member of the Hepadnaviridae family; members of this family of viruses have a narrow host range and predominately infect hepatocytes in their respective hosts. The extremely small and compact hepadnaviral genome, the unique arrangement of open reading frames, and a replication strategy utilizing reverse transcription of an RNA intermediate to generate the DNA genome are distinguishing features of the Hepadnaviridae. In this review, we provide a comprehensive description of HBV biology, summarize the model systems used for studying HBV infections, and highlight potential mechanisms that link a chronic HBV-infection to the development of HCC. For example, the HBV X protein (HBx), a key regulatory HBV protein that is important for HBV replication, is thought to play a cofactor role in the development of HBV-induced HCC, and we highlight the functions of HBx that may contribute to the development of HBV-associated HCC. PMID:28042609

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.

Inhibition of Bim Enhances Replication of Varicella-Zoster Virus and Delays Plaque Formation in Virus-Infected Cells

PubMed Central

Liu, XueQiao

2014-01-01

Programmed cell death (apoptosis) is an important host defense mechanism against intracellular pathogens, such as viruses. Accordingly, viruses have evolved multiple mechanisms to modulate apoptosis to enhance replication. Varicella-zoster virus (VZV) induces apoptosis in human fibroblasts and melanoma cells. We found that VZV triggered the phosphorylation of the proapoptotic proteins Bim and BAD but had little or no effect on other Bcl-2 family members. Since phosphorylation of Bim and BAD reduces their proapoptotic activity, this may prevent or delay apoptosis in VZV-infected cells. Phosphorylation of Bim but not BAD in VZV-infected cells was dependent on activation of the MEK/extracellular signal-regulated kinase (ERK) pathway. Cells knocked down for Bim showed delayed VZV plaque formation, resulting in longer survival of VZV-infected cells and increased replication of virus, compared with wild-type cells infected with virus. Conversely, overexpression of Bim resulted in earlier plaque formation, smaller plaques, reduced virus replication, and increased caspase 3 activity. Inhibition of caspase activity in VZV-infected cells overexpressing Bim restored levels of virus production similar to those seen with virus-infected wild-type cells. Previously we showed that VZV ORF12 activates ERK and inhibits apoptosis in virus-infected cells. Here we found that VZV ORF12 contributes to Bim and BAD phosphorylation. In summary, VZV triggers Bim phosphorylation; reduction of Bim levels results in longer survival of VZV-infected cells and increased VZV replication. PMID:24227856

Differential protein modulation in midguts of Aedes aegypti infected with chikungunya and dengue 2 viruses.

PubMed

Tchankouo-Nguetcheu, Stéphane; Khun, Huot; Pincet, Laurence; Roux, Pascal; Bahut, Muriel; Huerre, Michel; Guette, Catherine; Choumet, Valérie

2010-10-05

Arthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and chikungunya are responsible for a high rate of severe human diseases worldwide. The midgut of mosquitoes is the first barrier for pathogen transmission and is a target organ where arboviruses must replicate prior to infecting other organs. A proteomic approach was undertaken to characterize the key virus/vector interactions and host protein modifications that happen in the midgut for viral transmission to eventually take place. Using a proteomics differential approach with two-Dimensional Differential in-Gel Electrophoresis (2D-DIGE), we defined the protein modulations in the midgut of Aedes aegypti that were triggered seven days after an oral infection (7 DPI) with dengue 2 (DENV-2) and chikungunya (CHIKV) viruses. Gel profile comparisons showed that the level of 18 proteins was modulated by DENV-2 only and 12 proteins were modulated by CHIKV only. Twenty proteins were regulated by both viruses in either similar or different ways. Both viruses caused an increase of proteins involved in the generation of reactive oxygen species, energy production, and carbohydrate and lipid metabolism. Midgut infection by DENV-2 and CHIKV triggered an antioxidant response. CHIKV infection produced an increase of proteins involved in detoxification. Our study constitutes the first analysis of the protein response of Aedes aegypti's midgut infected with viruses belonging to different families. It shows that the differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins like antioxidant are probably involved in cell protection. On the other hand, we propose that the modulation of other proteins like transferrin, hsp60 and alpha glucosidase, may favour virus survival, replication and transmission, suggesting a subversion of the insect cell metabolism by the arboviruses.

Differential Protein Modulation in Midguts of Aedes aegypti Infected with Chikungunya and Dengue 2 Viruses

PubMed Central

Tchankouo-Nguetcheu, Stéphane; Khun, Huot; Pincet, Laurence; Roux, Pascal; Bahut, Muriel; Huerre, Michel; Guette, Catherine; Choumet, Valérie

2010-01-01

Background Arthropod borne virus infections cause several emerging and resurgent infectious diseases. Among the diseases caused by arboviruses, dengue and chikungunya are responsible for a high rate of severe human diseases worldwide. The midgut of mosquitoes is the first barrier for pathogen transmission and is a target organ where arboviruses must replicate prior to infecting other organs. A proteomic approach was undertaken to characterize the key virus/vector interactions and host protein modifications that happen in the midgut for viral transmission to eventually take place. Methodology and Principal Findings Using a proteomics differential approach with two-Dimensional Differential in-Gel Electrophoresis (2D-DIGE), we defined the protein modulations in the midgut of Aedes aegypti that were triggered seven days after an oral infection (7 DPI) with dengue 2 (DENV-2) and chikungunya (CHIKV) viruses. Gel profile comparisons showed that the level of 18 proteins was modulated by DENV-2 only and 12 proteins were modulated by CHIKV only. Twenty proteins were regulated by both viruses in either similar or different ways. Both viruses caused an increase of proteins involved in the generation of reactive oxygen species, energy production, and carbohydrate and lipid metabolism. Midgut infection by DENV-2 and CHIKV triggered an antioxidant response. CHIKV infection produced an increase of proteins involved in detoxification. Conclusion/Significance Our study constitutes the first analysis of the protein response of Aedes aegypti's midgut infected with viruses belonging to different families. It shows that the differentially regulated proteins in response to viral infection include structural, redox, regulatory proteins, and enzymes for several metabolic pathways. Some of these proteins like antioxidant are probably involved in cell protection. On the other hand, we propose that the modulation of other proteins like transferrin, hsp60 and alpha glucosidase, may favour virus survival, replication and transmission, suggesting a subversion of the insect cell metabolism by the arboviruses. PMID:20957153

Template Recognition Mechanisms by Replicase Proteins Differ between Bipartite Positive-Strand Genomic RNAs of a Plant Virus▿

PubMed Central

Iwakawa, Hiro-oki; Mine, Akira; Hyodo, Kiwamu; An, Mengnan; Kaido, Masanori; Mise, Kazuyuki; Okuno, Tetsuro

2011-01-01

Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3′ untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism. PMID:20980498

Membrane-Bound Tomato Mosaic Virus Replication Proteins Participate in RNA Synthesis and Are Associated with Host Proteins in a Pattern Distinct from Those That Are Not Membrane Bound

PubMed Central

Nishikiori, Masaki; Dohi, Koji; Mori, Masashi; Meshi, Tetsuo; Naito, Satoshi; Ishikawa, Masayuki

2006-01-01

Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity. PMID:16912296

Biological roles and functional mechanisms of arenavirus Z protein in viral replication.

PubMed

Wang, Jialong; Danzy, Shamika; Kumar, Naveen; Ly, Hinh; Liang, Yuying

2012-09-01

Arenaviruses can cause severe hemorrhagic fever diseases in humans, with limited prophylactic or therapeutic measures. A small RING-domain viral protein Z has been shown to mediate the formation of virus-like particles and to inhibit viral RNA synthesis, although its biological roles in an infectious viral life cycle have not been directly addressed. By taking advantage of the available reverse genetics system for a model arenavirus, Pichinde virus (PICV), we provide the direct evidence for the essential biological roles of the Z protein's conserved residues, including the G2 myristylation site, the conserved C and H residues of RING domain, and the poorly characterized C-terminal L79 and P80 residues. Dicodon substitutions within the late (L) domain (PSAPPYEP) of the PICV Z protein, although producing viable mutant viruses, have significantly reduced virus growth, a finding suggestive of an important role for the intact L domain in viral replication. Further structure-function analyses of both PICV and Lassa fever virus Z proteins suggest that arenavirus Z proteins have similar molecular mechanisms in mediating their multiple functions, with some interesting variations, such as the role of the G2 residue in blocking viral RNA synthesis. In summary, our studies have characterized the biological roles of the Z protein in an infectious arenavirus system and have shed important light on the distinct functions of its domains in virus budding and viral RNA regulation, the knowledge of which may lead to the development of novel antiviral drugs.

The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants

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

Hipp, Katharina; Rau, Peter; Schäfer, Benjamin

Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clonesmore » prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. - Highlights: • A potential cyclin interaction motif is conserved in geminivirus Rep proteins. • In ACMV Rep, this motif (RXL) is essential for rereplication of fission yeast DNA. • Mutating RXL abrogated viral infection completely in Nicotiana benthamiana. • Expression of a nanovirus Clink protein in yeast did not induce rereplication. • Plant viruses may have evolved multiple routes to exploit host DNA synthesis.« less

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication.

PubMed

Courtney, David G; Kennedy, Edward M; Dumm, Rebekah E; Bogerd, Hal P; Tsai, Kevin; Heaton, Nicholas S; Cullen, Bryan R

2017-09-13

Many viral RNAs are modified by methylation of the N 6 position of adenosine (m 6 A). m 6 A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m 6 A-modified RNAs, but the effects of m 6 A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m 6 A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m 6 A "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether m 6 A residues modulate IAV RNA function in cis, we mapped m 6 A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m 6 A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m 6 A residues in IAV transcripts enhance viral gene expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization of uncultivable bat influenza virus using a replicative synthetic virus.

PubMed

Zhou, Bin; Ma, Jingjiao; Liu, Qinfang; Bawa, Bhupinder; Wang, Wei; Shabman, Reed S; Duff, Michael; Lee, Jinhwa; Lang, Yuekun; Cao, Nan; Nagy, Abdou; Lin, Xudong; Stockwell, Timothy B; Richt, Juergen A; Wentworth, David E; Ma, Wenjun

2014-10-01

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.

Characterization of Uncultivable Bat Influenza Virus Using a Replicative Synthetic Virus

PubMed Central

Bawa, Bhupinder; Wang, Wei; Shabman, Reed S.; Duff, Michael; Lee, Jinhwa; Lang, Yuekun; Cao, Nan; Nagy, Abdou; Lin, Xudong; Stockwell, Timothy B.; Richt, Juergen A.; Wentworth, David E.; Ma, Wenjun

2014-01-01

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses. PMID:25275541

Role of zinc-finger anti-viral protein in host defense against Sindbis virus

PubMed Central

Kozaki, Tatsuya; Takahama, Michihiro; Misawa, Takuma; Matsuura, Yoshiharu; Saitoh, Tatsuya

2015-01-01

Accumulating evidence indicates that type I interferon (IFN) mediates the host protective response to RNA viruses. However, the anti-viral effector molecules involved in this response have not been fully identified. Here, we show that zinc-finger anti-viral protein (ZAP), an IFN-inducible gene, plays a critical role in the elimination of Sindbis virus (SINV) in vitro and in vivo. The loss of ZAP greatly enhances the replication of SINV but does not inhibit type I IFN production in primary mouse embryonic fibroblasts (MEFs). ZAP binds and destabilizes SINV RNA, thereby suppressing the replication of SINV. Type I IFN fails to suppress SINV replication in ZAP-deficient MEFs, whereas the ectopic expression of ZAP is sufficient to suppress the replication of SINV in MEFs lacking the expression of type I IFN and the IFN-inducible genes. ZAP-deficient mice are highly susceptible to SINV infection, although they produce sufficient amounts of type I IFN. Therefore, ZAP is an RNA-sensing anti-viral effector molecule that mediates the type-I-IFN-dependent host defense against SINV. PMID:25758257

Preparation of BFV Gag antiserum and preliminary study on cellular distribution of BFV.

PubMed

Wang, Jian; Guo, Hong-yan; Jia, Rui; Xu, Xuan; Tan, Juan; Geng, Yun-qi; Qiao, Wen-tao

2010-04-01

Viruses (e.g. Human immunodeficiency virus, Human simplex virus and Prototype foamy virus) are obligate intracellular parasites and therefore depend on the cellular machinery for cellular trafficking. Bovine foamy virus (BFV) is a member of the Spumaretrovirinae subfamily of Retroviruses, however, details of its cellular trafficking remain unknown. In this study, we cloned the BFV gag gene into prokaryotic expression vector pET28a and purified the denaturalized Gag protein. The protein was used to immunize BALB/c mouse to produce antiserum, which could specifically recognize the BFV Gag protein in BFV-infected cells through western blot assay. Additionally, these results demonstrated that both the optimal and suboptimal cleavage of Gag protein occur in BFV-infected cells. Subsequently, the Gag antiserum was used to investigate subcellular localization of BFV. In immunofluorescence microscopy assays, colocalization microtubules (MTs) and assembling viral particles were clearly observed, which implied that BFV may transport along cellular MTs in host cells. Furthermore, MTs-depolymerizing assay indicated MTs were required for the efficient replication of BFV. In conclusion, our study suggests that BFV has evolved the mechanism to hijack the cellular cytoskeleton for its replication.

Replication cycle of duck hepatitis A virus type 1 in duck embryonic hepatocytes

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

Yao, Fangke; Chen, Yun; Shi, Jintong

Duck hepatitis A virus type 1 (DHAV-1) is an important agent of duck viral hepatitis. Until recently, the replication cycle of DHAV-1 is still unknown. Here duck embryonic hepatocytes infected with DHAV-1 were collected at different time points, and dynamic changes of the relative DHAV-1 gene expression during replication were detected by real-time PCR. And the morphology of hepatocytes infected with DHAV was evaluated by electron microscope. The result suggested that the adsorption of DHAV-1 saturated at 90 min post-infection, and the virus particles with size of about 50 nm including more than 20 nm of vacuum drying gold weremore » observed on the infected cells surface. What's more, the replication lasted around 13 h after the early protein synthesis for about 5 h, and the release of DHAV-1 was in steady state after 32 h. The replication cycle will enrich the data for DVH control and provide the foundation for future studies. - Highlights: • This is the first description of the replication cycle of DHAV-1. • Firstly find that DHAV-1 adsorption saturated at 90 min post-infection. • The replication lasted around 13 h after early protein synthesis for about 5 h. • The release of DHAV-1 was in steady state after 32 h.« less

Distinct Contributions of Autophagy Receptors in Measles Virus Replication.

PubMed

Petkova, Denitsa S; Verlhac, Pauline; Rozières, Aurore; Baguet, Joël; Claviere, Mathieu; Kretz-Remy, Carole; Mahieux, Renaud; Viret, Christophe; Faure, Mathias

2017-05-22

Autophagy is a potent cell autonomous defense mechanism that engages the lysosomal pathway to fight intracellular pathogens. Several autophagy receptors can recognize invading pathogens in order to target them towards autophagy for their degradation after the fusion of pathogen-containing autophagosomes with lysosomes. However, numerous intracellular pathogens can avoid or exploit autophagy, among which is measles virus (MeV). This virus induces a complete autophagy flux, which is required to improve viral replication. We therefore asked how measles virus interferes with autophagy receptors during the course of infection. We report that in addition to NDP52/CALCOCO₂ and OPTINEURIN/OPTN, another autophagy receptor, namely T6BP/TAXIBP1, also regulates the maturation of autophagosomes by promoting their fusion with lysosomes, independently of any infection. Surprisingly, only two of these receptors, NDP52 and T6BP, impacted measles virus replication, although independently, and possibly through physical interaction with MeV proteins. Thus, our results suggest that a restricted set of autophagosomes is selectively exploited by measles virus to replicate in the course of infection.

Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line.

PubMed

Jiwaji, Meesbah; Short, James Roswell; Dorrington, Rosemary Ann

2016-10-01

Tetraviruses are small, positive (+ve)-sense ssRNA viruses that infect the midgut cells of lepidopteran larvae. Providence virus (PrV) is the only member of the family Carmotetraviridae (previously Tetraviridae). PrV particles exhibit the characteristic tetraviral T=4 icosahedral symmetry, but PrV is distinct from other tetraviruses with respect to genome organization and viral non-structural proteins. Currently, PrV is the only tetravirus known to infect and replicate in lepidopteran cell culture lines. In this report we demonstrate, using immunofluorescence microscopy, that PrV infects and replicates in a human tissue culture cell line (HeLa), producing infectious virus particles. We also provide evidence for PrV replication in vitro in insect, mammalian and plant cell-free systems. This study challenges the long-held view that tetraviruses have a narrow host range confined to one or a few lepidopteran species and highlights the need to consider the potential for apparently non-infectious viruses to be transferred to new hosts in the laboratory.

Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

PubMed Central

Yi, Zhigang; Sperzel, Lindsey; Nürnberger, Cindy; Bredenbeek, Peter J.; Lubick, Kirk J.; Best, Sonja M.; Stoyanov, Cristina T.; Law, Lok Man J.; Yuan, Zhenghong; Rice, Charles M.; MacDonald, Margaret R.

2011-01-01

Viruses in the Flavivirus genus of the Flaviviridae family are arthropod-transmitted and contribute to staggering numbers of human infections and significant deaths annually across the globe. To identify cellular factors with antiviral activity against flaviviruses, we screened a cDNA library using an iterative approach. We identified a mammalian Hsp40 chaperone protein (DNAJC14) that when overexpressed was able to mediate protection from yellow fever virus (YFV)-induced cell death. Further studies revealed that DNAJC14 inhibits YFV at the step of viral RNA replication. Since replication of bovine viral diarrhea virus (BVDV), a member of the related Pestivirus genus, is also known to be modulated by DNAJC14, we tested the effect of this host factor on diverse Flaviviridae family members. Flaviviruses, including the pathogenic Asibi strain of YFV, Kunjin, and tick-borne Langat virus, as well as a Hepacivirus, hepatitis C virus (HCV), all were inhibited by overexpression of DNAJC14. Mutagenesis showed that both the J-domain and the C-terminal domain, which mediates self-interaction, are required for anti-YFV activity. We found that DNAJC14 does not block YFV nor HCV NS2-3 cleavage, and using non-inhibitory mutants demonstrate that DNAJC14 is recruited to YFV replication complexes. Immunofluorescence analysis demonstrated that endogenous DNAJC14 rearranges during infection and is found in replication complexes identified by dsRNA staining. Interestingly, silencing of endogenous DNAJC14 results in impaired YFV replication suggesting a requirement for DNAJC14 in YFV replication complex assembly. Finally, the antiviral activity of overexpressed DNAJC14 occurs in a time- and dose-dependent manner. DNAJC14 overexpression may disrupt the proper stoichiometry resulting in inhibition, which can be overcome upon restoration of the optimal ratios due to the accumulation of viral nonstructural proteins. Our findings, together with previously published work, suggest that the members of the Flaviviridae family have evolved in unique and important ways to interact with this host Hsp40 chaperone molecule. PMID:21249176

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen

PubMed Central

2011-01-01

Background The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. Results We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. Conclusions We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy. PMID:22014111

Flavivirus NS3 and NS5 proteins interaction network: a high-throughput yeast two-hybrid screen.

PubMed

Le Breton, Marc; Meyniel-Schicklin, Laurène; Deloire, Alexandre; Coutard, Bruno; Canard, Bruno; de Lamballerie, Xavier; Andre, Patrice; Rabourdin-Combe, Chantal; Lotteau, Vincent; Davoust, Nathalie

2011-10-20

The genus Flavivirus encompasses more than 50 distinct species of arthropod-borne viruses, including several major human pathogens, such as West Nile virus, yellow fever virus, Japanese encephalitis virus and the four serotypes of dengue viruses (DENV type 1-4). Each year, flaviviruses cause more than 100 million infections worldwide, some of which lead to life-threatening conditions such as encephalitis or haemorrhagic fever. Among the viral proteins, NS3 and NS5 proteins constitute the major enzymatic components of the viral replication complex and are essential to the flavivirus life cycle. We report here the results of a high-throughput yeast two-hybrid screen to identify the interactions between human host proteins and the flavivirus NS3 and NS5 proteins. Using our screen results and literature curation, we performed a global analysis of the NS3 and NS5 cellular targets based on functional annotation with the Gene Ontology features. We finally created the first flavivirus NS3 and NS5 proteins interaction network and analysed the topological features of this network. Our proteome mapping screen identified 108 human proteins interacting with NS3 or NS5 proteins or both. The global analysis of the cellular targets revealed the enrichment of host proteins involved in RNA binding, transcription regulation, vesicular transport or innate immune response regulation. We proposed that the selective disruption of these newly identified host/virus interactions could represent a novel and attractive therapeutic strategy in treating flavivirus infections. Our virus-host interaction map provides a basis to unravel fundamental processes about flavivirus subversion of the host replication machinery and/or immune defence strategy.

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication.

PubMed

Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E; Miorin, Lisa; Johnson, Jeffrey R; Krogan, Nevan J; Basler, Christopher F; Freiberg, Alexander N; Rajsbaum, Ricardo

2017-09-15

Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV. Copyright © 2017 American Society for Microbiology.

The Host E3-Ubiquitin Ligase TRIM6 Ubiquitinates the Ebola Virus VP35 Protein and Promotes Virus Replication

PubMed Central

Bharaj, Preeti; Atkins, Colm; Luthra, Priya; Giraldo, Maria Isabel; Dawes, Brian E.; Miorin, Lisa; Johnson, Jeffrey R.; Krogan, Nevan J.; Basler, Christopher F.; Freiberg, Alexander N.

2017-01-01

ABSTRACT Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic virus that causes severe hemorrhagic fever in humans and is responsible for epidemics throughout sub-Saharan, central, and West Africa. The EBOV genome encodes VP35, an important viral protein involved in virus replication by acting as an essential cofactor of the viral polymerase as well as a potent antagonist of the host antiviral type I interferon (IFN-I) system. By using mass spectrometry analysis and coimmunoprecipitation assays, we show here that VP35 is ubiquitinated on lysine 309 (K309), a residue located on its IFN antagonist domain. We also found that VP35 interacts with TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family. We recently reported that TRIM6 promotes the synthesis of unanchored K48-linked polyubiquitin chains, which are not covalently attached to any protein, to induce efficient antiviral IFN-I-mediated responses. Consistent with this notion, VP35 also associated noncovalently with polyubiquitin chains and inhibited TRIM6-mediated IFN-I induction. Intriguingly, we also found that TRIM6 enhances EBOV polymerase activity in a minigenome assay and TRIM6 knockout cells have reduced replication of infectious EBOV, suggesting that VP35 hijacks TRIM6 to promote EBOV replication through ubiquitination. Our work provides evidence that TRIM6 is an important host cellular factor that promotes EBOV replication, and future studies will focus on whether TRIM6 could be targeted for therapeutic intervention against EBOV infection. IMPORTANCE EBOV belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans and other mammals with high mortality rates (40 to 90%). Because of its high pathogenicity and lack of licensed antivirals and vaccines, EBOV is listed as a tier 1 select-agent risk group 4 pathogen. An important mechanism for the severity of EBOV infection is its suppression of innate immune responses. The EBOV VP35 protein contributes to pathogenesis, because it serves as an essential cofactor of the viral polymerase as well as a potent antagonist of innate immunity. However, how VP35 function is regulated by host cellular factors is poorly understood. Here, we report that the host E3-ubiquitin ligase TRIM6 promotes VP35 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM6, as a potential target in the development of antiviral drugs against EBOV. PMID:28679761

Turnip mosaic virus Moves Systemically through Both Phloem and Xylem as Membrane-Associated Complexes1

PubMed Central

Zheng, Huanquan

2015-01-01

Plant viruses move systemically in plants through the phloem. They move as virions or as ribonucleic protein complexes, although it is not clear what these complexes are made of. The approximately 10-kb RNA genome of Turnip mosaic virus (TuMV) encodes a membrane protein, known as 6K2, that induces endomembrane rearrangements for the formation of viral replication factories. These factories take the form of vesicles that contain viral RNA (vRNA) and viral replication proteins. In this study, we report the presence of 6K2-tagged vesicles containing vRNA and the vRNA-dependent RNA polymerase in phloem sieve elements and in xylem vessels. Transmission electron microscopy observations showed the presence in the xylem vessels of vRNA-containing vesicles that were associated with viral particles. Stem-girdling experiments, which leave xylem vessels intact but destroy the surrounding tissues, confirmed that TuMV could establish a systemic infection of the plant by going through xylem vessels. Phloem sieve elements and xylem vessels from Potato virus X-infected plants also contained lipid-associated nonencapsidated vRNA, indicating that the presence of membrane-associated ribonucleic protein complexes in the phloem and xylem may not be limited to TuMV. Collectively, these studies indicate that viral replication factories could end up in the phloem and the xylem. PMID:25717035

Development of viable TAP-tagged dengue virus for investigation of host-virus interactions in viral replication.

PubMed

Poyomtip, Teera; Hodge, Kenneth; Matangkasombut, Ponpan; Sakuntabhai, Anavaj; Pisitkun, Trairak; Jirawatnotai, Siwanon; Chimnaronk, Sarin

2016-03-01

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for life-threatening dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The viral replication machinery containing the core non-structural protein 5 (NS5) is implicated in severe dengue symptoms but molecular details remain obscure. To date, studies seeking to catalogue and characterize interaction networks between viral NS5 and host proteins have been limited to the yeast two-hybrid system, computational prediction and co-immunoprecipitation (IP) of ectopically expressed NS5. However, these traditional approaches do not reproduce a natural course of infection in which a number of DENV NS proteins colocalize and tightly associate during the replication process. Here, we demonstrate the development of a recombinant DENV that harbours a TAP tag in NS5 to study host-virus interactions in vivo. We show that our engineered DENV was infective in several human cell lines and that the tags were stable over multiple viral passages, suggesting negligible structural and functional disturbance of NS5. We further provide proof-of-concept for the use of rationally tagged virus by revealing a high confidence NS5 interaction network in human hepatic cells. Our analysis uncovered previously unrecognized hnRNP complexes and several low-abundance fatty acid metabolism genes, which have been implicated in the viral life cycle. This study sets a new standard for investigation of host-flavivirus interactions.

A peptide derived from enzymatic digestion of globulins from amaranth shows strong affinity binding to the replication origin of Tomato yellow leaf curl virus reducing viral replication in Nicotiana benthamiana.

PubMed

Mendoza-Figueroa, J S; Kvarnheden, A; Méndez-Lozano, J; Rodríguez-Negrete, E-A; Arreguín-Espinosa de Los Monteros, R; Soriano-García, M

2018-02-01

Tomato yellow leaf curl virus (TYLCV; genus Begomovirus; family Geminiviridae) infects mainly plants of the family Solanaceae, and the infection induces curling and chlorosis of leaves, dwarfing of the whole plant, and reduced fruit production. Alternatives for direct control of TYLCV and other geminiviruses have been reported, for example, the use of esterified whey proteins, peptide aptamer libraries or artificial zinc finger proteins. The two latter alternatives affect directly the replication of TYLCV as well as of other geminiviruses because the replication structures and sequences are highly conserved within this virus family. Because peptides and proteins offer a potential solution for virus replication control, in this study we show the isolation, biochemical characterization and antiviral activity of a peptide derived from globulins of amaranth seeds (Amaranthus hypochondriacus) that binds to the replication origin sequence (OriRep) of TYLCV and affects viral replication with a consequent reduction of disease symptoms in Nicotiana benthamiana. Aromatic peptides obtained from papain digests of extracted globulins and albumins of amaranth were tested by intrinsic fluorescent titration and localized surface resonance plasmon to analyze their binding affinity to OriRep of TYLCV. The peptide AmPep1 (molecular weight 2.076 KDa) showed the highest affinity value (Kd = 1.8 nM) for OriRep. This peptide shares a high amino acid similarity with a part of an amaranth 11S globulin, and the strong affinity of AmPep1 could be explained by the presence of tryptophan and lysine facilitating interaction with the secondary structure of OriRep. In order to evaluate the effect of the peptide on in vitro DNA synthesis, rolling circle amplification (RCA) was performed using as template DNA from plants infected with TYLCV or another begomovirus, pepper huasteco yellow vein virus (PHYVV), and adding AmPep1 peptide at different concentrations. The results showed a decrease in DNA synthesis of both viruses at increasing concentrations of AmPep1. To further confirm the antiviral activity of the peptide in vivo, AmPep1 was infiltrated into leaves of N. benthamiana plants previously infected with TYLCV. Plants treated with AmPep1 showed a significant decrease in virus titer compared with untreated N. benthamiana plants as well as reduced symptom progression due to the effect of AmPep1 curtailing TYLCV replication in the plant. The peptide also showed antiviral activity in plants infected with PHYVV. This is the first report, in which a peptide is directly used for DNA virus control in plants, supplied as exogenous application and without generation of transgenic lines. Copyright © 2018 Elsevier Inc. All rights reserved.

Understanding the causes and consequences of measles virus persistence

PubMed Central

Griffin, Diane E.; Lin, Wen-Hsuan W.; Nelson, Ashley N.

2018-01-01

Measles is an acute systemic viral disease with initial amplification of infection in lymphoid tissue and subsequent spread over 10–14 days to multiple organs. Failure of the innate response to control initial measles virus (MeV) replication is associated with the ability of MeV to inhibit the induction of type I interferon and interferon-stimulated antiviral genes. Rather, the innate response is characterized by the expression of proteins regulated by nuclear factor kappa B and the inflammasome. With eventual development of the adaptive response, the rash appears with immune cell infiltration into sites of virus replication to initiate the clearance of infectious virus. However, MeV RNA is cleared much more slowly than recoverable infectious virus and remains present in lymphoid tissue for at least 6 months after infection. Persistence of viral RNA and protein suggests persistent low-level replication in lymphoid tissue that may facilitate maturation of the immune response, resulting in lifelong protection from reinfection, while persistence in other tissues (for example, the nervous system) may predispose to development of late disease such as subacute sclerosing panencephalitis. Further studies are needed to identify mechanisms of viral clearance and to understand the relationship between persistence and development of lifelong immunity. PMID:29560260

Understanding the causes and consequences of measles virus persistence.

PubMed

Griffin, Diane E; Lin, Wen-Hsuan W; Nelson, Ashley N

2018-01-01

Measles is an acute systemic viral disease with initial amplification of infection in lymphoid tissue and subsequent spread over 10-14 days to multiple organs. Failure of the innate response to control initial measles virus (MeV) replication is associated with the ability of MeV to inhibit the induction of type I interferon and interferon-stimulated antiviral genes. Rather, the innate response is characterized by the expression of proteins regulated by nuclear factor kappa B and the inflammasome. With eventual development of the adaptive response, the rash appears with immune cell infiltration into sites of virus replication to initiate the clearance of infectious virus. However, MeV RNA is cleared much more slowly than recoverable infectious virus and remains present in lymphoid tissue for at least 6 months after infection. Persistence of viral RNA and protein suggests persistent low-level replication in lymphoid tissue that may facilitate maturation of the immune response, resulting in lifelong protection from reinfection, while persistence in other tissues (for example, the nervous system) may predispose to development of late disease such as subacute sclerosing panencephalitis. Further studies are needed to identify mechanisms of viral clearance and to understand the relationship between persistence and development of lifelong immunity.

Fragile X mental retardation protein stimulates ribonucleoprotein assembly of influenza A virus

NASA Astrophysics Data System (ADS)

Zhou, Zhuo; Cao, Mengmeng; Guo, Yang; Zhao, Lili; Wang, Jingfeng; Jia, Xue; Li, Jianguo; Wang, Conghui; Gabriel, Gülsah; Xue, Qinghua; Yi, Yonghong; Cui, Sheng; Jin, Qi; Wang, Jianwei; Deng, Tao

2014-02-01

The ribonucleoprotein (RNP) of the influenza A virus is responsible for the transcription and replication of viral RNA in the nucleus. These processes require interplay between host factors and RNP components. Here, we report that the Fragile X mental retardation protein (FMRP) targets influenza virus RNA synthesis machinery and facilitates virus replication both in cell culture and in mice. We demonstrate that FMRP transiently associates with viral RNP and stimulates viral RNP assembly through RNA-mediated interaction with the nucleoprotein. Furthermore, the KH2 domain of FMRP mediates its association with the nucleoprotein. A point mutation (I304N) in the KH2 domain, identified from a Fragile X syndrome patient, disrupts the FMRP-nucleoprotein association and abolishes the ability of FMRP to participate in viral RNP assembly. We conclude that FMRP is a critical host factor used by influenza viruses to facilitate viral RNP assembly. Our observation reveals a mechanism of influenza virus RNA synthesis and provides insights into FMRP functions.

Novel Nonreplicating Vaccinia Virus Vector Enhances Expression of Heterologous Genes and Suppresses Synthesis of Endogenous Viral Proteins.

PubMed

Wyatt, Linda S; Xiao, Wei; Americo, Jeffrey L; Earl, Patricia L; Moss, Bernard

2017-06-06

Viruses are used as expression vectors for protein synthesis, immunology research, vaccines, and therapeutics. Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels. On the other hand, competition of approximately 200 viral genes with the target gene for expression and immune recognition may be disadvantageous. We describe a vaccinia virus (VACV) vector that uses an early promoter to express the bacteriophage T7 RNA polymerase; has the A23R intermediate transcription factor gene deleted, thereby restricting virus replication to complementing cells; and has a heterologous gene regulated by a T7 promoter. In noncomplementing cells, viral early gene expression and DNA replication occurred normally but synthesis of intermediate and late proteins was prevented. Nevertheless, the progeny viral DNA provided templates for abundant expression of heterologous genes regulated by a T7 promoter. Selective expression of the Escherichia coli lac repressor gene from an intermediate promoter reduced transcription of the heterologous gene specifically in complementing cells, where large amounts might adversely impact VACV replication. Expression of heterologous proteins mediated by the A23R deletion vector equaled that of a replicating VACV, was higher than that of a nonreplicating modified vaccinia virus Ankara (MVA) vector used for candidate vaccines in vitro and in vivo , and was similarly immunogenic in mice. Unlike the MVA vector, the A23R deletion vector still expresses numerous early genes that can restrict immunogenicity as demonstrated here by the failure of the prototype vector to induce interferon alpha. By deleting immunomodulatory genes, we anticipate further improvements in the system. IMPORTANCE Vaccines provide an efficient and effective way of preventing infectious diseases. Nevertheless, new and better vaccines are needed. Vaccinia virus, which was used successfully as a live vaccine to eradicate smallpox, has been further attenuated and adapted as a recombinant vector for immunization against other pathogens. However, since the initial description of this vector system, only incremental improvements largely related to safety have been implemented. Here we described novel modifications of the platform that increased expression of the heterologous target gene and decreased expression of endogenous vaccinia virus genes while providing safety by preventing replication of the candidate vaccine except in complementing cells used for vector propagation. Copyright © 2017 Wyatt et al.

Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain.

PubMed

Mochizuki, Tomofumi; Hirai, Katsuyuki; Kanda, Ayami; Ohnishi, Jun; Ohki, Takehiro; Tsuda, Shinya

2009-08-01

The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the second putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.

A Brief Review of West Nile Virus Biology.

PubMed

Londono-Renteria, Berlin; Colpitts, Tonya M

2016-01-01

West Nile virus (WNV) is an arbovirus with increased global incidence in the last decade. It is also a major cause of human encephalitis in the USA. WNV is an arthropod-transmitted virus that mainly affects birds but humans become infected as incidental dead-end hosts which can cause outbreaks in naïve populations. The main vectors of WNV are mosquitoes of the genus Culex, which preferentially feed on birds. As in many other arboviruses, the characteristics that allow Flaviviruses like WNV to replicate and transmit to different hosts are encrypted in their genome, which also contains information for the production of structural and nonstructural proteins needed for host cell infection. WNV and other Flaviviruses have developed different strategies to establish infection, replication, and successful transmission. Most of these strategies include the diversion of the host's immune responses away from the virus. In this review, we describe the molecular structure and protein function of WNV with emphasis on protein involvement in the modulation of antiviral immune responses.

Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain

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

Mochizuki, Tomofumi; Hirai, Katsuyuki; Kanda, Ayami

2009-08-01

The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the secondmore » putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.« less

HSP90 Protects the Human T-Cell Leukemia Virus Type 1 (HTLV-1) Tax Oncoprotein from Proteasomal Degradation To Support NF-κB Activation and HTLV-1 Replication

PubMed Central

Gao, Linlin

2013-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 genome encodes the Tax protein that plays essential regulatory roles in HTLV-1 replication and oncogenic transformation of T lymphocytes. Despite intensive study of Tax, how Tax interfaces with host signaling pathways to regulate virus replication and drive T-cell proliferation and immortalization remains poorly understood. To gain new insight into the mechanisms of Tax function and regulation, we used tandem affinity purification and mass spectrometry to identify novel cellular Tax-interacting proteins. This screen identified heat shock protein 90 (HSP90) as a new binding partner of Tax. The interaction between HSP90 and Tax was validated by coimmunoprecipitation assays, and colocalization between the two proteins was observed by confocal microscopy. Treatment of HTLV-1-transformed cells with the HSP90 inhibitor 17-DMAG elicited proteasomal degradation of Tax in the nuclear matrix with concomitant inhibition of NF-κB and HTLV-1 long terminal repeat (LTR) activation. Knockdown of HSP90 by lentiviral shRNAs similarly provoked a loss of Tax protein in HTLV-1-transformed cells. Finally, treatment of HTLV-1-transformed cell lines with 17-DMAG suppressed HTLV-1 replication and promoted apoptotic cell death. Taken together, our results reveal that Tax is a novel HSP90 client protein and HSP90 inhibitors may exert therapeutic benefits for ATL and HAM/TSP patients. PMID:24109220

Tomato yellow leaf curl virus C4 protein is a determinant of disease phenotype in tomato

USDA-ARS?s Scientific Manuscript database

Tomato yellow leaf curl virus (TYLCV) is a monopartite begomovirus. Its genome contains six open reading frames, with V1 and V2 in sense, and C1 to C4 in complementary orientation. The functions of V1 and V2 are for coat protein and pre-coat, respectively. C1 is for virus replication, C2 for trans-a...

Timing Is Everything: Coordinated Control of Host Shutoff by Influenza A Virus NS1 and PA-X Proteins

PubMed Central

Khaperskyy, Denys A.

2015-01-01

Like all viruses, influenza viruses (IAVs) use host translation machinery to decode viral mRNAs. IAVs ensure efficient translation of viral mRNAs through host shutoff, a process whereby viral proteins limit the accumulation of host proteins through subversion of their biogenesis. Despite its small genome, the virus deploys multiple host shutoff mechanisms at different stages of infection, thereby ensuring successful replication while limiting the communication of host antiviral responses. In this Gem, we review recent data on IAV host shutoff proteins, frame the outstanding questions in the field, and propose a temporally coordinated model of IAV host shutoff. PMID:25878098

Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes.

PubMed

Sharma, Manish; Bhattacharyya, Sankar; Nain, Minu; Kaur, Manpreet; Sood, Vikas; Gupta, Vishal; Khasa, Renu; Abdin, Malik Z; Vrati, Sudhanshu; Kalia, Manjula

2014-09-01

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.

Hepatitis C virus inhibitor synergism suggests multistep interactions between heat-shock protein 90 and hepatitis C virus replication

PubMed Central

Kubota, Naoko; Nomoto, Masataka; Hwang, Gi-Wook; Watanabe, Toshihiko; Kohara, Michinori; Wakita, Takaji; Naganuma, Akira; Kuge, Shusuke

2016-01-01

AIM: To address the effect of heat-shock protein 90 (HSP90) inhibitors on the release of the hepatitis C virus (HCV), a cell culture-derived HCV (JFH1/HCVcc) from Huh-7 cells was examined. METHODS: We quantified both the intracellular and extracellular (culture medium) levels of the components (RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A (CsA) or interferon. Finally, we statistically examined the combined effect of radicicol and CsA using the combination index (CI) and graphical representation proposed by Chou and Talalay. RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor (CsA or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy (CI < 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release. PMID:26925202

Antiviral activity of silymarin against chikungunya virus

PubMed Central

Lani, Rafidah; Hassandarvish, Pouya; Chiam, Chun Wei; Moghaddam, Ehsan; Chu, Justin Jang Hann; Rausalu, Kai; Merits, Andres; Higgs, Stephen; Vanlandingham, Dana; Abu Bakar, Sazaly; Zandi, Keivan

2015-01-01

The mosquito-borne chikungunya virus (CHIKV) causes chikungunya fever, with clinical presentations such as severe back and small joint pain, and debilitating arthritis associated with crippling pains that persist for weeks and even years. Although there are several studies to evaluate the efficacy of drugs against CHIKV, the treatment for chikungunya fever is mainly symptom-based and no effective licensed vaccine or antiviral are available. Here, we investigated the antiviral activity of three types of flavonoids against CHIKV in vitro replication. Three compounds: silymarin, quercetin and kaempferol were evaluated for their in vitro antiviral activities against CHIKV using a CHIKV replicon cell line and clinical isolate of CHIKV of Central/East African genotype. A cytopathic effect inhibition assay was used to determine their activities on CHIKV viral replication and quantitative reverse transcription PCR was used to calculate virus yield. Antiviral activity of effective compound was further investigated by evaluation of CHIKV protein expression using western blotting for CHIKV nsP1, nsP3, and E2E1 proteins. Briefly, silymarin exhibited significant antiviral activity against CHIKV, reducing both CHIKV replication efficiency and down-regulating production of viral proteins involved in replication. This study may have important consequence for broaden the chance of getting the effective antiviral for CHIKV infection. PMID:26078201

Efficient Translation of Epstein-Barr Virus (EBV) DNA Polymerase Contributes to the Enhanced Lytic Replication Phenotype of M81 EBV.

PubMed

Church, Trenton Mel; Verma, Dinesh; Thompson, Jacob; Swaminathan, Sankar

2018-03-15

Epstein-Barr virus (EBV) is linked to the development of both lymphoid and epithelial malignancies worldwide. The M81 strain of EBV, isolated from a Chinese patient with nasopharyngeal carcinoma (NPC), demonstrates spontaneous lytic replication and high-titer virus production in comparison to the prototype B95-8 EBV strain. Genetic comparisons of M81 and B95-8 EBVs were previously been performed in order to determine if the hyperlytic property of M81 is associated with sequence differences in essential lytic genes. EBV SM is an RNA-binding protein expressed during early lytic replication that is essential for virus production. We compared the functions of M81 SM and B95-8 SM and demonstrate that polymorphisms in SM do not contribute to the lytic phenotype of M81 EBV. However, the expression level of the EBV DNA polymerase protein was much higher in M81- than in B95-8-infected cells. The relative deficiency in the expression of B95-8 DNA polymerase was related to the B95-8 genome deletion, which truncates the BALF5 3' untranslated region (UTR). Similarly, the insertion of bacmid DNA into the widely used recombinant B95-8 bacmid creates an inefficient BALF5 3' UTR. We further showed that the while SM is required for and facilitates the efficient expression of both M81 and B95-8 mRNAs regardless of the 3' UTR, the BALF5 3' UTR sequence is important for BALF5 protein translation. These data indicate that the enhanced lytic replication and virus production of M81 compared to those of B95-8 are partly due to the robust translation of EBV DNA polymerase required for viral DNA replication due to a more efficient BALF5 3' UTR in M81. IMPORTANCE Epstein-Barr virus (EBV) infects more than 90% of the human population, but the incidence of EBV-associated tumors varies greatly in different parts of the world. Thus, understanding the connection between genetic polymorphisms from patient isolates of EBV, gene expression phenotypes, and disease is important and may help in developing antiviral therapy. This study examines potential causes of the enhanced lytic replicative properties of M81 EBV isolated from a nasopharyngeal carcinoma (NPC) patient and provides new evidence for the role of the BALF5 gene 3' UTR sequence in DNA polymerase protein expression during lytic replication. Variation in the gene structure of the DNA polymerase gene may therefore contribute to lytic virus reactivation and pathogenesis. Copyright © 2018 American Society for Microbiology.

Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication.

PubMed

Esser-Nobis, Katharina; Harak, Christian; Schult, Philipp; Kusov, Yuri; Lohmann, Volker

2015-08-01

Hepatitis A virus (HAV) and hepatitis C virus (HCV) are two positive-strand RNA viruses sharing a similar biology, but causing opposing infection outcomes, with HAV always being cleared and HCV establishing persistence in the majority of infections. To gain deeper insight into determinants of replication, persistence, and treatment, we established a homogenous cell-culture model allowing a thorough comparison of RNA replication of both viruses. By screening different human liver-derived cell lines with subgenomic reporter replicons of HAV as well as of different HCV genotypes, we found that Huh7-Lunet cells supported HAV- and HCV-RNA replication with similar efficiency and limited interference between both replicases. HAV and HCV replicons were similarly sensitive to interferon (IFN), but differed in their ability to establish persistent replication in cell culture. In contrast to HCV, HAV replicated independently from microRNA-122 and phosphatidylinositol 4-kinase IIIα and β (PI4KIII). Both viruses were efficiently inhibited by cyclosporin A and NIM811, a nonimmunosuppressive analog thereof, suggesting an overlapping dependency on cyclophilins for replication. However, analysis of a broader set of inhibitors revealed that, in contrast to HCV, HAV does not depend on cyclophilin A, but rather on adenosine-triphosphate-binding cassette transporters and FK506-binding proteins. Finally, silibinin, but not its modified intravenous formulation, efficiently inhibited HAV genome replication in vitro, suggesting oral silibinin as a potential therapeutic option for HAV infections. We established a cell-culture model enabling comparative studies on RNA replication of HAV and HCV in a homogenous cellular background with comparable replication efficiency. We thereby identified new host cell targets and potential treatment options for HAV and set the ground for future studies to unravel determinants of clearance and persistence. © 2015 by the American Association for the Study of Liver Diseases.

Coordination of Hepatitis C Virus Assembly by Distinct Regulatory Regions in Nonstructural Protein 5A

PubMed Central

Zayas, Margarita; Long, Gang; Madan, Vanesa; Bartenschlager, Ralf

2016-01-01

Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles. PMID:26727512

A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

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

Mourez, Thomas; APHP, GH Saint-Louis-Lariboisiere, Laboratoire de Bacteriologie-Virologie, F-75010 Paris; Universite Paris 7 Denis Diderot, F-75010 Paris

2011-10-25

We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimericmore » particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.« less

Cell lines that support replication of a novel herpes simplex virus 1 U{sub L}31 deletion mutant can properly target U{sub L}34 protein to the nuclear rim in the absence of U{sub L}31

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

Liang Li; Tanaka, Michiko; Kawaguchi, Yasushi

2004-11-10

Previous results indicated that the herpes simplex virus 1 (HSV-1) U{sub L}31 gene is necessary and sufficient for localization of the U{sub L}34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U{sub L}31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Veromore » cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U{sub L}31 gene. The replication of the U{sub L}31 deletion virus was restored on U{sub L}31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U{sub L}34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U{sub L}34 protein localized at the nuclear membrane in rabbit skin cells, and U{sub L}31 complementing CV1 cells infected with the U{sub L}31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U{sub L}34 protein to the nuclear membrane. We speculate that this function partially complements that of U{sub L}31 and may explain why U{sub L}31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.« less

Gene I mutants of peanut chlorotic streak virus, a caulimovirus, replicate in plants but do not move from cell to cell.

PubMed Central

Ducasse, D A; Mushegian, A R; Shepherd, R J

1995-01-01

Gene I of peanut chlorotic streak virus (PCISV), a caulimovirus, is homologous to gene I of other caulimoviruses and may encode a protein for virus movement. To evaluate the function of gene I, several mutations were created in this gene of an infectious, partially redundant clone of PCISV. Constructs with an in-frame deletion and a single amino acid substitution in gene I were not infectious. To test for replication of these mutants in primarily infected cells, an immunosorbent PCR technique was devised. Virus particles formed by mutants in plants were recovered by binding to antivirus antibodies on a solid matrix and DNase treated to discriminate against residual inoculum, and DNA of trapped virions was subjected to PCR amplification. Gene I mutants were shown to direct formation of encapsidated DNA as revealed by a PCR product. Control gene V mutants (reverse transcriptase essential for replication) did not yield a PCR product. Quantitative PCR allowed estimation of the proportion of cells initially infected by gene I mutants and the amount of extractable virus per cell. It is concluded that PCISV gene I encodes a movement protein and that the immunoselection-PCR technique is useful in studying subliminal virus infection in plants. PMID:7543587

Identification, Characterization, and Application of the Replicon Region of the Halophilic Temperate Sphaerolipovirus SNJ1.

PubMed

Wang, Yuchen; Sima, Linshan; Lv, Jie; Huang, Suiyuan; Liu, Ying; Wang, Jiao; Krupovic, Mart; Chen, Xiangdong

2016-07-15

The temperate haloarchaeal virus SNJ1 displays lytic and lysogenic life cycles. During the lysogenic cycle, the virus resides in its host, Natrinema sp. strain J7-1, in the form of an extrachromosomal circular plasmid, pHH205. In this study, a 3.9-kb region containing seven predicted genes organized in two operons was identified as the minimal replicon of SNJ1. Only RepA, encoded by open reading frame 11-12 (ORF11-12), was found to be essential for replication, and its expression increased during the lytic cycle. Sequence analysis suggested that RepA is a distant homolog of HUH endonucleases, a superfamily that includes rolling-circle replication initiation proteins from various viruses and plasmids. In addition to RepA, two genetic elements located within both termini of the 3.9-kb replicon were also required for SNJ1 replication. SNJ1 genome and SNJ1 replicon-based shuttle vectors were present at 1 to 3 copies per chromosome. However, the deletion of ORF4 significantly increased the SNJ1 copy number, suggesting that the product of ORF4 is a negative regulator of SNJ1 abundance. Shuttle vectors based on the SNJ1 replicon were constructed and validated for stable expression of heterologous proteins, both in J7 derivatives and in Natrinema pallidum JCM 8980(T), suggesting their broad applicability as genetic tools for Natrinema species. Archaeal viruses exhibit striking morphological diversity and unique gene content. In this study, the minimal replicon of the temperate haloarchaeal virus SNJ1 was identified. A number of ORFs and genetic elements controlling virus genome replication, maintenance, and copy number were characterized. In addition, based on the replicon, a novel expression shuttle vector has been constructed and validated for protein expression and purification in Natrinema sp. CJ7 and Natrinema pallidum JCM 8980(T) This study not only provided mechanistic and functional insights into SNJ1 replication but also led to the development of useful genetic tools to investigate SNJ1 and other viruses infecting Natrinema species as well as their hosts. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Recent advances in the identification of the host factors involved in dengue virus replication.

PubMed

Wang, Yi; Zhang, Ping

2017-02-01

Dengue virus (DENV) belongs to the genus Flavivirus of the family Flaviviridae and it is primarily transmitted via Aedes aegypti and Aedes albopictus mosquitoes. The life cycle of DENV includes attachment, endocytosis, protein translation, RNA synthesis, assembly, egress, and maturation. Recent researches have indicated that a variety of host factors, including cellular proteins and microRNAs, positively or negatively regulate the DENV replication process. This review summarizes the latest findings (from 2014 to 2016) in the identification of the host factors involved in the DENV life cycle and Dengue infection.

Immunogenic HSV-mediated oncolysis shapes the antitumor immune response and contributes to therapeutic efficacy.

PubMed

Workenhe, Samuel T; Simmons, Graydon; Pol, Jonathan G; Lichty, Brian D; Halford, William P; Mossman, Karen L

2014-01-01

Within the oncolytic virus field, the extent of virus replication that is essential for immune stimulation to control tumor growth remains unresolved. Using infected cell protein 0 (ICP0)-defective oncolytic Herpes simplex virus type 1 (HSV-1) and HSV-2 viruses (dICP0 and dNLS) that show differences in their in vitro replication and cytotoxicity, we investigated the inherent features of oncolytic HSV viruses that are required for potent antitumor activity. In vitro, the HSV-2 vectors showed rapid cytotoxicity despite lower viral burst sizes compared to HSV-1 vectors. In vivo, although both of the dICP0 vectors initially replicated to a similar level, HSV-1 dICP0 was rapidly cleared from the tumors. In spite of this rapid clearance, HSV-1 dICP0 treatment conferred significant survival benefit. HSV-1 dICP0-treated tumors showed significantly higher levels of danger-associated molecular patterns that correlated with higher numbers of antigen-presenting cells within the tumor and increased antigen-specific CD8+ T-cell levels in the peripheral blood. This study suggests that, at least in the context of oncolytic HSV, the initial stages of immunogenic virus replication leading to activation of antitumor immunity are more important than persistence of a replicating virus within the tumor. This knowledge provides important insight for the design of therapeutically successful oncolytic viruses.

Maribavir Inhibits Epstein-Barr Virus Transcription through the EBV Protein Kinase

PubMed Central

Whitehurst, Christopher B.; Sanders, Marcia K.; Law, Mankit; Wang, Fu-Zhang; Xiong, Jie; Dittmer, Dirk P.

2013-01-01

Maribavir (MBV) inhibits Epstein-Barr virus (EBV) replication and the enzymatic activity of the viral protein kinase BGLF4. MBV also inhibits expression of multiple EBV transcripts during EBV lytic infection. Here we demonstrate, with the use of a BGLF4 knockout virus, that effects of MBV on transcription take place primarily through inhibition of BGLF4. MBV inhibits viral genome copy numbers and infectivity to levels similar to and exceeding levels produced by BGLF4 knockout virus. PMID:23449792

Characterization and Construction of Functional cDNA Clones of Pariacoto Virus, the First Alphanodavirus Isolated outside Australasia

PubMed Central

Johnson, Karyn N.; Zeddam, Jean-Louis; Ball, L. Andrew

2000-01-01

Pariacoto virus (PaV) was recently isolated in Peru from the Southern armyworm (Spodoptera eridania). PaV particles are isometric, nonenveloped, and about 30 nm in diameter. The virus has a bipartite RNA genome and a single major capsid protein with a molecular mass of 39.0 kDa, features that support its classification as a Nodavirus. As such, PaV is the first Alphanodavirus to have been isolated from outside Australasia. Here we report that PaV replicates in wax moth larvae and that PaV genomic RNAs replicate when transfected into cultured baby hamster kidney cells. The complete nucleotide sequences of both segments of the bipartite RNA genome were determined. The larger genome segment, RNA1, is 3,011 nucleotides long and contains a 973-amino-acid open reading frame (ORF) encoding protein A, the viral contribution to the RNA replicase. During replication, a 414-nucleotide long subgenomic RNA (RNA3) is synthesized which is coterminal with the 3′ end of RNA1. RNA3 contains a small ORF which could encode a protein of 90 amino acids similar to the B2 protein of other alphanodaviruses. RNA2 contains 1,311 nucleotides and encodes the 401 amino acids of the capsid protein precursor α. The amino acid sequences of the PaV capsid protein and the replicase subunit share 41 and 26% identity with homologous proteins of Flock house virus, the best characterized of the alphanodaviruses. These and other sequence comparisons indicate that PaV is evolutionarily the most distant of the alphanodaviruses described to date, consistent with its novel geographic origin. Although the PaV capsid precursor is cleaved into the two mature capsid proteins β and γ, the amino acid sequence at the cleavage site, which is Asn/Ala in all other alphanodaviruses, is Asn/Ser in PaV. To facilitate the investigation of PaV replication in cultured cells, we constructed plasmids that transcribed full-length PaV RNAs with authentic 5′ and 3′ termini. Transcription of these plasmids in cells recreated the replication of PaV RNA1 and RNA2, synthesis of subgenomic RNA3, and translation of viral proteins A and α. PMID:10799587

Characterization and construction of functional cDNA clones of Pariacoto virus, the first Alphanodavirus isolated outside Australasia.

PubMed

Johnson, K N; Zeddam, J L; Ball, L A

2000-06-01

Pariacoto virus (PaV) was recently isolated in Peru from the Southern armyworm (Spodoptera eridania). PaV particles are isometric, nonenveloped, and about 30 nm in diameter. The virus has a bipartite RNA genome and a single major capsid protein with a molecular mass of 39.0 kDa, features that support its classification as a Nodavirus. As such, PaV is the first Alphanodavirus to have been isolated from outside Australasia. Here we report that PaV replicates in wax moth larvae and that PaV genomic RNAs replicate when transfected into cultured baby hamster kidney cells. The complete nucleotide sequences of both segments of the bipartite RNA genome were determined. The larger genome segment, RNA1, is 3,011 nucleotides long and contains a 973-amino-acid open reading frame (ORF) encoding protein A, the viral contribution to the RNA replicase. During replication, a 414-nucleotide long subgenomic RNA (RNA3) is synthesized which is coterminal with the 3' end of RNA1. RNA3 contains a small ORF which could encode a protein of 90 amino acids similar to the B2 protein of other alphanodaviruses. RNA2 contains 1,311 nucleotides and encodes the 401 amino acids of the capsid protein precursor alpha. The amino acid sequences of the PaV capsid protein and the replicase subunit share 41 and 26% identity with homologous proteins of Flock house virus, the best characterized of the alphanodaviruses. These and other sequence comparisons indicate that PaV is evolutionarily the most distant of the alphanodaviruses described to date, consistent with its novel geographic origin. Although the PaV capsid precursor is cleaved into the two mature capsid proteins beta and gamma, the amino acid sequence at the cleavage site, which is Asn/Ala in all other alphanodaviruses, is Asn/Ser in PaV. To facilitate the investigation of PaV replication in cultured cells, we constructed plasmids that transcribed full-length PaV RNAs with authentic 5' and 3' termini. Transcription of these plasmids in cells recreated the replication of PaV RNA1 and RNA2, synthesis of subgenomic RNA3, and translation of viral proteins A and alpha.

Investigation of Molecular Mechanism of JC virus Viroporin Activity.

PubMed

Suzuki, Tadaki

2015-01-01

Viroporins are small and hydrophobic viral proteins that form pores on host cell membranes, and their expression can increase the permeability of cellular membranes and the production of progeny virus particles. JC virus (JCV) is the causative agent of progressive multifocal leukoenchephalopathy (PML). We demonstrate that JCV Agno, which is the small and hydrophobic protein, andincreases the plasma membrane permeability and virion release, acts as a viroporin. We also demonstrate that an interaction of Agno with a host cellular protein regulates the viroporin activity of Agno. These findings indicate a new paradigm in virus-host interactions regulating viroporin activity and viral replication.

Replication Cycle and Molecular Biology of the West Nile Virus

PubMed Central

Brinton, Margo A.

2013-01-01

West Nile virus (WNV) is a member of the genus Flavivirus in the family Flaviviridae. Flaviviruses replicate in the cytoplasm of infected cells and modify the host cell environment. Although much has been learned about virion structure and virion-endosomal membrane fusion, the cell receptor(s) used have not been definitively identified and little is known about the early stages of the virus replication cycle. Members of the genus Flavivirus differ from members of the two other genera of the family by the lack of a genomic internal ribosomal entry sequence and the creation of invaginations in the ER membrane rather than double-membrane vesicles that are used as the sites of exponential genome synthesis. The WNV genome 3' and 5' sequences that form the long distance RNA-RNA interaction required for minus strand initiation have been identified and contact sites on the 5' RNA stem loop for NS5 have been mapped. Structures obtained for many of the viral proteins have provided information relevant to their functions. Viral nonstructural protein interactions are complex and some may occur only in infected cells. Although interactions between many cellular proteins and virus components have been identified, the functions of most of these interactions have not been delineated. PMID:24378320

Zika Virus Antagonizes Type I Interferon Responses during Infection of Human Dendritic Cells

PubMed Central

Maddur, Mohan S.; O’Neal, Justin T.; Fedorova, Nadia B.; Puri, Vinita; Pulendran, Bali; Suthar, Mehul S.

2017-01-01

Zika virus (ZIKV) is an emerging mosquito-borne flavivirus that is causally linked to severe neonatal birth defects, including microcephaly, and is associated with Guillain-Barre syndrome in adults. Dendritic cells (DCs) are an important cell type during infection by multiple mosquito-borne flaviviruses, including dengue virus, West Nile virus, Japanese encephalitis virus, and yellow fever virus. Despite this, the interplay between ZIKV and DCs remains poorly defined. Here, we found human DCs supported productive infection by a contemporary Puerto Rican isolate with considerable variability in viral replication, but not viral binding, between DCs from different donors. Historic isolates from Africa and Asia also infected DCs with distinct viral replication kinetics between strains. African lineage viruses displayed more rapid replication kinetics and infection magnitude as compared to Asian lineage viruses, and uniquely induced cell death. Infection of DCs with both contemporary and historic ZIKV isolates led to minimal up-regulation of T cell co-stimulatory and MHC molecules, along with limited secretion of inflammatory cytokines. Inhibition of type I interferon (IFN) protein translation was observed during ZIKV infection, despite strong induction at the RNA transcript level and up-regulation of other host antiviral proteins. Treatment of human DCs with RIG-I agonist potently restricted ZIKV replication, while type I IFN had only modest effects. Mechanistically, we found all strains of ZIKV antagonized type I IFN-mediated phosphorylation of STAT1 and STAT2. Combined, our findings show that ZIKV subverts DC immunogenicity during infection, in part through evasion of type I IFN responses, but that the RLR signaling pathway is still capable of inducing an antiviral state, and therefore may serve as an antiviral therapeutic target. PMID:28152048

Oral or parenteral administration of replication-deficient adenoviruses expressing the measles virus haemagglutinin and fusion proteins: protective immune responses in rodents.

PubMed

Fooks, A R; Jeevarajah, D; Lee, J; Warnes, A; Niewiesk, S; ter Meulen, V; Stephenson, J R; Clegg, J C

1998-05-01

The genes encoding the measles virus (MV) haemagglutinin (H) and fusion (F) proteins were placed under the control of the human cytomegalovirus immediate early promoter in a replication-deficient adenovirus vector. Immunofluorescence and radioimmune precipitation demonstrated the synthesis of each protein and biological activity was confirmed by the detection of haemadsorption and fusion activities in infected cells. Oral as well as parenteral administration of the H-expressing recombinant adenovirus elicited a significant protective response in mice challenged with MV. While the F-expressing adenovirus failed to protect mice, cotton rats immunized with either the H- or F-expressing recombinant showed reduced MV replication in the lungs. Antibodies elicited in mice following immunization with either recombinant had no in vitro neutralizing activity, suggesting a protective mechanism involving a cell-mediated immune response. This study demonstrates the feasibility of using oral administration of adenovirus recombinants to induce protective responses to heterologous proteins.

DEAD-box RNA helicase DDX3X inhibits DENV replication via regulating type one interferon pathway.

PubMed

Li, Guanghao; Feng, Tingting; Pan, Wen; Shi, Xiaohong; Dai, Jianfeng

2015-01-02

Dengue virus (DENV) is a mosquito-borne virus that threatens approximately 2.5 billion people worldwide. Vaccines against DENV are currently unavailable. DEAD-box RNA helicases (DDXs) have been reported to participate in viral replication and host innate immune response. In the present study, we analyzed the role of 40 DDX proteins during DENV replication. Among these proteins, DDX3X showed antiviral effect against DENV infection. Viral replication significantly increased in DDX3X-silenced cells compared with the controls. The interferon (IFN)-β transcription level decreased during the early stage of DENV infection in DDX3X-silenced cells compared with that in the controls. DDX3X could stimulate IFN-β transcription through the IRF3 and the NFκB branches in DENV-infected cells. Our data imply that DDX3X, a member of DEAD-box RNA helicase, is necessary for IFN production and could inhibit DENV replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

PubMed

Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean

2002-07-01

The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

The Bombyx mori nucleopolyhedrovirus (BmNPV) ODV-E56 envelope protein is also a per os infectivity factor.

PubMed

Xiang, Xingwei; Chen, Lin; Guo, Aiqin; Yu, Shaofang; Yang, Rui; Wu, Xiaofeng

2011-01-01

The Bombyx mori nucleopolyhedrovirus (BmNPV) odv-e56 gene is a late gene and encodes an occlusion-derived virus (ODV)-specific envelope protein, ODV-E56. To determine its role in the BmNPV life cycle, an odv-e56 null virus, BmE56D, was constructed through homologous recombination. A repaired virus was also constructed, named BmE56DR. The production of budded virion (BV) and polyhedra, the replication of viral DNA, and the morphological of infected BmN cells were analyzed, revealing no significant difference among the BmE56D, the wild-type (WT), and the BmE56DR virus. Larval bioassays demonstrated that injection of BmE56D BV into the hemocoel could kill B. mori larvae as efficiently as repaired and WT viruses, however BmE56D was unable to infect the B. mori larvae when inoculated per os. Thus, these results indicated that ODV-E56 envelope protein of BmNPV is also a per os infectivity factor (PIF), but is not essential for virus replication. Copyright © 2010 Elsevier B.V. All rights reserved.

Hepatitis C Virus Infection Induces Autophagy as a Prosurvival Mechanism to Alleviate Hepatic ER-Stress Response

PubMed Central

Dash, Srikanta; Chava, Srinivas; Aydin, Yucel; Chandra, Partha K.; Ferraris, Pauline; Chen, Weina; Balart, Luis A.; Wu, Tong; Garry, Robert F.

2016-01-01

Hepatitis C virus (HCV) infection frequently leads to chronic liver disease, liver cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms by which HCV infection leads to chronic liver disease and HCC are not well understood. The infection cycle of HCV is initiated by the attachment and entry of virus particles into a hepatocyte. Replication of the HCV genome inside hepatocytes leads to accumulation of large amounts of viral proteins and RNA replication intermediates in the endoplasmic reticulum (ER), resulting in production of thousands of new virus particles. HCV-infected hepatocytes mount a substantial stress response. How the infected hepatocyte integrates the viral-induced stress response with chronic infection is unknown. The unfolded protein response (UPR), an ER-associated cellular transcriptional response, is activated in HCV infected hepatocytes. Over the past several years, research performed by a number of laboratories, including ours, has shown that HCV induced UPR robustly activates autophagy to sustain viral replication in the infected hepatocyte. Induction of the cellular autophagy response is required to improve survival of infected cells by inhibition of cellular apoptosis. The autophagy response also inhibits the cellular innate antiviral program that usually inhibits HCV replication. In this review, we discuss the physiological implications of the HCV-induced chronic ER-stress response in the liver disease progression. PMID:27223299

Human Heat shock protein 40 (Hsp40/DnaJB1) promotes influenza A virus replication by assisting nuclear import of viral ribonucleoproteins.

PubMed

Batra, Jyoti; Tripathi, Shashank; Kumar, Amrita; Katz, Jacqueline M; Cox, Nancy J; Lal, Renu B; Sambhara, Suryaprakash; Lal, Sunil K

2016-01-11

A unique feature of influenza A virus (IAV) life cycle is replication of the viral genome in the host cell nucleus. The nuclear import of IAV genome is an indispensable step in establishing virus infection. IAV nucleoprotein (NP) is known to mediate the nuclear import of viral genome via its nuclear localization signals. Here, we demonstrate that cellular heat shock protein 40 (Hsp40/DnaJB1) facilitates the nuclear import of incoming IAV viral ribonucleoproteins (vRNPs) and is important for efficient IAV replication. Hsp40 was found to interact with NP component of IAV RNPs during early stages of infection. This interaction is mediated by the J domain of Hsp40 and N-terminal region of NP. Drug or RNAi mediated inhibition of Hsp40 resulted in reduced nuclear import of IAV RNPs, diminished viral polymerase function and attenuates overall viral replication. Hsp40 was also found to be required for efficient association between NP and importin alpha, which is crucial for IAV RNP nuclear translocation. These studies demonstrate an important role for cellular chaperone Hsp40/DnaJB1 in influenza A virus life cycle by assisting nuclear trafficking of viral ribonucleoproteins.

Human Heat shock protein 40 (Hsp40/DnaJB1) promotes influenza A virus replication by assisting nuclear import of viral ribonucleoproteins

PubMed Central

Batra, Jyoti; Tripathi, Shashank; Kumar, Amrita; Katz, Jacqueline M.; Cox, Nancy J.; Lal, Renu B.; Sambhara, Suryaprakash; Lal, Sunil K.

2016-01-01

A unique feature of influenza A virus (IAV) life cycle is replication of the viral genome in the host cell nucleus. The nuclear import of IAV genome is an indispensable step in establishing virus infection. IAV nucleoprotein (NP) is known to mediate the nuclear import of viral genome via its nuclear localization signals. Here, we demonstrate that cellular heat shock protein 40 (Hsp40/DnaJB1) facilitates the nuclear import of incoming IAV viral ribonucleoproteins (vRNPs) and is important for efficient IAV replication. Hsp40 was found to interact with NP component of IAV RNPs during early stages of infection. This interaction is mediated by the J domain of Hsp40 and N-terminal region of NP. Drug or RNAi mediated inhibition of Hsp40 resulted in reduced nuclear import of IAV RNPs, diminished viral polymerase function and attenuates overall viral replication. Hsp40 was also found to be required for efficient association between NP and importin alpha, which is crucial for IAV RNP nuclear translocation. These studies demonstrate an important role for cellular chaperone Hsp40/DnaJB1 in influenza A virus life cycle by assisting nuclear trafficking of viral ribonucleoproteins. PMID:26750153

Cyclooxygenase activity is important for efficient replication of mouse hepatitis virus at an early stage of infection

PubMed Central

Raaben, Matthijs; Einerhand, Alexandra WC; Taminiau, Lucas JA; van Houdt, Michel; Bouma, Janneke; Raatgeep, Rolien H; Büller, Hans A; de Haan, Cornelis AM; Rossen, John WA

2007-01-01

Cyclooxygenases (COXs) play a significant role in many different viral infections with respect to replication and pathogenesis. Here we investigated the role of COXs in the mouse hepatitis coronavirus (MHV) infection cycle. Blocking COX activity by different inhibitors or by RNA interference affected MHV infection in different cells. The COX inhibitors reduced MHV infection at a post-binding step, but early in the replication cycle. Both viral RNA and viral protein synthesis were affected with subsequent loss of progeny virus production. Thus, COX activity appears to be required for efficient MHV replication, providing a potential target for anti-coronaviral therapy. PMID:17555580

A novel life cycle modeling system for Ebola virus shows a genome length-dependent role of VP24 in virus infectivity.

PubMed

Watt, Ari; Moukambi, Felicien; Banadyga, Logan; Groseth, Allison; Callison, Julie; Herwig, Astrid; Ebihara, Hideki; Feldmann, Heinz; Hoenen, Thomas

2014-09-01

Work with infectious Ebola viruses is restricted to biosafety level 4 (BSL4) laboratories, presenting a significant barrier for studying these viruses. Life cycle modeling systems, including minigenome systems and transcription- and replication-competent virus-like particle (trVLP) systems, allow modeling of the virus life cycle under BSL2 conditions; however, all current systems model only certain aspects of the virus life cycle, rely on plasmid-based viral protein expression, and have been used to model only single infectious cycles. We have developed a novel life cycle modeling system allowing continuous passaging of infectious trVLPs containing a tetracistronic minigenome that encodes a reporter and the viral proteins VP40, VP24, and GP1,2. This system is ideally suited for studying morphogenesis, budding, and entry, in addition to genome replication and transcription. Importantly, the specific infectivity of trVLPs in this system was ∼ 500-fold higher than that in previous systems. Using this system for functional studies of VP24, we showed that, contrary to previous reports, VP24 only very modestly inhibits genome replication and transcription when expressed in a regulated fashion, which we confirmed using infectious Ebola viruses. Interestingly, we also discovered a genome length-dependent effect of VP24 on particle infectivity, which was previously undetected due to the short length of monocistronic minigenomes and which is due at least partially to a previously unknown function of VP24 in RNA packaging. Based on our findings, we propose a model for the function of VP24 that reconciles all currently available data regarding the role of VP24 in nucleocapsid assembly as well as genome replication and transcription. Ebola viruses cause severe hemorrhagic fevers in humans, with no countermeasures currently being available, and must be studied in maximum-containment laboratories. Only a few of these laboratories exist worldwide, limiting our ability to study Ebola viruses and develop countermeasures. Here we report the development of a novel reverse genetics-based system that allows the study of Ebola viruses without maximum-containment laboratories. We used this system to investigate the Ebola virus protein VP24, showing that, contrary to previous reports, it only modestly inhibits virus genome replication and transcription but is important for packaging of genomes into virus particles, which constitutes a previously unknown function of VP24 and a potential antiviral target. We further propose a comprehensive model for the function of VP24 in nucleocapsid assembly. Importantly, on the basis of this approach, it should easily be possible to develop similar experimental systems for other viruses that are currently restricted to maximum-containment laboratories. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Analysis of the genome of the sexually transmitted insect virus Hz-2V

USDA-ARS?s Scientific Manuscript database

Hz-2V is an insect DNA virus closely related to the baculoviruses that grow to high titers in insect cells and produces high yields of virus progeny. The capacity of this virus to replicate to high titers in insect cells may allow the use of this virus for production of large amount of proteins. Th...

Cyclosporin A Associated Helicase-Like Protein Facilitates the Association of Hepatitis C Virus RNA Polymerase with Its Cellular Cyclophilin B

PubMed Central

Sahara, Hiroeki; Iwabata, Kazuki; Sunoki, Takashi; Kuramochi, Kouji; Takakusagi, Kaori; Miyashita, Hiroki; Sato, Noriyuki; Tanabe, Atsushi; Shimotohno, Kunitada; Kobayashi, Susumu; Sakaguchi, Kengo; Sugawara, Fumio

2011-01-01

Background Cyclosporin A (CsA) is well known as an immunosuppressive drug useful for allogeneic transplantation. It has been reported that CsA inhibits hepatitis C virus (HCV) genome replication, which indicates that cellular targets of CsA regulate the viral replication. However, the regulation mechanisms of HCV replication governed by CsA target proteins have not been fully understood. Principal Findings Here we show a chemical biology approach that elucidates a novel mechanism of HCV replication. We developed a phage display screening to investigate compound-peptide interaction and identified a novel cellular target molecule of CsA. This protein, named CsA associated helicase-like protein (CAHL), possessed RNA-dependent ATPase activity that was negated by treatment with CsA. The downregulation of CAHL in the cells resulted in a decrease of HCV genome replication. CAHL formed a complex with HCV-derived RNA polymerase NS5B and host-derived cyclophilin B (CyPB), known as a cellular cofactor for HCV replication, to regulate NS5B-CyPB interaction. Conclusions We found a cellular factor, CAHL, as CsA associated helicase-like protein, which would form trimer complex with CyPB and NS5B of HCV. The strategy using a chemical compound and identifying its target molecule by our phage display analysis is useful to reveal a novel mechanism underlying cellular and viral physiology. PMID:21559518

Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.

PubMed

Ikegami, Tetsuro; Won, Sungyong; Peters, C J; Makino, Shinji

2006-03-01

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.

Rift Valley fever virus NSs protein promotes post-transcriptional downregulation of protein kinase PKR and inhibits eIF2alpha phosphorylation.

PubMed

Ikegami, Tetsuro; Narayanan, Krishna; Won, Sungyong; Kamitani, Wataru; Peters, C J; Makino, Shinji

2009-02-01

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) is a negative-stranded RNA virus with a tripartite genome. RVFV is transmitted by mosquitoes and causes fever and severe hemorrhagic illness among humans, and fever and high rates of abortions in livestock. A nonstructural RVFV NSs protein inhibits the transcription of host mRNAs, including interferon-beta mRNA, and is a major virulence factor. The present study explored a novel function of the RVFV NSs protein by testing the replication of RVFV lacking the NSs gene in the presence of actinomycin D (ActD) or alpha-amanitin, both of which served as a surrogate of the host mRNA synthesis suppression function of the NSs. In the presence of the host-transcriptional inhibitors, the replication of RVFV lacking the NSs protein, but not that carrying NSs, induced double-stranded RNA-dependent protein kinase (PKR)-mediated eukaryotic initiation factor (eIF)2alpha phosphorylation, leading to the suppression of host and viral protein translation. RVFV NSs promoted post-transcriptional downregulation of PKR early in the course of the infection and suppressed the phosphorylated eIF2alpha accumulation. These data suggested that a combination of RVFV replication and NSs-induced host transcriptional suppression induces PKR-mediated eIF2alpha phosphorylation, while the NSs facilitates efficient viral translation by downregulating PKR and inhibiting PKR-mediated eIF2alpha phosphorylation. Thus, the two distinct functions of the NSs, i.e., the suppression of host transcription, including that of type I interferon mRNAs, and the downregulation of PKR, work together to prevent host innate antiviral functions, allowing efficient replication and survival of RVFV in infected mammalian hosts.

2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation.

PubMed

Lai, Jeffrey K F; Sam, I-Ching; Verlhac, Pauline; Baguet, Joël; Eskelinen, Eeva-Liisa; Faure, Mathias; Chan, Yoke Fun

2017-07-04

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N -ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.

2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation

PubMed Central

Lai, Jeffrey K. F.; Sam, I-Ching; Verlhac, Pauline; Baguet, Joël; Faure, Mathias

2017-01-01

Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection. PMID:28677644

Hepatitis Delta Virus: Replication Strategy and Upcoming Therapeutic Options for a Neglected Human Pathogen

PubMed Central

Lempp, Florian A.; Urban, Stephan

2017-01-01

The human Hepatitis Delta Virus (HDV) is unique among all viral pathogens. Encoding only one protein (Hepatitis Delta Antigen; HDAg) within its viroid-like self-complementary RNA, HDV constitutes the smallest known virus in the animal kingdom. To disseminate in its host, HDV depends on a helper virus, the human Hepatitis B virus (HBV), which provides the envelope proteins required for HDV assembly. HDV affects an estimated 15–20 million out of the 240 million chronic HBV-carriers and disperses unequally in disparate geographical regions of the world. The disease it causes (chronic Hepatitis D) presents as the most severe form of viral hepatitis, leading to accelerated progression of liver dysfunction including cirrhosis and hepatocellular carcinoma and a high mortality rate. The lack of approved drugs interfering with specific steps of HDV replication poses a high burden for gaining insights into the molecular biology of the virus and, consequently, the development of specific novel medications that resiliently control HDV replication or, in the best case, functionally cure HDV infection or HBV/HDV co-infection. This review summarizes our current knowledge of HBV molecular biology, presents an update on novel cell culture and animal models to study the virus and provides updates on the clinical development of the three developmental drugs Lonafarnib, REP2139-Ca and Myrcludex B. PMID:28677645

Interplay of PA-X and NS1 Proteins in Replication and Pathogenesis of a Temperature-Sensitive 2009 Pandemic H1N1 Influenza A Virus.

PubMed

Nogales, Aitor; Rodriguez, Laura; DeDiego, Marta L; Topham, David J; Martínez-Sobrido, Luis

2017-09-01

Influenza A viruses (IAVs) cause seasonal epidemics and occasional pandemics, representing a serious public health concern. It has been described that one mechanism used by some IAV strains to escape the host innate immune responses and modulate virus pathogenicity involves the ability of the PA-X and NS1 proteins to inhibit the host protein synthesis in infected cells. It was reported that for the 2009 pandemic H1N1 IAV (pH1N1) only the PA-X protein had this inhibiting capability, while the NS1 protein did not. In this work, we have evaluated, for the first time, the combined effect of PA-X- and NS1-mediated inhibition of general gene expression on virus pathogenesis, using a temperature-sensitive, live-attenuated 2009 pandemic H1N1 IAV (pH1N1 LAIV). We found that viruses containing PA-X and NS1 proteins that simultaneously have (PA WT + /NS1 MUT + ) or do not have (PA MUT - /NS1 WT - ) the ability to block host gene expression showed reduced pathogenicity in vivo However, a virus where the ability to inhibit host protein expression was switched between PA-X and NS1 (PA MUT - /NS1 MUT + ) presented pathogenicity similar to that of a virus containing both wild-type proteins (PA WT + /NS1 WT - ). Our findings suggest that inhibition of host protein expression is subject to a strict balance, which can determine the successful progression of IAV infection. Importantly, knowledge obtained from our studies could be used for the development of new and more effective vaccine approaches against IAV. IMPORTANCE Influenza A viruses (IAVs) are one of the most common causes of respiratory infections in humans, resulting in thousands of deaths annually. Furthermore, IAVs can cause unpredictable pandemics of great consequence when viruses not previously circulating in humans are introduced into humans. The defense machinery provided by the host innate immune system limits IAV replication; however, to counteract host antiviral activities, IAVs have developed different inhibition mechanisms, including prevention of host gene expression mediated by the viral PA-X and NS1 proteins. Here, we provide evidence demonstrating that optimal control of host protein synthesis by IAV PA-X and/or NS1 proteins is required for efficient IAV replication in the host. Moreover, we demonstrate the feasibility of genetically controlling the ability of IAV PA-X and NS1 proteins to inhibit host immune responses, providing an approach to develop more effective vaccines to combat disease caused by this important respiratory pathogen. Copyright © 2017 American Society for Microbiology.

Norovirus Genome Circularization and Efficient Replication Are Facilitated by Binding of PCBP2 and hnRNP A1

PubMed Central

López-Manríquez, Eduardo; Vashist, Surender; Ureña, Luis; Goodfellow, Ian; Chavez, Pedro; Mora-Heredia, José Eduardo; Cancio-Lonches, Clotilde; Garrido, Efraín

2013-01-01

Sequences and structures within the terminal genomic regions of plus-strand RNA viruses are targets for the binding of host proteins that modulate functions such as translation, RNA replication, and encapsidation. Using murine norovirus 1 (MNV-1), we describe the presence of long-range RNA-RNA interactions that were stabilized by cellular proteins. The proteins potentially responsible for the stabilization were selected based on their ability to bind the MNV-1 genome and/or having been reported to be involved in the stabilization of RNA-RNA interactions. Cell extracts were preincubated with antibodies against the selected proteins and used for coprecipitation reactions. Extracts treated with antibodies to poly(C) binding protein 2 (PCBP2) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 significantly reduced the 5′-3′ interaction. Both PCBP2 and hnRNP A1 recombinant proteins stabilized the 5′-3′ interactions and formed ribonucleoprotein complexes with the 5′ and 3′ ends of the MNV-1 genomic RNA. Mutations within the 3′ complementary sequences (CS) that disrupt the 5′-3′-end interactions resulted in a significant reduction of the viral titer, suggesting that the integrity of the 3′-end sequence and/or the lack of complementarity with the 5′ end is important for efficient virus replication. Small interfering RNA-mediated knockdown of PCBP2 or hnRNP A1 resulted in a reduction in virus yield, confirming a role for the observed interactions in efficient viral replication. PCBP2 and hnRNP A1 induced the circularization of MNV-1 RNA, as revealed by electron microscopy. This study provides evidence that PCBP2 and hnRNP A1 bind to the 5′ and 3′ ends of the MNV-1 viral RNA and contribute to RNA circularization, playing a role in the virus life cycle. PMID:23946460

Human Immunodeficiency Virus Type 1 Employs the Cellular Dynein Light Chain 1 Protein for Reverse Transcription through Interaction with Its Integrase Protein

PubMed Central

Jayappa, Kallesh Danappa; Ao, Zhujun; Wang, Xiaoxia; Mouland, Andrew J.; Shekhar, Sudhanshu; Yang, Xi

2015-01-01

ABSTRACT In this study, we examined the requirement for host dynein adapter proteins such as dynein light chain 1 (DYNLL1), dynein light chain Tctex-type 1 (DYNLT1), and p150Glued in early steps of human immunodeficiency virus type 1 (HIV-1) replication. We found that the knockdown (KD) of DYNLL1, but not DYNLT1 or p150Glued, resulted in significantly lower levels of HIV-1 reverse transcription in cells. Following an attempt to determine how DYNLL1 could impact HIV-1 reverse transcription, we detected the DYNLL1 interaction with HIV-1 integrase (IN) but not with capsid (CA), matrix (MA), or reverse transcriptase (RT) protein. Furthermore, by mutational analysis of putative DYNLL1 interaction motifs in IN, we identified the motifs 52GQVD and 250VIQD in IN as essential for DYNLL1 interaction. The DYNLL1 interaction-defective IN mutant HIV-1 (HIV-1INQ53A/Q252A) exhibited impaired reverse transcription. Through further investigations, we have also detected relatively smaller amounts of particulate CA in DYNLL1-KD cells or in infections with HIV-1INQ53A/Q252A mutant virus. Overall, our study demonstrates the novel interaction between HIV-1 IN and cellular DYNLL1 proteins and suggests the requirement of this virus-cell interaction for proper uncoating and efficient reverse transcription of HIV-1. IMPORTANCE Host cellular DYNLL1, DYNLT1, and p150Glued proteins have been implicated in the replication of several viruses. However, their roles in HIV-1 replication have not been investigated. For the first time, we demonstrated that during viral infection, HIV-1 IN interacts with DYNLL1, and their interaction was found to have a role in proper uncoating and efficient reverse transcription of HIV-1. Thus, interaction of IN and DYNLL1 may be a potential target for future anti-HIV therapy. Moreover, while our study has evaluated the involvement of IN in HIV-1 uncoating and reverse transcription, it also predicts a possible mechanism by which IN contributes to these early viral replication steps. PMID:25568209

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.

Virus-Like Particles That Can Deliver Proteins and RNA | NCI Technology Transfer Center | TTC

Cancer.gov

The present invention describes novel virus-like particles (VLPs) that are capable of binding to and replicating within a target mammalian cell, including human cells. The claimed VLPs are safer than viral delivery because they are incapable of re-infecting target cells. The National Cancer Institute's Protein Expression Laboratory seeks parties interested in licensing the novel delivery of RNA to mammalian cells using virus-like particles.

Inhibition of aldolase A blocks biogenesis of ATP and attenuates Japanese encephalitis virus production.

PubMed

Tien, Chih-Feng; Cheng, Shih-Ching; Ho, Yen-Peng; Chen, Yi-Shiuan; Hsu, Jung-Hsin; Chang, Ruey-Yi

2014-01-10

Viral replication depends on host proteins to supply energy and replication accessories for the sufficient production of viral progeny. In this study, we identified fructose-bisphosphate aldolase A as a binding partner of Japanese encephalitis virus (JEV) untranslated regions (UTRs) on the antigenome via RNA affinity capture and mass spectrometry. Direct interaction of aldolase A with JEV RNAs was confirmed by gel mobility shift assay and colocalization with active replication of double-stranded RNA in JEV-infected cells. Infection of JEV caused an increase in aldolase A expression of up to 33%. Knocking down aldolase A reduced viral translation, genome replication, and viral production significantly. Furthermore, JEV infection consumed 50% of cellular ATP, and the ATP level decreased by 70% in the aldolase A-knockdown cells. Overexpression of aldolase A in aldolase A-knockdown cells increased ATP levels significantly. Taken together, these results indicate that JEV replication requires aldolase A and consumes ATP. This is the first report of direct involvement of a host metabolic enzyme, aldolase A protein, in JEV replication. Copyright © 2013 Elsevier Inc. All rights reserved.

Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation

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

Eyre, Nicholas S., E-mail: nicholas.eyre@adelaide.edu.au; Centre for Cancer Biology, SA Pathology, Adelaide; Hampton-Smith, Rachel J.

Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using ‘APEX2’-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using amore » customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα. - Highlights: • NS5A residues Ser-222, Ser-235 and Thr-348 are phosphorylated during HCV infection. • Phosphorylation of Ser-235 is essential to HCV RNA replication. • Mutation of Ser-235 alters replication compartment localization and morphology. • Phosphatidylinositol-4 kinase III alpha is important for Ser-235 phosphorylation.« less

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.

Identification, cloning, and expression analysis of three putative Lymantria dispar nuclear polyhedrosis virus immediate early genes

Treesearch

James M. Slavicek; Nancy Hayes-Plazolles

1991-01-01

Viral immediate early gene products are usually regulatory proteins that control expression of other viral genes at the transcriptional level or are proteins that are part of the viral DNA replication complex. The identification and functional characterization of the immediate early gene products of Lymantria dispar nuclear polyhedrosis virus (LdNPV...

Triticum mosaic virus exhibits limited population variation yet shows evidence of parallel evolution after replicated serial passage in wheat

USDA-ARS?s Scientific Manuscript database

An infectious cDNA clone of Triticum mosaic virus (TriMV) (genus Poacevirus; family Potyviridae) was used to establish three independent lineages in wheat to examine intra-host population diversity levels within protein 1 (P1) and coat protein (CP) cistrons over time. Genetic variation was assessed ...

Effect of temperature on wheat streak mosaic disease development in winter wheat

USDA-ARS?s Scientific Manuscript database

Temperature is one of the key factors that influence viral disease development in plants. In this study, temperature effect on Wheat streak mosaic virus (WSMV) replication and in planta movement was determined using a green fluorescent protein (GFP)-tagged virus in two winter wheat cultivars. Virus-...

Evaluation of Measles Vaccine Virus as a Vector to Deliver Respiratory Syncytial Virus Fusion Protein or Epstein-Barr Virus Glycoprotein gp350

PubMed Central

Mok, Hoyin; Cheng, Xing; Xu, Qi; Zengel, James R; Parhy, Bandita; Zhao, Jackie; Wang, C. Kathy; Jin, Hong

2012-01-01

Live attenuated recombinant measles vaccine virus (MV) Edmonston-Zagreb (EZ) strain was evaluated as a viral vector to express the ectodomains of fusion protein of respiratory syncytial virus (RSV F) or glycoprotein 350 of Epstein-Barr virus (EBV gp350) as candidate vaccines for prophylaxis of RSV and EBV. The glycoprotein gene was inserted at the 1st or the 3rd position of the measles virus genome and the recombinant viruses were generated. Insertion of the foreign gene at the 3rd position had a minimal impact on viral replication in vitro. RSV F or EBV gp350 protein was secreted from infected cells. In cotton rats, EZ-RSV F and EZ-EBV gp350 induced MV- and insert-specific antibody responses. In addition, both vaccines also induced insert specific interferon gamma (IFN-γ) secreting T cell response. EZ-RSV F protected cotton rats from pulmonary replication of RSV A2 challenge infection. In rhesus macaques, although both EZ-RSV F and EZ-EBV gp350 induced MV specific neutralizing antibody responses, only RSV F specific antibody response was detected. Thus, the immunogenicity of the foreign antigens delivered by measles vaccine virus is dependent on the nature of the insert and the animal models used for vaccine evaluation. PMID:22383906

Canonical and Non-Canonical Autophagy in HIV-1 Replication Cycle

PubMed Central

Leymarie, Olivier; Lepont, Leslie; Berlioz-Torrent, Clarisse

2017-01-01

Autophagy is a lysosomal-dependent degradative process essential for maintaining cellular homeostasis, and is a key player in innate and adaptive immune responses to intracellular pathogens such as human immunodeficiency virus type 1 (HIV-1). In HIV-1 target cells, autophagy mechanisms can (i) selectively direct viral proteins and viruses for degradation; (ii) participate in the processing and presentation of viral-derived antigens through major histocompatibility complexes; and (iii) contribute to interferon production in response to HIV-1 infection. As a consequence, HIV-1 has evolved different strategies to finely regulate the autophagy pathway to favor its replication and dissemination. HIV-1 notably encodes accessory genes encoding Tat, Nef and Vpu proteins, which are able to perturb and hijack canonical and non-canonical autophagy mechanisms. This review outlines the current knowledge on the complex interplay between autophagy and HIV-1 replication cycle, providing an overview of the autophagy-mediated molecular processes deployed both by infected cells to combat the virus and by HIV-1 to evade antiviral response. PMID:28946621

Analysis of cis and trans Requirements for DNA Replication at the Right-End Hairpin of the Human Bocavirus 1 Genome

PubMed Central

Shen, Weiran; Deng, Xuefeng; Zou, Wei; Engelhardt, John F.; Yan, Ziying

2016-01-01

ABSTRACT Parvoviruses are single-stranded DNA viruses that use the palindromic structures at the ends of the viral genome for their replication. The mechanism of parvovirus replication has been studied mostly in the dependoparvovirus adeno-associated virus 2 (AAV2) and the protoparvovirus minute virus of mice (MVM). Here, we used human bocavirus 1 (HBoV1) to understand the replication mechanism of bocaparvovirus. HBoV1 is pathogenic to humans, causing acute respiratory tract infections, especially in young children under 2 years old. By using the duplex replicative form of the HBoV1 genome in human embryonic kidney 293 (HEK293) cells, we identified the HBoV1 minimal replication origin at the right-end hairpin (OriR). Mutagenesis analyses confirmed the putative NS1 binding and nicking sites within the OriR. Of note, unlike the large nonstructural protein (Rep78/68 or NS1) of other parvoviruses, HBoV1 NS1 did not specifically bind OriR in vitro, indicating that other viral and cellular components or the oligomerization of NS1 is required for NS1 binding to the OriR. In vivo studies demonstrated that residues responsible for NS1 binding and nicking are within the origin-binding domain. Further analysis identified that the small nonstructural protein NP1 is required for HBoV1 DNA replication at OriR. NP1 and other viral nonstructural proteins (NS1 to NS4) colocalized within the viral DNA replication centers in both OriR-transfected cells and virus-infected cells, highlighting a direct involvement of NP1 in viral DNA replication at OriR. Overall, our study revealed the characteristics of HBoV1 DNA replication at OriR, suggesting novel characteristics of autonomous parvovirus DNA replication. IMPORTANCE Human bocavirus 1 (HBoV1) causes acute respiratory tract infections in young children. The duplex HBoV1 genome replicates in HEK293 cells and produces progeny virions that are infectious in well-differentiated airway epithelial cells. A recombinant AAV2 vector pseudotyped with an HBoV1 capsid has been developed to efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to human airway epithelia. Here, we identified both cis-acting elements and trans-acting proteins that are required for HBoV1 DNA replication at the right-end hairpin in HEK293 cells. We localized the minimal replication origin, which contains both NS1 nicking and binding sites, to a 46-nucleotide sequence in the right-end hairpin. The identification of these essential elements of HBoV1 DNA replication acting both in cis and in trans will provide guidance to develop antiviral strategies targeting viral DNA replication at the right-end hairpin and to design next-generation recombinant HBoV1 vectors, a promising tool for gene therapy of lung diseases. PMID:27334591

Analysis of cis and trans Requirements for DNA Replication at the Right-End Hairpin of the Human Bocavirus 1 Genome.

PubMed

Shen, Weiran; Deng, Xuefeng; Zou, Wei; Engelhardt, John F; Yan, Ziying; Qiu, Jianming

2016-09-01

Parvoviruses are single-stranded DNA viruses that use the palindromic structures at the ends of the viral genome for their replication. The mechanism of parvovirus replication has been studied mostly in the dependoparvovirus adeno-associated virus 2 (AAV2) and the protoparvovirus minute virus of mice (MVM). Here, we used human bocavirus 1 (HBoV1) to understand the replication mechanism of bocaparvovirus. HBoV1 is pathogenic to humans, causing acute respiratory tract infections, especially in young children under 2 years old. By using the duplex replicative form of the HBoV1 genome in human embryonic kidney 293 (HEK293) cells, we identified the HBoV1 minimal replication origin at the right-end hairpin (OriR). Mutagenesis analyses confirmed the putative NS1 binding and nicking sites within the OriR. Of note, unlike the large nonstructural protein (Rep78/68 or NS1) of other parvoviruses, HBoV1 NS1 did not specifically bind OriR in vitro, indicating that other viral and cellular components or the oligomerization of NS1 is required for NS1 binding to the OriR. In vivo studies demonstrated that residues responsible for NS1 binding and nicking are within the origin-binding domain. Further analysis identified that the small nonstructural protein NP1 is required for HBoV1 DNA replication at OriR. NP1 and other viral nonstructural proteins (NS1 to NS4) colocalized within the viral DNA replication centers in both OriR-transfected cells and virus-infected cells, highlighting a direct involvement of NP1 in viral DNA replication at OriR. Overall, our study revealed the characteristics of HBoV1 DNA replication at OriR, suggesting novel characteristics of autonomous parvovirus DNA replication. Human bocavirus 1 (HBoV1) causes acute respiratory tract infections in young children. The duplex HBoV1 genome replicates in HEK293 cells and produces progeny virions that are infectious in well-differentiated airway epithelial cells. A recombinant AAV2 vector pseudotyped with an HBoV1 capsid has been developed to efficiently deliver the cystic fibrosis transmembrane conductance regulator gene to human airway epithelia. Here, we identified both cis-acting elements and trans-acting proteins that are required for HBoV1 DNA replication at the right-end hairpin in HEK293 cells. We localized the minimal replication origin, which contains both NS1 nicking and binding sites, to a 46-nucleotide sequence in the right-end hairpin. The identification of these essential elements of HBoV1 DNA replication acting both in cis and in trans will provide guidance to develop antiviral strategies targeting viral DNA replication at the right-end hairpin and to design next-generation recombinant HBoV1 vectors, a promising tool for gene therapy of lung diseases. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Evaluation of hepatitis B virus replication and proteomic analysis of HepG2.2.15 cell line after cyclosporine A treatment.

PubMed

Xie, Hai-Yang; Xia, Wei-Liang; Zhang, Chun-Chao; Wu, Li-Ming; Ji, Hao-Feng; Cheng, Yu; Zheng, Shu-Sen

2007-07-01

The effect of cyclosporine A (CsA) on hepatitis B virus (HBV) replication was investigated, and proteomics expression differentiation after CsA treatment was studied in order to provide clues to explore the effect of CsA on HBV replication. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cytotoxicity of CsA. The HBV replication level in the HBV genomic DNA transfected HepG2.2.15 cell line was determined by an ELISA analysis of hepatitis B surface antigens (HBsAg) and Hepatitis B e antigens (HBeAg) in culture supernatant, while the intracellular HBV DNA replication level was analyzed by slot blot hybridization. Two-dimensional electrophoresis was used to investigate the alteration of protein expression in HepG2.2.15 after CsA treatment in vitro. The differentially-expressed proteins were identified by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry combined with an online database search. CsA was able to inhibit the expression of HBsAg, HBeAg, and HBV DNA replication in vitro in a dose-dependent manner. A proteomics analysis indicated that the expression of 17 proteins changed significantly in the CsA treatment group compared to the control group. Eleven of the 17 proteins were identified, including the overexpression of eukaryotic translation initiation factors (eIF) 3k, otubain 1, 14.3.3 protein, eIF2-1 alpha, eIF5A, and the tyrosine 3/tryptophan 5-mono-oxygenase activation protein in CsA-treated HepG2.2.15 cells. The downregulation of the ferritin light subunit, erythrocyte cytosolic protein of 51 kDa (ECP-51), stathmin 1/oncoprotein, adenine phosphoribosyl-transferase, and the position of a tumor protein, translationally controlled 1, was shifted, suggesting it had undergone posttranslational modifications. Our study identified the inhibitory effect of CsA on HBV replication, and found that a group of proteins may be responsible for this inhibitory effect.

Genome-wide profiling of microRNAs reveals novel insights into the interactions between H9N2 avian influenza virus and avian dendritic cells.

PubMed

Lin, Jian; Xia, Jing; Zhang, Tian; Zhang, Keyun; Yang, Qian

2018-05-10

The antigen-presenting ability of dendritic cells (DCs) plays an important and irreplaceable role in recognising and clearing viruses. Antiviral responses must rapidly defend against infection while minimising inflammatory damage, but the mechanisms that regulate the magnitude of response within an infected cell are not well understood. MicroRNAs (microRNAs), small non-coding RNAs, can regulate mouse or avian DCs to inhibit the infection and replication of avian influenza virus (AIV). Here, we performed a global analysis to understand how avian DCs respond to H9N2 AIV and provide a potential mechanism to explain how avian microRNAs can defend against H9N2 AIV replication. First, we found that both active and inactive H9N2 AIV enhanced the ability of DCs to present antigens and activate T lymphocytes. Next, total microarray analyses suggested that H9N2 AIV stimulation involved protein localisation, nucleotide binding, leucocyte transendothelial migration and MAPK signalling. Moreover, we constructed 551 transcription factor (TF)-miRNA-mRNA loops based on the above analyses. Furthermore, we found that the haemagglutinin (HA) fragment, neither H5N1-HA or H9N2-HA, could not activate DCs, while truncated HA greatly increased the immune function of DCs by activating ERK and STAT3 signalling pathways. Lastly, our results not only suggested that gga-miR1644 targets muscleblind-like protein 2 (MBNL2) to enhance the ability of avian DCs to inhibit virus replication, but also suggested that gga-miR6675 targets the nuclear localisation sequence of polymerase basic protein 1 (PB1) to trigger the silencing of PB1 genes, resulting in the inhibition of H9N2 AIV replication. Altogether, our innovative study will shed new light on the role of avian microRNAs in evoking avian DCs and inhibiting virus replication.

Characterization of a baculovirus lacking the DBP (DNA-binding protein) gene

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

Vanarsdall, Adam L.; Mikhailov, Victor S.; N.K. Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Moscow 117808

2007-08-01

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) encodes two proteins that possess properties typical of single-stranded DNA-binding proteins (SSBs), late expression factor-3 (LEF-3), and a protein referred to as DNA-binding protein (DBP). Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. Therefore, to better understand the functional role of DBP in viral replication, a DBP knockout virus was generated from an AcMNPV bacmid and analyzed. The results of a growth curve analysis indicated that the dbpmore » knockout construct was unable to produce budded virus indicating that dbp is essential. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early (LEF-3), late (VP39), and very late (P10) proteins in cells transfected with the dbp knockout construct. To investigate the role of DBP in DNA replication, a real-time PCR-based assay was employed and showed that, although viral DNA synthesis occurred in cells transfected with the dbp knockout, the levels were less than that of the control virus suggesting that DBP is required for normal levels of DNA synthesis or for stability of nascent viral DNA. In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Furthermore, analysis of DBP from infected cells indicated that similar to LEF-3, DBP was tightly bound to viral chromatin. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp knockout revealed that DBP is required for the production of normal-appearing nucleocapsids and for the generation of the virogenic stroma.« less

Evolutionary dynamics of giant viruses and their virophages.

PubMed

Wodarz, Dominik

2013-07-01

Giant viruses contain large genomes, encode many proteins atypical for viruses, replicate in large viral factories, and tend to infect protists. The giant virus replication factories can in turn be infected by so called virophages, which are smaller viruses that negatively impact giant virus replication. An example is Mimiviruses that infect the protist Acanthamoeba and that are themselves infected by the virophage Sputnik. This study examines the evolutionary dynamics of this system, using mathematical models. While the models suggest that the virophage population will evolve to increasing degrees of giant virus inhibition, it further suggests that this renders the virophage population prone to extinction due to dynamic instabilities over wide parameter ranges. Implications and conditions required to avoid extinction are discussed. Another interesting result is that virophage presence can fundamentally alter the evolutionary course of the giant virus. While the giant virus is predicted to evolve toward increasing its basic reproductive ratio in the absence of the virophage, the opposite is true in its presence. Therefore, virophages can not only benefit the host population directly by inhibiting the giant viruses but also indirectly by causing giant viruses to evolve toward weaker phenotypes. Experimental tests for this model are suggested.

Evolutionary dynamics of giant viruses and their virophages

PubMed Central

Wodarz, Dominik

2013-01-01

Giant viruses contain large genomes, encode many proteins atypical for viruses, replicate in large viral factories, and tend to infect protists. The giant virus replication factories can in turn be infected by so called virophages, which are smaller viruses that negatively impact giant virus replication. An example is Mimiviruses that infect the protist Acanthamoeba and that are themselves infected by the virophage Sputnik. This study examines the evolutionary dynamics of this system, using mathematical models. While the models suggest that the virophage population will evolve to increasing degrees of giant virus inhibition, it further suggests that this renders the virophage population prone to extinction due to dynamic instabilities over wide parameter ranges. Implications and conditions required to avoid extinction are discussed. Another interesting result is that virophage presence can fundamentally alter the evolutionary course of the giant virus. While the giant virus is predicted to evolve toward increasing its basic reproductive ratio in the absence of the virophage, the opposite is true in its presence. Therefore, virophages can not only benefit the host population directly by inhibiting the giant viruses but also indirectly by causing giant viruses to evolve toward weaker phenotypes. Experimental tests for this model are suggested. PMID:23919155

Specific functions of the Rep and Rep׳ proteins of porcine circovirus during copy-release and rolling-circle DNA replication.

PubMed

Cheung, Andrew K

2015-07-01

The roles of two porcine circovirus replication initiator proteins, Rep and Rep׳, in generating copy-release and rolling-circle DNA replication intermediates were determined. Rep uses the supercoiled closed-circular genome (ccc) to initiate leading-strand synthesis (identical to copy-release replication) and generates the single-stranded circular (ssc) genome from the displaced DNA strand. In the process, a minus-genome primer (MGP) necessary for complementary-strand synthesis, from ssc to ccc, is synthesized. Rep׳ cleaves the growing nascent-strand to regenerate the parent ccc molecule. In the process, a Rep׳-DNA hybrid containing the right palindromic sequence (at the origin of DNA replication) is generated. Analysis of the virus particle showed that it is composed of four components: ssc, MGP, capsid protein and a novel Rep-related protein (designated Protein-3). Copyright © 2015. Published by Elsevier Inc.

Identification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and Demonstration that it Interacts with ICP8, the Major DNA Binding Protein of Herpes Simplex Virus

DTIC Science & Technology

1992-10-20

Identification of ORFs HSV DNA binding proteins • 1 3 3 5 7 7 11 17 18 22 reps and its role in HSV replication 23 Biochemical properties . . 23...Figure 1 . 2. 3 • 4. 5. 6. 7. 8. Structural model of the herpesvirus virion Schematic diagram of HSV pathogenesis . Diagram of the main...vaccinia virus- 13. Autoradiogram of an immunoblot of HSV - 1 -infected cell proteins harvested at various times postinfec- 85 tioD probed with anti-UL42

Nucleoprotein of influenza A virus negatively impacts antiapoptotic protein API5 to enhance E2F1-dependent apoptosis and virus replication.

PubMed

Mayank, A K; Sharma, S; Nailwal, H; Lal, S K

2015-12-17

Apoptosis of host cells profoundly influences virus propagation and dissemination, events that are integral to influenza A virus (IAV) pathogenesis. The trigger for activation of apoptosis is regulated by an intricate interplay between cellular and viral proteins, with a strong bearing on IAV replication. Though the knowledge of viral proteins and mechanisms employed by IAV to induce apoptosis has advanced considerably of late, we know relatively little about the repertoire of host factors targeted by viral proteins. Thus, identification of cellular proteins that are hijacked by the virus will help us not only to understand the molecular underpinnings of IAV-induced apoptosis, but also to design future antiviral therapies. Here we show that the nucleoprotein (NP) of IAV directly interacts with and suppresses the expression of API5, a host antiapoptotic protein that antagonizes E2F1-dependent apoptosis. siRNA-mediated depletion of API5, in NP-overexpressed as well as IAV-infected cells, leads to upregulation of apoptotic protease activating factor 1 (APAF1), a downstream modulator of E2F1-mediated apoptosis, and cleavage of caspases 9 and 3, although a reciprocal pattern of these events was observed on ectopic overexpression of API5. In concordance with these observations, annexin V and 7AAD staining assays exhibit downregulation of early and late apoptosis in IAV-infected or NP-transfected cells on overexpression of API5. Most significantly, while overexpression of API5 decreases viral titers, cellular NP protein as well as mRNA levels in IAV-infected A549 cells, silencing of API5 expression causes a steep rise in the same parameters. From the data reported in this manuscript, we propose a proapoptotic role for NP in IAV pathogenesis, whereby it suppresses expression of antiapoptotic factor API5, thus potentiating the E2F1-dependent apoptotic pathway and ensuring viral replication.

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

Live cell imaging of interactions between replicase and capsid protein of Brome mosaic virus using Bimolecular Fluorescence Complementation: implications for replication and genome packaging.

PubMed

Chaturvedi, Sonali; Rao, A L N

2014-09-01

In Brome mosaic virus, it was hypothesized that a physical interaction between viral replicase and capsid protein (CP) is obligatory to confer genome packaging specificity. Here we tested this hypothesis by employing Bimolecular Fluorescent Complementation (BiFC) as a tool for evaluating protein-protein interactions in living cells. The efficacy of BiFC was validated by a known interaction between replicase protein 1a (p1a) and protein 2a (p2a) at the endoplasmic reticulum (ER) site of viral replication. Additionally, co-expression in planta of a bona fide pair of interacting protein partners of p1a and p2a had resulted in the assembly of a functional replicase. Subsequent BiFC assays in conjunction with mCherry labeled ER as a fluorescent cellular marker revealed that CP physically interacts with p2a, but not p1a, and this CP:p2a interaction occurs at the cytoplasmic phase of the ER. The significance of the CP:p2a interaction in BMV replication and genome packaging is discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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.

Diverse Effects of Cyclosporine on Hepatitis C Virus Strain Replication

PubMed Central

Ishii, Naoto; Watashi, Koichi; Hishiki, Takayuki; Goto, Kaku; Inoue, Daisuke; Hijikata, Makoto; Wakita, Takaji; Kato, Nobuyuki; Shimotohno, Kunitada

2006-01-01

Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents. PMID:16611911

Disruption of a stem-loop structure located upstream of pseudoknot domain in Tobacco mosaic virus enhanced its infectivity and viral RNA accumulation.

PubMed

Guo, Song; Wong, Sek-Man

2018-06-01

A predicted stem-loop structure of 25 nucleotides, located in the coat protein (CP) gene and 3'-UTR sequences of Tobacco mosaic virus (TMV), was validated previously (Guo et al., 2015). In this study, both disrupted stem-loop and nucleotide deletion mutants of TMV replicated more rapidly in Nicotiana benthamiana protoplasts. The TMV mutant with a complete mirrored stem-loop structure showed similar level of viral RNA accumulation as TMV. Recovering the stem-loop structure also resulted in a similar replication level as TMV. All these mutants induced necrosis in N. benthamiana and assembled into typical rigid rod-shaped virions. TMV mutant without the stem-loop structure induced more local lesions in Chenopodium quinoa. When the putative stem-loop structure in Tomato mosaic virus (ToMV) was disrupted, the mutant also showed an enhanced virus replication. This suggests that the stem-loop structure of TMV is a new cis-acting element with a role in virus replication. Copyright © 2018 Elsevier Inc. All rights reserved.

Mutations in the Cytoplasmic Domain of the Newcastle Disease Virus Fusion Protein Confer Hyperfusogenic Phenotypes Modulating Viral Replication and Pathogenicity

PubMed Central

Samal, Sweety; Khattar, Sunil K.; Paldurai, Anandan; Palaniyandi, Senthilkumar; Zhu, Xiaoping; Collins, Peter L.

2013-01-01

The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis. PMID:23843643

Mutations in the cytoplasmic domain of the Newcastle disease virus fusion protein confer hyperfusogenic phenotypes modulating viral replication and pathogenicity.

PubMed

Samal, Sweety; Khattar, Sunil K; Paldurai, Anandan; Palaniyandi, Senthilkumar; Zhu, Xiaoping; Collins, Peter L; Samal, Siba K

2013-09-01

The Newcastle disease virus (NDV) fusion protein (F) mediates fusion of viral and host cell membranes and is a major determinant of NDV pathogenicity. In the present study, we demonstrate the effects of functional properties of F cytoplasmic tail (CT) amino acids on virus replication and pathogenesis. Out of a series of C-terminal deletions in the CT, we were able to rescue mutant viruses lacking two or four residues (rΔ2 and rΔ4). We further rescued viral mutants with individual amino acid substitutions at each of these four terminal residues (rM553A, rK552A, rT551A, and rT550A). In addition, the NDV F CT has two conserved tyrosine residues (Y524 and Y527) and a dileucine motif (LL536-537). In other paramyxoviruses, these residues were shown to affect fusion activity and are central elements in basolateral targeting. The deletion of 2 and 4 CT amino acids and single tyrosine substitution resulted in hyperfusogenic phenotypes and increased viral replication and pathogenesis. We further found that in rY524A and rY527A viruses, disruption of the targeting signals did not reduce the expression on the apical or basolateral surface in polarized Madin-Darby canine kidney cells, whereas in double tyrosine mutant, it was reduced on both the apical and basolateral surfaces. Interestingly, in rL536A and rL537A mutants, the F protein expression was more on the apical than on the basolateral surface, and this effect was more pronounced in the rL537A mutant. We conclude that these wild-type residues in the NDV F CT have an effect on regulating F protein biological functions and thus modulating viral replication and pathogenesis.

The Influenza Virus M2 Protein Cytoplasmic Tail Interacts with the M1 Protein and Influences Virus Assembly at the Site of Virus Budding ▿

PubMed Central

Chen, Benjamin J.; Leser, George P.; Jackson, David; Lamb, Robert A.

2008-01-01

The cytoplasmic tail of the influenza A virus M2 proton-selective ion channel has been shown to be important for virus replication. Previous analysis of M2 cytoplasmic tail truncation mutants demonstrated a defect in incorporation of viral RNA (vRNA) into virions, suggesting a role for M2 in the recruitment of M1-vRNA complexes. To further characterize the effect of the M2 cytoplasmic tail mutations on virus assembly and budding, we constructed a series of alanine substitution mutants of M2 with mutations in the cytoplasmic tail, from residues 71 to 97. Mutant proteins M2-Mut1 and M2-Mut2, with mutations of residues 71 to 73 and 74 to 76, respectively, appeared to have the greatest effect on virus-like particle and virus budding, showing a defect in M1 incorporation. Mutant viruses containing M2-Mut1 and M2-Mut2 failed to replicate in multistep growth analyses on wild-type (wt) MDCK cells and were able to form plaques only on MDCK cells stably expressing wt M2 protein. Compared to wt M2 protein, M2-Mut1 and M2-Mut2 were unable to efficiently coimmunoprecipitate with M1. Furthermore, statistical analysis of planar sheets of membrane from cells infected by virus containing M2-Mut1 revealed a reduction in M1-hemagglutinin (HA) and M2-HA clustering as well as a severe loss of clustering between M1 and M2. These results suggest an essential, direct interaction between the cytoplasmic tail of M2 and M1 that promotes the recruitment of the internal viral proteins and vRNA to the plasma membrane for efficient virus assembly to occur. PMID:18701586

Two synthetic Sp1-binding sites functionally substitute for the 21-base-pair repeat region to activate simian virus 40 growth in CV-1 cells.

PubMed Central

Lednicky, J; Folk, W R

1992-01-01

The 21-bp repeat region of simian virus 40 (SV40) activates viral transcription and DNA replication and contains binding sites for many cellular proteins, including Sp1, LSF, ETF, Ap2, Ap4, GT-1B, H16, and p53, and for the SV40 large tumor antigen. We have attempted to reduce the complexity of this region while maintaining its growth-promoting capacity. Deletion of the 21-bp repeat region from the SV40 genome delays the expression of viral early proteins and DNA replication and reduces virus production in CV-1 cells. Replacement of the 21-bp repeat region with two copies of DNA sequence motifs bound with high affinities by Sp1 promotes SV40 growth in CV-1 cells to nearly wild-type levels, but substitution by motifs bound less avidly by Sp1 or bound by other activator proteins does not restore growth. This indicates that Sp1 or a protein with similar sequence specificity is primarily responsible for the function of the 21-bp repeat region. We speculate about how Sp1 activates both SV40 transcription and DNA replication. Images PMID:1328672

Innate immune responses against foot-and-mouth disease virus: current understanding and future directions.

PubMed

Summerfield, Artur; Guzylack-Piriou, Laurence; Harwood, Lisa; McCullough, Kenneth C

2009-03-15

Foot-and-mouth disease (FMD) represents one of the most economically important diseases of farm animals. The basis for the threat caused by this virus is the high speed of replication, short incubation time, high contagiousness, and high mutation rate resulting in constant antigenic changes. Thus, although protective immune responses against FMD virus (FMDV) can be efficacious, the rapidity of virus replication and spread can outpace immune defence development and overrun the immune system. FMDV can also evade innate immune responses through its ability to shut down cellular protein synthesis, including IFN type I, in susceptible epithelial cells. This is important for virus evolution, as FMDV is quite sensitive to the action of IFN. Despite this, innate immune responses are probably induced in vivo, although detailed studies on this subject are lacking. Accordingly, this interaction of FMDV with cells of the innate immune system is of particular interest. Dendritic cells (DC) can be infected by FMDV and support viral RNA replication, and viral protein synthesis but the latter is inefficient or abortive, leading most often to incomplete replication and progeny virus release. As a result DC can be activated, and particularly in the case of plasmacytoid DC (pDC), this is manifest in terms of IFN-alpha release. Our current state of knowledge on innate immune responses induced by FMDV is still only at a relatively early stage of understanding. As we progress, the investigations in this area will help to improve the design of current vaccines and the development of novel control strategies against FMD.

Interferon signaling in Peromyscus leucopus confers a potent and specific restriction to vector-borne flaviviruses.

PubMed

Izuogu, Adaeze O; McNally, Kristin L; Harris, Stephen E; Youseff, Brian H; Presloid, John B; Burlak, Christopher; Munshi-South, Jason; Best, Sonja M; Taylor, R Travis

2017-01-01

Tick-borne flaviviruses (TBFVs), including Powassan virus and tick-borne encephalitis virus cause encephalitis or hemorrhagic fevers in humans with case-fatality rates ranging from 1-30%. Despite severe disease in humans, TBFV infection of natural rodent hosts has little noticeable effect. Currently, the basis for resistance to disease is not known. We hypothesize that the coevolution of flaviviruses with their respective hosts has shaped the evolution of potent antiviral factors that suppress virus replication and protect the host from lethal infection. In the current study, we compared virus infection between reservoir host cells and related susceptible species. Infection of primary fibroblasts from the white-footed mouse (Peromyscus leucopus, a representative host) with a panel of vector-borne flaviviruses showed up to a 10,000-fold reduction in virus titer compared to control Mus musculus cells. Replication of vesicular stomatitis virus was equivalent in P. leucopus and M. musculus cells suggesting that restriction was flavivirus-specific. Step-wise comparison of the virus infection cycle revealed a significant block to viral RNA replication, but not virus entry, in P. leucopus cells. To understand the role of the type I interferon (IFN) response in virus restriction, we knocked down signal transducer and activator of transcription 1 (STAT1) or the type I IFN receptor (IFNAR1) by RNA interference. Loss of IFNAR1 or STAT1 significantly relieved the block in virus replication in P. leucopus cells. The major IFN antagonist encoded by TBFV, nonstructural protein 5, was functional in P. leucopus cells, thus ruling out ineffective viral antagonism of the host IFN response. Collectively, this work demonstrates that the IFN response of P. leucopus imparts a strong and virus-specific barrier to flavivirus replication. Future identification of the IFN-stimulated genes responsible for virus restriction specifically in P. leucopus will yield mechanistic insight into efficient control of virus replication and may inform the development of antiviral therapeutics.

Sindbis virus proteins nsP1 and nsP2 contain homology to nonstructural proteins from several RNA plant viruses.

PubMed Central

Ahlquist, P; Strauss, E G; Rice, C M; Strauss, J H; Haseloff, J; Zimmern, D

1985-01-01

Although the genetic organization of tobacco mosaic virus (TMV) differs considerably from that of the tripartite viruses (alfalfa mosaic virus [AlMV] and brome mosaic virus [BMV]), all of these RNA plant viruses share three domains of homology among their nonstructural proteins. One such domain, common to the AlMV and BMV 2a proteins and the readthrough portion of TMV p183, is also homologous to the readthrough protein nsP4 of Sindbis virus (Haseloff et al., Proc. Natl. Acad. Sci. U.S.A. 81:4358-4362, 1984). Two more domains are conserved among the AlMV and BMV 1a proteins and TMV p126. We show here that these domains have homology with portions of the Sindbis proteins nsP1 and nsP2, respectively. These results strengthen the view that the four viruses share mechanistic similarities in their replication strategies and may be evolutionarily related. These results also suggest that either the AlMV 1a, BMV 1a, and TMV p126 proteins are multifunctional or Sindbis proteins nsP1 and nsP2 function together as subunits in a single complex. PMID:3968720

Inhibition mechanisms of baicalin and its phospholipid complex against DHAV-1 replication.

PubMed

Chen, Yun; Yuan, Wenjuan; Yang, Yuhui; Yao, Fangke; Ming, Ke; Liu, Jiaguo

2018-06-15

Duck hepatitis A virus type 1 (DHAV-1) is a serious infectious virus of ducklings. Recent study showed baicalin (BA) and baicalin phospholipid complex (BAPC) possessed anti-DHAV-1 effect. However, the antiviral mechanism is not clear. Therefore, the aim of the present work is to study influences and mechanisms of BA and BAPC on DHAV-1. The effects of BA and BAPC on DHAV-1 replication were analyzed by CCK-8 and RT-qPCR methods. And the results showed BA inhibited the replication of DHAV-1, and BAPC was more effective. Then, the influences of BA and BAPC on DHAV-1 protein translation and RNA synthesis were detected by western blot and RT-qPCR. Both BA and BAPC inhibited the protein translation, and BAPC did better. Furthermore, BAPC also inhibited the RNA synthesis. Afterwards, DHAV-1 IRES activity, DHAV-1 3D protein stability, and cellular Hsp70 expression were studied to in-depth understand the inhibition effects of BA and BAPC on DHAV-1 replication. The results indicated BA and BAPC dropped the protein translation via suppressing DHAV-1 IRES activity. Additionally, BAPC dropped the RNA synthesis via reducing the 3D protein stability and inhibiting cellular Hsp70 expression.

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

Expression Dynamics of Innate Immunity in Influenza Virus-Infected Swine

PubMed Central

Montoya, María; Foni, Emanuela; Solórzano, Alicia; Razzuoli, Elisabetta; Baratelli, Massimiliano; Bilato, Dania; Córdoba, Lorena; del Burgo, Maria Angeles Martín; Martinez, Jorge; Martinez-Orellana, Pamela; Chiapponi, Chiara; Perlin, David S.; del Real, Gustavo; Amadori, Massimo

2017-01-01

The current circulating swine influenza virus (IV) subtypes in Europe (H1N1, H1N2, and H3N2) are associated with clinical outbreaks of disease. However, we showed that pigs could be susceptible to other IV strains that are able to cross the species barrier. In this work, we extended our investigations into whether different IV strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. For this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a H3N2 Swine IV and four different H3N8 IV strains circulating in different animal species. Pigs had been clinically inspected and four subjects/group were sacrificed at 3, 6, and 21 days post infection. In the present study, all groups but mock exhibited antibody responses to IV nucleoprotein protein. Pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. Interestingly, pigs infected with avian and seal H3N8 strains also showed moderate lesions and viral replication, whereas equine and canine IVs did not cause overt pathological signs, and replication was barely detectable. Swine IV infection induced interferon (IFN)-alpha and interleukin-6 responses in bronchoalveolar fluids (BALF) at day 3 post infection, as opposed to the other non-swine-adapted virus strains. However, IFN-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of IFN-alpha genes. Remarkably, the Equine strain gave rise to a Serum Amyloid A response in BALF despite little if any replication. Each virus strain could be associated with expression of cytokine genes and/or proteins after infection. These responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system. PMID:28484702

Dynamics of virus shedding and in situ confirmation of chelonid herpesvirus 5 in Hawaiian green turtles with Fibropapillomatosis

USGS Publications Warehouse

Work, Thierry M.; Dagenais, Julie; Balazs, George H.; Schettle, Nelli; Ackermann, Mathias

2015-01-01

Cancers in humans and animals can be caused by viruses, but virus-induced tumors are considered to be poor sites for replication of intact virions (lytic replication). Fibropapillomatosis (FP) is a neoplastic disease associated with a herpesvirus, chelonid herpesvirus 5 (ChHV5), that affects green turtles globally. ChHV5 probably replicates in epidermal cells of tumors, because epidermal intranuclear inclusions (EIIs) contain herpesvirus-like particles. However, although EIIs are a sign of herpesvirus replication, they have not yet been firmly linked to ChHV5. Moreover, the dynamics of viral shedding in turtles are unknown, and there are no serological reagents to confirm actual presence of the specific ChHV5 virus in tissues. The investigators analyzed 381 FP tumors for the presence of EIIs and found that overall, about 35% of green turtles had lytic replication in skin tumors with 7% of tumors showing lytic replication. A few (11%) turtles accounted for more than 30% cases having lytic viral replication, and lytic replication was more likely in smaller tumors. To confirm that turtles were actively replicating ChHV5, a prerequisite for shedding, the investigators used antiserum raised against F-VP26, a predicted capsid protein of ChHV5 that localizes to the host cell nucleus during viral replication. This antiserum revealed F-VP26 in EIIs of tumors, thus confirming the presence of replicating ChHV5. In this light, it is proposed that unlike other virus-induced neoplastic diseases, FP is a disease that may depend on superspreaders, a few highly infectious individuals growing numerous small tumors permissive to viral production, for transmission of ChHV5.

Dynamics of Virus Shedding and In Situ Confirmation of Chelonid Herpesvirus 5 in Hawaiian Green Turtles With Fibropapillomatosis.

PubMed

Work, T M; Dagenais, J; Balazs, G H; Schettle, N; Ackermann, M

2015-11-01

Cancers in humans and animals can be caused by viruses, but virus-induced tumors are considered to be poor sites for replication of intact virions (lytic replication). Fibropapillomatosis (FP) is a neoplastic disease associated with a herpesvirus, chelonid herpesvirus 5 (ChHV5), that affects green turtles globally. ChHV5 probably replicates in epidermal cells of tumors, because epidermal intranuclear inclusions (EIIs) contain herpesvirus-like particles. However, although EIIs are a sign of herpesvirus replication, they have not yet been firmly linked to ChHV5. Moreover, the dynamics of viral shedding in turtles are unknown, and there are no serological reagents to confirm actual presence of the specific ChHV5 virus in tissues. The investigators analyzed 381 FP tumors for the presence of EIIs and found that overall, about 35% of green turtles had lytic replication in skin tumors with 7% of tumors showing lytic replication. A few (11%) turtles accounted for more than 30% cases having lytic viral replication, and lytic replication was more likely in smaller tumors. To confirm that turtles were actively replicating ChHV5, a prerequisite for shedding, the investigators used antiserum raised against F-VP26, a predicted capsid protein of ChHV5 that localizes to the host cell nucleus during viral replication. This antiserum revealed F-VP26 in EIIs of tumors, thus confirming the presence of replicating ChHV5. In this light, it is proposed that unlike other virus-induced neoplastic diseases, FP is a disease that may depend on superspreaders, a few highly infectious individuals growing numerous small tumors permissive to viral production, for transmission of ChHV5. © The Author(s) 2014.

Inhibition of hepatitis C virus replication through adenosine monophosphate-activated protein kinase-dependent and -independent pathways.

PubMed

Nakashima, Kenji; Takeuchi, Kenji; Chihara, Kazuyasu; Hotta, Hak; Sada, Kiyonao

2011-11-01

Persistent infection with hepatitis C virus (HCV) is closely correlated with type 2 diabetes. In this study, replication of HCV at different glucose concentrations was investigated by using J6/JFH1-derived cell-adapted HCV in Huh-7.5 cells and the mechanism of regulation of HCV replication by AMP-activated protein kinase (AMPK) as an energy sensor of the cell analyzed. Reducing the glucose concentration in the cell culture medium from 4.5 to 1.0 g/L resulted in suppression of HCV replication, along with activation of AMPK. Whereas treatment of cells with AMPK activator 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) suppressed HCV replication, compound C, a specific AMPK inhibitor, prevented AICAR's effect, suggesting that AICAR suppresses the replication of HCV by activating AMPK in Huh-7.5 cells. In contrast, compound C induced further suppression of HCV replication when the cells were cultured in low glucose concentrations or with metformin. These results suggest that low glucose concentrations and metformin have anti-HCV effects independently of AMPK activation. © 2011 The Societies and Blackwell Publishing Asia Pty Ltd.

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.

Genome sequence of foot-and-mouth disease virus outside the 3A region is also responsible for virus replication in bovine cells.

PubMed

Ma, Xueqing; Li, Pinghua; Sun, Pu; Lu, Zengjun; Bao, Huifang; Bai, Xingwen; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

2016-07-15

The deletion of residues 93-102 in non-structure protein 3A of foot-and-mouth disease virus (FMDV) is associated with the inability of FMDV to grow in bovine cells and attenuated virulence in cattle.Whereas, a previously reported FMDV strain O/HKN/21/70 harboring 93-102 deletion in 3A protein grew equally well in bovine and swine cells. This suggests that changes inFMDV genome sequence, in addition to 93-102 deletion in 3A, may also affectthe viral growth phenotype in bovine cellsduring infection and replication.However, it is nuclear that changes in which region (inside or outside of 3A region) influences FMDV growth phenotype in bovine cells.In this study, to determine the region in FMDV genomeaffecting viral growth phenotype in bovine cells, we constructed chimeric FMDVs, rvGZSB-HKN3A and rvHN-HKN3A, by introducing the 3A coding region of O/HKN/21/70 into the context of O/SEA/Mya-98 strain O/GZSB/2011 and O Cathay topotype strain O/HN/CHA/93, respectively, since O/GZSB/2011 containing full-length 3A protein replicated well in bovine and swine cells, and O/HN/CHA/93 harboring 93-102 deletion in 3A protein grew poorly in bovine cells.The chimeric virusesrvGZSB-HKN3A and rvHN-HKN3A displayed growth properties and plaque phenotypes similar to those of the parental virus rvGZSB and rv-HN in BHK-21 and primary fetal porcine kidney (FPK) cells. However, rvHN-HKN3A and rv-HN replicated poorly in primary fetal bovine kidney (FBK) cells with no visible plaques, and rvGZSB-HKN3A exhibited lower growth rate and smaller plaque size phenotypes than those of the parental virus in FBK cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the difference present in FMDV genome sequence outside the 3A coding region also have influence on FMDV replication ability in bovine cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Marek's disease virus protein kinase gene identified within the short unique region of the viral genome is not essential for viral replication in cell culture and vaccine-induced immunity in chickens.

PubMed

Sakaguchi, M; Urakawa, T; Hirayama, Y; Miki, N; Yamamoto, M; Zhu, G S; Hirai, K

1993-07-01

The open reading frame (ORF) of 1206 bp within the short unique region (Us) of Marek's disease virus type 1 (MDV1) shows significant homology with the herpes simplex virus type 1 US3 gene encoding protein kinase (PK). The lacZ gene of Escherichia coli was inserted within the ORF, designated MDV1-US3, of MDV1 K544 strain DNA by homologous recombination. The plaque-purified recombinant MDV1 stably expressed the beta-galactosidase encoded by the inserted lacZ gene in infected cells and replicated well as the parental K544 strain. Antibodies against both MDV1 antigen and beta-galactosidase were detected in the sera of chickens immunized with recombinant MDV1. Chickens vaccinated with the recombinant MDV1 were protected from challenge with virulent MDV1. The MDV1 US3 gene expressed by a baculovirus vector encoded a 44-kDa protein. Mouse antisera against the 44-kDa protein reacted with two proteins of 44 and 45 kDa in extracts of cells infected with MDV1 but not with MDV types 2 or 3. The PK activity was detected in immune complexes of the anti-44-kDa sera with extracts of cells infected with MDV1 but not with the recombinant MDV1. Thus, PK encoded from the MDV1-US3 is not essential for virus replication in cell culture and vaccine-induced immunity.

Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy

PubMed Central

Lei, Janet; Osen, Wolfram; Gardyan, Adriane; Hotz-Wagenblatt, Agnes; Wei, Guochao; Gissmann, Lutz; Eichmüller, Stefan; Löchelt, Martin

2015-01-01

The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy. PMID:26397953

Recombinant human parainfluenza virus type 2 with mutations in V that permit cellular interferon signaling are not attenuated in non-human primates

PubMed Central

Schaap-Nutt, Anne; D’Angelo, Christopher; Amaro-Carambot, Emerito; Nolan, Sheila M.; Davis, Stephanie; Wise, Shenelle-Marie; Higgins, Caraline; Bradley, Konrad; Kim, Olivia; Mayor, Reina; Skiadopoulos, Mario H.; Collins, Peter L.; Murphy, Brian R.; Schmidt, Alexander C.

2010-01-01

The HPIV2 V protein inhibits type I interferon (IFN) induction and signaling. To manipulate the V protein, whose coding sequence overlaps that of the polymerase-associated phosphoprotein (P), without altering the P protein, we generated an HPIV2 virus in which P and V are expressed from separate genes (rHPIV2-P+V). rHPIV2-P+V replicated like HPIV2-WT in vitro and in non-human primates. HPIV2-P+V was modified by introducing two separate mutations into the V protein to create rHPIV2-L101E/L102E and rHPIV2-Δ122–127. In contrast to HPIV2-WT, both mutant viruses were unable to degrade STAT2, leaving virus-infected cells susceptible to IFN. Neither mutant, nor HPIV2-WT, induced significant amounts of IFN-β in infected cells. Surprisingly, neither rHPIV2-L101E/L102E nor rHPIV2-Δ122–127 was attenuated in two species of non-human primates. This indicates that loss of HPIV2's ability to inhibit IFN signaling is insufficient to attenuate virus replication in vivo as long as IFN induction is still inhibited. PMID:20667570

Host cell interactome of PA protein of H5N1 influenza A virus in chicken cells.

PubMed

Wang, Qiao; Li, Qinghe; Liu, Ranran; Zheng, Maiqing; Wen, Jie; Zhao, Guiping

2016-03-16

Influenza A virus (IAV) heavily depends on viral-host protein interactions in order to replicate and spread. Identification of host factors that interact with viral proteins plays crucial roles in understanding the mechanism of IAV infection. Here we report the interaction landscape of H5N1 IAV PA protein in chicken cells through the use of affinity purification and mass spectrometry. PA protein was expressed in chicken cells and PA interacting complexes were captured by co-immunoprecipitation and analyzed by mass spectrometry. A total of 134 proteins were identified as PA-host interacting factors. Protein complexes including the minichromosome maintenance complex (MCM), 26S proteasome and the coat protein I (COPI) complex associated with PA in chicken cells, indicating the essential roles of these functional protein complexes during the course of IAV infection. Gene Ontology and pathway enrichment analysis both showed strong enrichment of PA interacting proteins in the category of DNA replication, covering genes such as PCNA, MCM2, MCM3, MCM4, MCM5 and MCM7. This study has uncovered the comprehensive interactome of H5N1 IAV PA protein in its chicken host and helps to establish the foundation for further investigation into the newly identified viral-host interactions. Influenza A virus (IAV) is a great threat to public health and avian production. However, the manner in which avian IAV recruits the host cellular machinery for replication and how the host antagonizes the IAV infection was previously poorly understood. Here we present the viral-host interactome of the H5N1 IAV PA protein and reveal the comprehensive association of host factors with PA. Copyright © 2016 Elsevier B.V. All rights reserved.

Protein Interactions during the Flavivirus and Hepacivirus Life Cycle*

PubMed Central

Bruening, Janina; Weigel, Bettina; Pietschmann, Thomas

2017-01-01

Protein–protein interactions govern biological functions in cells, in the extracellular milieu, and at the border between cells and extracellular space. Viruses are small intracellular parasites and thus rely on protein interactions to produce progeny inside host cells and to spread from cell to cell. Usage of host proteins by viruses can have severe consequences e.g. apoptosis, metabolic disequilibria, or altered cell proliferation and mobility. Understanding protein interactions during virus infection can thus educate us on viral infection and pathogenesis mechanisms. Moreover, it has led to important clinical translations, including the development of new therapeutic and vaccination strategies. Here, we will discuss protein interactions of members of the Flaviviridae family, which are small enveloped RNA viruses. Dengue virus, Zika virus and hepatitis C virus belong to the most prominent human pathogenic Flaviviridae. With a genome of roughly ten kilobases encoding only ten viral proteins, Flaviviridae display intricate mechanisms to engage the host cell machinery for their purpose. In this review, we will highlight how dengue virus, hepatitis C virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, yellow fever virus, and Zika virus proteins engage host proteins and how this knowledge helps elucidate Flaviviridae infection. We will specifically address the protein composition of the virus particle as well as the protein interactions during virus entry, replication, particle assembly, and release from the host cell. Finally, we will give a perspective on future challenges in Flaviviridae interaction proteomics and why we believe these challenges should be met. PMID:28077444

Modifications to the Foot-and-Mouth Disease Virus 2A Peptide: Influence on Polyprotein Processing and Virus Replication.

PubMed

Kjær, Jonas; Belsham, Graham J

2018-04-15

Foot-and-mouth disease virus (FMDV) has a positive-sense single-stranded RNA (ssRNA) genome that includes a single, large open reading frame encoding a polyprotein. The cotranslational "cleavage" of this polyprotein at the 2A/2B junction is mediated by the 2A peptide (18 residues in length) using a nonproteolytic mechanism termed "ribosome skipping" or "StopGo." Multiple variants of the 2A polypeptide with this property among the picornaviruses share a conserved C-terminal motif [D(V/I)E(S/T)NPG↓P]. The impact of 2A modifications within this motif on FMDV protein synthesis, polyprotein processing, and virus viability were investigated. Amino acid substitutions are tolerated at residues E 14 , S 15 , and N 16 within the 2A sequences of infectious FMDVs despite their reported "cleavage" efficiencies at the 2A/2B junction of only ca. 30 to 50% compared to that of the wild type (wt). In contrast, no viruses containing substitutions at residue P 17 , G 18 , or P 19 , which displayed little or no "cleavage" activity in vitro , were rescued, but wt revertants were obtained. The 2A substitutions impaired the replication of an FMDV replicon. Using transient-expression assays, it was shown that certain amino acid substitutions at residues E 14 , S 15 , N 16 , and P 19 resulted in partial "cleavage" of a protease-free polyprotein, indicating that these specific residues are not essential for cotranslational "cleavage." Immunofluorescence studies, using full-length FMDV RNA transcripts encoding mutant 2A peptides, indicated that the 2A peptide remained attached to adjacent proteins, presumably 2B. These results show that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity does not appear to be essential for the viability of FMDV. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes one of the most economically important diseases of farm animals. Cotranslational "cleavage" of the FMDV polyprotein precursor at the 2A/2B junction, termed StopGo, is mediated by the short 2A peptide through a nonproteolytic mechanism which leads to release of the nascent protein and continued translation of the downstream sequence. Improved understanding of this process will not only give a better insight into how this peptide influences the FMDV replication cycle but may also assist the application of this sequence in biotechnology for the production of multiple proteins from a single mRNA. Our data show that single amino acid substitutions in the 2A peptide can have a major influence on viral protein synthesis, virus viability, and polyprotein processing. They also indicate that efficient "cleavage" at the 2A/2B junction is required for optimal virus replication. However, maximal StopGo activity is not essential for the viability of FMDV. Copyright © 2018 American Society for Microbiology.

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

Chan, Shih-Ching; Lo, Shih-Yen; Graduate Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan

Research highlights: {yields} Lipid rafts are known to play an important role in virus entry and virus assembly of many viruses. {yields} However, HCV is the first example of the association of lipid raft with viral RNA replication. {yields} Our results in this manuscript demonstrate that purified HCV RCs with associated lipid raft membrane appeared as distinct particles of around 0.7 um under EM and AFM. {yields} Knockdown of proteins associated with lipid raft suppressed the HCV replication and reduced the number of these particles. {yields} To our knowledge, structures of HCV RCs were demonstrated at its first time inmore » this manuscript. -- Abstract: Hepatitis C viral RNA synthesis has been demonstrated to occur on a lipid raft membrane structure. Lipid raft membrane fraction purified by membrane flotation analysis was observed using transmission electron microscopy and atomic force microscopy. Particles around 0.7 um in size were found in lipid raft membrane fraction purified from hepatitis C virus (HCV) replicon but not their parental HuH7 cells. HCV NS5A protein was associated with these specialized particles. After several cycles of freezing-thawing, these particles would fuse into larger sizes up to 10 um. Knockdown of seven proteins associated with lipid raft (VAPA, COPG, RAB18, COMT, CDC42, DPP4, and KDELR2) of HCV replicon cells reduced the observed number of these particles and suppressed the HCV replication. Results in this study indicated that HCV replication complexes with associated lipid raft membrane form distinct particle structures of around 0.7 um as observed from transmission electron microscopy and atomic force microscopy.« less

Enhanced growth of influenza A virus by coinfection with human parainfluenza virus type 2.

PubMed

Goto, Hideo; Ihira, Hironobu; Morishita, Keiichi; Tsuchiya, Mitsuki; Ohta, Keisuke; Yumine, Natsuko; Tsurudome, Masato; Nishio, Machiko

2016-06-01

It has been reported that dual or multiple viruses can coinfect epithelial cells of the respiratory tract. However, little has been reported on in vitro interactions of coinfected viruses. To explore how coinfection of different viruses affects their biological property, we examined growth of influenza A virus (IAV) and human parainfluenza virus type 2 (hPIV2) during coinfection of Vero cells. We found that IAV growth was enhanced by coinfection with hPIV2. The enhanced growth of IAV was not reproduced by coinfection with an hPIV2 mutant with reduced cell fusion activity, or by ectopic expression of the V protein of hPIV2. In contrast, induction of cell fusion by ectopic expression of the hPIV2 HN and F proteins augments IAV growth. hPIV2 coinfection supported IAV growth in cells originated from the respiratory epithelium. The enhancement correlated closely with cell fusion ability of hPIV2 in those cells. These results indicate that cell fusion induced by hPIV2 infection is beneficial to IAV replication and that enhanced viral replication by coinfection with different viruses can modify their pathological consequences.

Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements.

PubMed

Prchal, Jan; Junkova, Petra; Strmiskova, Miroslava; Lipov, Jan; Hynek, Radovan; Ruml, Tomas; Hrabal, Richard

2011-09-01

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification. Copyright © 2011 Elsevier Inc. All rights reserved.

Partial characterisation of citrus leaf blotch virus, a new virus from Nagami kumquat.

PubMed

Galipienso, L; Vives, M C; Moreno, P; Milne, R G; Navarro, L; Guerri, J

2001-01-01

Citrus leaf blotch virus (CLBV) was purified from leaves of Nagami kumquat SRA-153 that showed bud union crease when propagated on Troyer citrange. Virions were filamentous particles (960 x 14 nm) containing a 42 kDa protein and a single-stranded RNA (ssRNA) of about 9,000 nt (Mr 3 x 10(6)). Infected tissue contained three species of double-stranded RNA (dsRNA) of Mr 6, 4.5 and 3.4 x 10(6). The nucleotide sequence of several complementary DNA (cDNA) clones showed significant similarities with replication-related proteins from plant filamentous viruses in several genera. A digoxigenin-labelled probe from one of these cDNA clones hybridised in Northern blots with ssRNA from virions and with the three dsRNA species, suggesting that the ssRNA is the genomic RNA of the virus, the largest dsRNA is its replicative form, and the two smaller dsRNAs probably replicative forms of 5' co-terminal subgenomic RNAs. CLBV was also detected in several citrus cultivars from Spain and Japan including Navelina sweet orange field trees propagated on Troyer citrange showing bud union crease; however, no virus could be detected in other citrus trees with similar symptoms. This indicates that CLBV is not restricted to kumquat SRA-153, but its involvement in causing the bud union disorder remains unclear.

Lassa and Mopeia virus replication in human monocytes/macrophages and in endothelial cells: different effects on IL-8 and TNF-alpha gene expression.

PubMed

Lukashevich, I S; Maryankova, R; Vladyko, A S; Nashkevich, N; Koleda, S; Djavani, M; Horejsh, D; Voitenok, N N; Salvato, M S

1999-12-01

Cells of the mononuclear and endothelial lineages are targets for viruses which cause hemorrhagic fevers (HF) such as the filoviruses Marburg and Ebola, and the arenaviruses Lassa and Junin. A recent model of Marburg HF pathogenesis proposes that virus directly causes endothelial cell damage and macrophage release of TNF-alpha which increases the permeability of endothelial monolayers [Feldmann et al. , 1996]. We show that Lassa virus replicates in human monocytes/macrophages and endothelial cells without damaging them. Human endothelial cells (HUVEC) are highly susceptible to infection by both Lassa and Mopeia (a non-pathogenic Lassa-related arenavirus). Whereas monocytes must differentiate into macrophages before supporting even low level production of these viruses, the virus yields in the culture medium of infected HUVEC cells reach more than 7 log10 PFU/ml without cellular damage. In contrast to filovirus, Lassa virus replication in monocytes/macrophages fails to stimulate TNF-alpha gene expression and even down-regulates LPS-stimulated TNF-alpha mRNA synthesis. The expression of IL-8, a prototypic proinflammatory CXC chemokine, was also suppressed in Lassa virus infected monocytes/macrophages and HUVEC on both the protein and mRNA levels. This contrasts with Mopeia virus infection of HUVEC in which neither IL-8 mRNA nor protein are reduced. The cumulative down-regulation of TNF-alpha and IL-8 expression could explain the absence of inflammatory and effective immune responses in severe cases of Lassa HF. Copyright 1999 Wiley-Liss, Inc.

Caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis.

PubMed

Richard, A; Tulasne, D

2012-03-08

Viral infection constitutes an unwanted intrusion that needs to be eradicated by host cells. On one hand, one of the first protective barriers set up to prevent viral replication, spread or persistence involves the induction of apoptotic cell death that aims to limit the availability of the cellular components for viral amplification. On the other hand, while they completely depend on the host molecular machinery, viruses also need to evade the cellular responses that are meant to destroy them. The existence of numerous antiapoptotic products within the viral kingdom proves that apoptosis constitutes a major threat that should better be bypassed. Among the different strategies developed to deal with apoptosis, one is based on what viruses do best: backfiring the cell on itself. Several unrelated viruses have been described to take advantage of apoptosis induction by expressing proteins targeted by caspases, the key effectors of apoptotic cell death. Caspase cleavage of these proteins results in various consequences, from logical apoptosis inhibition to more surprising enhancement or attenuation of viral replication. The present review aims at discussing the characterization and relevance of this post-translational modification that adds a new complexity in the already intricate host-apoptosis-virus triangle.

Strand-like structures and the nonstructural proteins 5, 3 and 1 are present in the nucleus of mosquito cells infected with dengue virus.

PubMed

Reyes-Ruiz, José M; Osuna-Ramos, Juan F; Cervantes-Salazar, Margot; Lagunes Guillen, Anel E; Chávez-Munguía, Bibiana; Salas-Benito, Juan S; Del Ángel, Rosa M

2018-02-01

Dengue virus (DENV) is an arbovirus, which replicates in the endoplasmic reticulum. Although replicative cycle takes place in the cytoplasm, some viral proteins such as NS5 and C are translocated to the nucleus during infection in mosquitoes and mammalian cells. To localized viral proteins in DENV-infected C6/36 cells, an immunofluorescence (IF) and immunoelectron microscopy (IEM) analysis were performed. Our results indicated that C, NS1, NS3 and NS5 proteins were found in the nucleus of DENV-infected C6/36 cells. Additionally, complex structures named strand-like structures (Ss) were observed in the nucleus of infected cells. Interestingly, the NS5 protein was located in these structures. Ss were absent in mock-infected cells, suggesting that DENV induces their formation in the nucleus of infected mosquito cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Ectromelia virus lacking the E3L ortholog is replication-defective and nonpathogenic but does induce protective immunity in a mouse strain susceptible to lethal mousepox.

PubMed

Frey, Tiffany R; Forsyth, Katherine S; Sheehan, Maura M; De Haven, Brian C; Pevarnik, Julia G; Hand, Erin S; Pizzorno, Marie C; Eisenlohr, Laurence C; Hersperger, Adam R

2018-05-01

All known orthopoxviruses, including ectromelia virus (ECTV), contain a gene in the E3L family. The protein product of this gene, E3, is a double-stranded RNA-binding protein. It can impact host range and is used by orthopoxviruses to combat cellular defense pathways, such as PKR and RNase L. In this work, we constructed an ECTV mutant with a targeted disruption of the E3L open reading frame (ECTVΔE3L). Infection with this virus resulted in an abortive replication cycle in all cell lines tested. We detected limited transcription of late genes but no significant translation of these mRNAs. Notably, the replication defects of ECTVΔE3L were rescued in human and mouse cells lacking PKR. ECTVΔE3L was nonpathogenic in BALB/c mice, a strain susceptible to lethal mousepox disease. However, infection with ECTVΔE3L induced protective immunity upon subsequent challenge with wild-type virus. In summary, E3L is an essential gene for ECTV. Copyright © 2018 Elsevier Inc. All rights reserved.

Infection of epithelial cells with dengue virus promotes the expression of proteins favoring the replication of certain viral strains.

PubMed

Martínez-Betancur, Viviana; Marín-Villa, Marcel; Martínez-Gutierrez, Marlén

2014-08-01

Dengue virus (DENV) is the causative agent of dengue and severe dengue. To understand better the dengue virus-host interaction, it is important to determine how the expression of cellular proteins is modified due to infection. Therefore, a comparison of protein expression was conducted in Vero cells infected with two different DENV strains, both serotype 2: DENV-2/NG (associated with dengue) and DENV-2/16681 (associated with severe dengue). The viability of the infected cells was determined, and neither strain induced cell death at 48 hr. In addition, the viral genomes and infectious viral particles were quantified, and the genome of the DENV-2/16681 strain was determined to have a higher replication rate compared with the DENV-2/NG strain. Finally, the proteins from infected and uninfected cultures were separated using two-dimensional gel electrophoresis, and the differentially expressed proteins were identified by mass spectrometry. Compared with the uninfected controls, the DENV-2/NG- and DENV-2/16681-infected cultures had five and six differentially expressed proteins, respectively. The most important results were observed when the infected cultures were compared to each other (DENV-2/NG vs. DENV-2/16681), and 18 differentially expressed proteins were identified. Based on their cellular functions, many of these proteins were linked to the increase in the replication efficiency of DENV. Among the proteins were calreticulin, acetyl coenzyme A, acetyl transferase, and fatty acid-binding protein. It was concluded that the infection of Vero cells with DENV-2/NG or DENV-2/16681 differentially modifies the expression of certain proteins, which can, in turn, facilitate infection. © 2013 Wiley Periodicals, Inc.

A new mechanism of interferon's antiviral action: Induction of autophagy, essential for paramyxovirus replication, is inhibited by the interferon stimulated gene, TDRD7.

PubMed

Subramanian, Gayatri; Kuzmanovic, Teodora; Zhang, Ying; Peter, Cara Beate; Veleeparambil, Manoj; Chakravarti, Ritu; Sen, Ganes C; Chattopadhyay, Saurabh

2018-01-01

The interferon (IFN) system represents the first line of defense against a wide range of viruses. Virus infection rapidly triggers the transcriptional induction of IFN-β and IFN Stimulated Genes (ISGs), whose protein products act as viral restriction factors by interfering with specific stages of virus life cycle, such as entry, transcription, translation, genome replication, assembly and egress. Here, we report a new mode of action of an ISG, IFN-induced TDRD7 (tudor domain containing 7) inhibited paramyxovirus replication by inhibiting autophagy. TDRD7 was identified as an antiviral gene by a high throughput screen of an ISG shRNA library for blocking IFN's protective effect against Sendai virus (SeV) replication. The antiviral activity of TDRD7 against SeV, human parainfluenza virus 3 and respiratory syncytial virus was confirmed by its genetic ablation or ectopic expression in several types of mouse and human cells. TDRD7's antiviral action was mediated by its ability to inhibit autophagy, a cellular catabolic process which was robustly induced by SeV infection and required for its replication. Mechanistic investigation revealed that TDRD7 interfered with the activation of AMP-dependent kinase (AMPK), an enzyme required for initiating autophagy. AMPK activity was required for efficient replication of several paramyxoviruses, as demonstrated by its genetic ablation or inhibition of its activity by TDRD7 or chemical inhibitors. Therefore, our study has identified a new antiviral ISG with a new mode of action.

Structure and Function of the N-Terminal Domain of the Vesicular Stomatitis Virus RNA Polymerase

PubMed Central

Qiu, Shihong; Ogino, Minako; Luo, Ming

2015-01-01

ABSTRACT Viruses have various mechanisms to duplicate their genomes and produce virus-specific mRNAs. Negative-strand RNA viruses encode their own polymerases to perform each of these processes. For the nonsegmented negative-strand RNA viruses, the polymerase is comprised of the large polymerase subunit (L) and the phosphoprotein (P). L proteins from members of the Rhabdoviridae, Paramyxoviridae, and Filoviridae share sequence and predicted secondary structure homology. Here, we present the structure of the N-terminal domain (conserved region I) of the L protein from a rhabdovirus, vesicular stomatitis virus, at 1.8-Å resolution. The strictly and strongly conserved residues in this domain cluster in a single area of the protein. Serial mutation of these residues shows that many of the amino acids are essential for viral transcription but not for mRNA capping. Three-dimensional alignments show that this domain shares structural homology with polymerases from other viral families, including segmented negative-strand RNA and double-stranded RNA (dsRNA) viruses. IMPORTANCE Negative-strand RNA viruses include a diverse set of viral families that infect animals and plants, causing serious illness and economic impact. The members of this group of viruses share a set of functionally conserved proteins that are essential to their replication cycle. Among this set of proteins is the viral polymerase, which performs a unique set of reactions to produce genome- and subgenome-length RNA transcripts. In this article, we study the polymerase of vesicular stomatitis virus, a member of the rhabdoviruses, which has served in the past as a model to study negative-strand RNA virus replication. We have identified a site in the N-terminal domain of the polymerase that is essential to viral transcription and that shares sequence homology with members of the paramyxoviruses and the filoviruses. Newly identified sites such as that described here could prove to be useful targets in the design of new therapeutics against negative-strand RNA viruses. PMID:26512087

Overexpression of Promyelocytic Leukemia Protein Precludes the Dispersal of ND10 Structures and Has No Effect on Accumulation of Infectious Herpes Simplex Virus 1 or Its Proteins

PubMed Central

Lopez, Pascal; Jacob, Robert J.; Roizman, Bernard

2002-01-01

A key early event in the replication of herpes simplex virus 1 (HSV-1) is the localization of infected-cell protein no. 0 (ICP0) in nuclear structures knows as ND10 or promyelocytic leukemia oncogenic domains (PODs). This is followed by dispersal of ND10 constituents such as the promyelocytic leukemia protein (PML), CREB-binding protein (CBP), and Daxx. Numerous experiments have shown that this dispersal is mediated by ICP0. PML is thought to be the organizing structural component of ND10. To determine whether the virus targets PML because it is inimical to viral replication, telomerase-immortalized human foreskin fibroblasts and HEp-2 cells were transduced with wild-type baculovirus or a baculovirus expressing the Mr 69,000 form of PML. The transduced cultures were examined for expression and localization of PML in mock-infected and HSV-1-infected cells. The results obtained from studies of cells overexpressing PML were as follows. (i) Transduced cells accumulate large amounts of unmodified and SUMO-I-modified PML. (ii) Mock-infected cells exhibited enlarged ND10 structures containing CBP and Daxx in addition to PML. (iii) In infected cells, ICP0 colocalized with PML in ND10 early in infection, but the two proteins did not overlap or were juxtaposed in orderly structures. (iv) The enlarged ND10 structures remained intact at least until 12 h after infection and retained CBP and Daxx in addition to PML. (v) Overexpression of PML had no effect on the accumulation of viral proteins representative of α, β, or γ groups and had no effect on the accumulation of infectious virus in cells infected with wild-type virus or a mutant (R7910) from which the α0 genes had been deleted. These results indicate the following: (i) PML overexpressed in transduced cells cannot be differentiated from endogenous PML with respect to sumoylation and localization in ND10 structures. (ii) PML does not affect viral replication or the changes in the localization of ICP0 through infection. (iii) Disaggregation of ND10 structures is not an obligatory event essential for viral replication. PMID:12186918

A single amino acid change, Q114R, in the cleavage-site sequence of Newcastle disease virus fusion protein attenuates viral replication and pathogenicity.

PubMed

Samal, Sweety; Kumar, Sachin; Khattar, Sunil K; Samal, Siba K

2011-10-01

A key determinant of Newcastle disease virus (NDV) virulence is the amino acid sequence at the fusion (F) protein cleavage site. The NDV F protein is synthesized as an inactive precursor, F(0), and is activated by proteolytic cleavage between amino acids 116 and 117 to produce two disulfide-linked subunits, F(1) and F(2). The consensus sequence of the F protein cleavage site of virulent [(112)(R/K)-R-Q-(R/K)-R↓F-I(118)] and avirulent [(112)(G/E)-(K/R)-Q-(G/E)-R↓L-I(118)] strains contains a conserved glutamine residue at position 114. Recently, some NDV strains from Africa and Madagascar were isolated from healthy birds and have been reported to contain five basic residues (R-R-R-K-R↓F-I/V or R-R-R-R-R↓F-I/V) at the F protein cleavage site. In this study, we have evaluated the role of this conserved glutamine residue in the replication and pathogenicity of NDV by using the moderately pathogenic Beaudette C strain and by making Q114R, K115R and I118V mutants of the F protein in this strain. Our results showed that changing the glutamine to a basic arginine residue reduced viral replication and attenuated the pathogenicity of the virus in chickens. The pathogenicity was further reduced when the isoleucine at position 118 was substituted for valine.

The phosphoprotein genes of measles viruses from subacute sclerosing panencephalitis cases encode functional as well as non-functional proteins and display reduced editing.

PubMed

Millar, E L; Rennick, L J; Weissbrich, B; Schneider-Schaulies, J; Duprex, W P; Rima, B K

2016-01-04

Products expressed from the second (P/V/C) gene are important in replication and abrogating innate immune responses during acute measles virus (MV) infection. Thirteen clone sets were derived from the P/V/C genes of measles virus (MV) RNA extracted from brains of a unique collection of seven cases of subacute sclerosing panencephalitis (SSPE) caused by persistent MV in the central nervous system (CNS). Whether these functions are fully maintained when MV replicates in the CNS has not been previously determined. Co-transcriptional editing of the P mRNAs by non-template insertion of guanine (G) nucleotides, which generates mRNAs encoding the viral V protein, occurs much less frequently (9%) in the SSPE derived samples than during the acute infection (30-50%). Thus it is likely that less V protein, which is involved in combatting the innate immune response, is produced. The P genes in MV from SSPE cases were not altered by biased hypermutation but exhibited a high degree of variation within each case. Most but not all SSPE derived phospho-(P) proteins were functional in mini genome replication/transcription assays. An eight amino acid truncation of the carboxyl-terminus made the P protein non-functional while the insertion of an additional glycine residue by insertion of G nucleotides at the editing site had no effect on protein function. Copyright © 2015 Elsevier B.V. All rights reserved.

Ring finger protein 39 genetic variants associate with HIV-1 plasma viral loads and its replication in cell culture.

PubMed

Lin, Ying-Ju; Chen, Chia-Yen; Jeang, Kuan-Teh; Liu, Xiang; Wang, Jen-Hsien; Hung, Chien-Hui; Tsang, Hsinyi; Lin, Ting-Hsu; Liao, Chiu-Chu; Huang, Shao-Mei; Lin, Cheng-Wen; Ho, Mao-Wang; Chien, Wen-Kuei; Chen, Jin-Hua; Ho, Tsung-Jung; Tsai, Fuu-Jen

2014-01-01

The human immunodeficiency virus (HIV-1) exploits host proteins to complete its life cycle. Genome-wide siRNA approaches suggested that host proteins affect HIV-1 replication. However, the results barely overlapped. RING finger protein 39 (RNF39) has been identified from genome-wide association studies. However, its function during HIV-1 replication remains unclear. We investigated the relationship between common RNF39 genetic variants and HIV-1 viral loads. The effect of RNF39 protein knockdown or overexpression on HIV-1 replication was then investigated in different cell lines. Two genetic variants were associated with HIV-1 viral loads. Patients with the ht1-GG/GG haplotype presented lower RNF39 expression levels and lower HIV-1 viral load. RNF39 knockdown inhibited HIV-1 expression. RNF39 protein may be involved in HIV-1 replication as observed in genetic studies on patients with HIV-1 and in in vitro cell cultures.

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

PubMed Central

Herfst, Sander; Bestebroer, Theo M.; Vaes, Vincent P.; van der Hoeven, Barbara; Koster, Abraham J.; Kremers, Gert-Jan; Scott, Dana P.; Gultyaev, Alexander P.; Sorell, Erin M.; de Graaf, Miranda; Bárcena, Montserrat; Rimmelzwaan, Guus F.; Fouchier, Ron A.

2015-01-01

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses. PMID:26241861

Bidirectional enhancing activities between human T cell leukemia-lymphoma virus type I and human cytomegalovirus in human term syncytiotrophoblast cells cultured in vitro.

PubMed

Tóth, F D; Aboagye-Mathiesen, G; Szabó, J; Liu, X; Mosborg-Petersen, P; Kiss, J; Hager, H; Zdravkovic, M; Andirkó, I; Aranyosi, J

1995-12-01

The syncytiotrophoblast layer of the human placenta has an important role in limiting transplacental viral spread from mother to fetus. Human cytomegalovirus (HCMV) is capable of establishing a latent infection in syncytiotrophoblast cells, with restriction of gene expression to immediate-early and early proteins. We analyzed the extent of replication of human T cell leukemia-lymphoma virus type I (HTLV-I) in human term syncytiotrophoblasts infected with HTLV-I alone or coinfected with HTLV-I and HCMV. Although syncytiotrophoblasts could be infected with cell-free HTLV-I, no viral protein expression was found in the singly infected cells. On the contrary, coinfection of the cells with HTLV-I and HCMV resulted in simultaneous replication of both viruses. Bidirectional enhancing activities between HTLV-I and HCMV were mediated primarily by the Tax and immediate-early proteins, respectively. The stimulatory effect of HTLV-I Tax on HCMV replication appeared to be mediated partly by tumor necrosis factor beta and transforming growth factor beta-1. We observed formation of pseudotypes with HTLV-I nucleocapsids within HCMV envelopes, whereas HCMV was not pseudotyped by HTLV-I envelopes in dually infected syncytiotrophoblast cells. Our data suggest that in vivo dual infection of syncytiotrophoblast cells with HTLV-I and HCMV may facilitate the transplacental transmission of both viruses.

Diversity, Replication, Pathogenicity and Cell Biology of Crimean Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2006-10-01

recombinant protein produced and purified in E . coli was able to bind to ssRNA in a dot blot filter binding assay. In order to identify domains in the N...Fig. 1. RNA binding domains of the N protein of CCHFV. The depicted GST-fusion proteins were expressed in E . coli and purified using a...detected and NSm protein produced after cleavage of the glycoprotein precursor in virus infected cells. The NSm is stable and transported to the Golgi

Novel cis-acting replication element in the adeno-associated virus type 2 genome is involved in amplification of integrated rep-cap sequences.

PubMed

Nony, P; Tessier, J; Chadeuf, G; Ward, P; Giraud, A; Dugast, M; Linden, R M; Moullier, P; Salvetti, A

2001-10-01

This study identifies a region of the adeno-associated virus type 2 (AAV-2) rep gene (nucleotides 190 to 540 of wild-type AAV-2) as a cis-acting Rep-dependent element able to promote the replication of transiently transfected plasmids. This viral element is also shown to be involved in the amplification of integrated sequences in the presence of adenovirus and Rep proteins.

Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

PubMed

Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

2018-02-28

Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of MV within the cell. Further studies demonstrated that annexin A2 interacts with the MV matrix (M) protein and mediates the localization of the M protein at the plasma membrane where MV particles are formed. The M protein lines the inner surface of the MV envelope membrane and plays a role in MV particle formation. Our results provide useful information for the understanding of the MV replication process and potential development of anti-viral agents. Copyright © 2018 American Society for Microbiology.

Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.

PubMed

Wang, Weiping; Manna, David; Simmons, Daniel T

2007-05-01

The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.

The vaccinia virus I3L gene product is localized to a complex endoplasmic reticulum-associated structure that contains the viral parental DNA.

PubMed

Welsch, Sonja; Doglio, Laura; Schleich, Sibylle; Krijnse Locker, Jacomine

2003-05-01

The vaccinia virus (VV) I3L gene product is a single-stranded DNA-binding protein made early in infection that localizes to the cytoplasmic sites of viral DNA replication (S. C. Rochester and P. Traktman, J. Virol. 72:2917-2926, 1998). Surprisingly, when replication was blocked, the protein localized to distinct cytoplasmic spots (A. Domi and G. Beaud, J. Gen. Virol. 81:1231-1235, 2000). Here these I3L-positive spots were characterized in more detail. By using an anti-I3L peptide antibody we confirmed that the protein localized to the cytoplasmic sites of viral DNA replication by both immunofluorescence and electron microscopy (EM). Before replication had started or when replication was inhibited with hydroxyurea or cytosine arabinoside, I3L localized to distinct cytoplasmic punctate structures of homogeneous size. We show that these structures are not incoming cores or cytoplasmic sites of VV early mRNA accumulation. Instead, morphological and quantitative data indicate that they are specialized sites where the parental DNA accumulates after its release from incoming viral cores. By EM, these sites appeared as complex, electron-dense structures that were intimately associated with the cellular endoplasmic reticulum (ER). By double labeling of cryosections we show that they contain DNA and a viral early protein, the gene product of E8R. Since E8R is a membrane protein that is able to bind to DNA, the localization of this protein to the I3L puncta suggests that they are composed of membranes. The results are discussed in relation to our previous data showing that the process of viral DNA replication also occurs in close association with the ER.

Differential effect of acute and persistent Junin virus infections on the nucleo-cytoplasmic trafficking and expression of heterogeneous nuclear ribonucleoproteins type A and B.

PubMed

Maeto, Cynthia A; Knott, María E; Linero, Florencia N; Ellenberg, Paula C; Scolaro, Luis A; Castilla, Viviana

2011-09-01

Heterogeneous nuclear ribonucleoproteins A and B (hnRNPs A/B), cellular RNA-binding proteins that participate in splicing, trafficking, translation and turnover of mRNAs, have been implicated in the life cycles of several cytoplasmic RNA viruses. Here, we demonstrate that silencing of hnRNPs A1 and A2 significantly reduces the replication of the arenavirus Junín virus (JUNV), the aetiological agent of Argentine haemorrhagic fever. While acute JUNV infection did not modify total levels of expression of hnRNPs A/B in comparison with uninfected cells, non-cytopathic persistent infection exhibited low levels of these cell proteins. Furthermore, acutely infected cells showed a cytoplasmic relocalization of overexpressed hnRNP A1, probably related to the involvement of this protein in virus replicative cycle. This cytoplasmic accumulation was also observed in cells expressing viral nucleoprotein (N), and co-immunoprecipitation studies revealed the interaction between hnRNP A1 and N protein. By contrast, a predominantly nuclear distribution of overexpressed hnRNP A1 was found during persistent infection, even in the presence of endogenous or overexpressed N protein, indicating a differential modulation of nucleo-cytoplasmic trafficking in acute and persistent JUNV infections.

Identification of human hnRNP C1/C2 as a dengue virus NS1-interacting protein

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

Noisakran, Sansanee; Medical Molecular Biology Unit, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Adulyadejvikrom Building; Sengsai, Suchada

Dengue virus nonstructural protein 1 (NS1) is a key glycoprotein involved in the production of infectious virus and the pathogenesis of dengue diseases. Very little is known how NS1 interacts with host cellular proteins and functions in dengue virus-infected cells. This study aimed at identifying NS1-interacting host cellular proteins in dengue virus-infected cells by employing co-immunoprecipitation, two-dimensional gel electrophoresis, and mass spectrometry. Using lysates of dengue virus-infected human embryonic kidney cells (HEK 293T), immunoprecipitation with an anti-NS1 monoclonal antibody revealed eight isoforms of dengue virus NS1 and a 40-kDa protein, which was subsequently identified by quadrupole time-of-flight tandem mass spectrometrymore » (Q-TOF MS/MS) as human heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Further investigation by co-immunoprecipitation and co-localization confirmed the association of hnRNP C1/C2 and dengue virus NS1 proteins in dengue virus-infected cells. Their interaction may have implications in virus replication and/or cellular responses favorable to survival of the virus in host cells.« less

Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus.

PubMed

Ma, Xueqing; Li, Pinghua; Sun, Pu; Bai, Xingwen; Bao, Huifang; Lu, Zengjun; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

2015-04-01

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94-101, 93-101, and 93-102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93-102 residues to 8 aa residues at positions 94-101 in 3A protein. Copyright © 2015 Elsevier B.V. All rights reserved.

SUMO1 depletion prevents lipid droplet accumulation and HCV replication.

PubMed

Akil, Abdellah; Wedeh, Ghaith; Zahid Mustafa, Mohammad; Gassama-Diagne, Ama

2016-01-01

Infection by hepatitis C virus (HCV) is a major public-health problem. Chronic infection often leads to cirrhosis, steatosis, and hepatocellular carcinoma. The life cycle of HCV depends on the host cell machinery and involves intimate interaction between viral and host proteins. However, the role of host proteins in the life cycle of HCV remains poorly understood. Here, we identify the small ubiquitin-related modifier (SUMO1) as a key host factor required for HCV replication. We performed a series of cell biology and biochemistry experiments using the HCV JFH-1 (Japanese fulminate hepatitis 1) genotype 2a strain, which produces infectious particles and recapitulates all the steps of the HCV life cycle. We observed that SUMO1 is upregulated in Huh7.5 infected cells. Reciprocally, SUMO1 was found to regulate the expression of viral core protein. Moreover, knockdown of SUMO1 using specific siRNA influenced the accumulation of lipid droplets and reduced HCV replication as measured by qRT-PCR. Thus, we identify SUMO1 as a key host factor required for HCV replication. To our knowledge, this is the first report showing that SUMO1 regulates lipid droplets in the context of viral infection. Our report provides a meaningful insight into how HCV replicates and interacts with host proteins and is of significant importance for the field of HCV and RNA viruses.

Accumulation of Herpes Simplex Virus Type 1 Early and Leaky-Late Proteins Correlates with Apoptosis Prevention in Infected Human HEp-2 Cells

PubMed Central

Aubert, Martine; Rice, Stephen A.; Blaho, John A.

2001-01-01

We previously reported that a recombinant ICP27-null virus stimulated, but did not prevent, apoptosis in human HEp-2 cells during infection (M. Aubert and J. A. Blaho, J. Virol. 73:2803–2813, 1999). In the present study, we used a panel of 15 recombinant ICP27 mutant viruses to determine which features of herpes simplex virus type 1 (HSV-1) replication are required for the apoptosis-inhibitory activity. Each virus was defined experimentally as either apoptotic, partially apoptotic, or nonapoptotic based on infected HEp-2 cell morphologies, percentages of infected cells with condensed chromatin, and patterns of specific cellular death factor processing. Viruses d27-1, d1-5, d1-2, M11, M15, M16, n504R, n406R, n263R, and n59R are apoptotic or partially apoptotic in HEp-2 cells and severely defective for growth in Vero cells. Viruses d2-3, d3-4, d4-5, d5-6, and d6-7 are nonapoptotic, demonstrating that ICP27 contains a large amino-terminal region, including its RGG box RNA binding domain, which is not essential for apoptosis prevention. Accumulations of viral TK, VP16, and gD but not gC, ICP22, or ICP4 proteins correlated with prevention of apoptosis during the replication of these viruses. Of the nonapoptotic viruses, d4-5 did not produce gC, indicating that accumulation of true late gene products is not necessary for the prevention process. Analyses of viral DNA synthesis in HEp-2 cells indicated that apoptosis prevention by HSV-1 requires that the infection proceeds to the stage in which viral DNA replication takes place. Infections performed in the presence of the drug phosphonoacetic acid confirmed that the process of viral DNA synthesis and the accumulation of true late (γ2) proteins are not required for apoptosis prevention. Based on our results, we conclude that the accumulation of HSV-1 early (β) and leaky-late (γ1) proteins correlates with the prevention of apoptosis in infected HEp-2 cells. PMID:11134315

Proteolytic Processing of Turnip Yellow Mosaic Virus Replication Proteins and Functional Impact on Infectivity▿

PubMed Central

Jakubiec, Anna; Drugeon, Gabrièle; Camborde, Laurent; Jupin, Isabelle

2007-01-01

Turnip yellow mosaic virus (TYMV), a positive-strand RNA virus belonging to the alphavirus-like supergroup, encodes its nonstructural replication proteins as a 206K precursor with domains indicative of methyltransferase (MT), proteinase (PRO), NTPase/helicase (HEL), and polymerase (POL) activities. Subsequent processing of 206K generates a 66K protein encompassing the POL domain and uncharacterized 115K and 85K proteins. Here, we demonstrate that TYMV proteinase mediates an additional cleavage between the PRO and HEL domains of the polyprotein, generating the 115K protein and a 42K protein encompassing the HEL domain that can be detected in plant cells using a specific antiserum. Deletion and substitution mutagenesis experiments and sequence comparisons indicate that the scissile bond is located between residues Ser879 and Gln880. The 85K protein is generated by a host proteinase and is likely to result from nonspecific proteolytic degradation occurring during protein sample extraction or analysis. We also report that TYMV proteinase has the ability to process substrates in trans in vivo. Finally, we examined the processing of the 206K protein containing native, mutated, or shuffled cleavage sites and analyzed the effects of cleavage mutations on viral infectivity and RNA synthesis by performing reverse-genetics experiments. We present evidence that PRO/HEL cleavage is critical for productive virus infection and that the impaired infectivity of PRO/HEL cleavage mutants is due mainly to defective synthesis of positive-strand RNA. PMID:17686855

Cytoskeletal protein transformation in HIV-1-infected macrophage giant cells.

PubMed

Kadiu, Irena; Ricardo-Dukelow, Mary; Ciborowski, Pawel; Gendelman, Howard E

2007-05-15

The mechanisms linking HIV-1 replication, macrophage biology, and multinucleated giant cell formation are incompletely understood. With the advent of functional proteomics, the characterization, regulation, and transformation of HIV-1-infected macrophage-secreted proteins can be ascertained. To these ends, we performed proteomic analyses of culture fluids derived from HIV-1 infected monocyte-derived macrophages. Robust reorganization, phosphorylation, and exosomal secretion of the cytoskeletal proteins profilin 1 and actin were observed in conjunction with productive viral replication and giant cell formation. Actin and profilin 1 recruitment to the macrophage plasma membrane paralleled virus-induced cytopathicity, podosome formation, and cellular fusion. Poly-l-proline, an inhibitor of profilin 1-mediated actin polymerization, inhibited cytoskeletal transformations and suppressed, in part, progeny virion production. These data support the idea that actin and profilin 1 rearrangement along with exosomal secretion affect viral replication and cytopathicity. Such events favor the virus over the host cell and provide insights into macrophage defense mechanisms used to contain viral growth and how they may be affected during progressive HIV-1 infection.

A Proline-Rich N-Terminal Region of the Dengue Virus NS3 Is Crucial for Infectious Particle Production.

PubMed

Gebhard, Leopoldo G; Iglesias, Néstor G; Byk, Laura A; Filomatori, Claudia V; De Maio, Federico A; Gamarnik, Andrea V

2016-06-01

Dengue virus is currently the most important insect-borne viral human pathogen. Viral nonstructural protein 3 (NS3) is a key component of the viral replication machinery that performs multiple functions during viral replication and participates in antiviral evasion. Using dengue virus infectious clones and reporter systems to dissect each step of the viral life cycle, we examined the requirements of different domains of NS3 on viral particle assembly. A thorough site-directed mutagenesis study based on solvent-accessible surface areas of NS3 revealed that, in addition to being essential for RNA replication, different domains of dengue virus NS3 are critically required for production of infectious viral particles. Unexpectedly, point mutations in the protease, interdomain linker, or helicase domain were sufficient to abolish infectious particle formation without affecting translation, polyprotein processing, or RNA replication. In particular, we identified a novel proline-rich N-terminal unstructured region of NS3 that contains several amino acid residues involved in infectious particle formation. We also showed a new role for the interdomain linker of NS3 in virion assembly. In conclusion, we present a comprehensive genetic map of novel NS3 determinants for viral particle assembly. Importantly, our results provide evidence of a central role of NS3 in the coordination of both dengue virus RNA replication and particle formation. Dengue virus is an important human pathogen, and its prominence is expanding globally; however, basic aspects of its biology are still unclear, hindering the development of effective therapeutic and prophylactic treatments. Little is known about the initial steps of dengue and other flavivirus particle assembly. This process involves a complex interplay between viral and cellular components, making it an attractive antiviral target. Unpredictably, we identified spatially separated regions of the large NS3 viral protein as determinants for dengue virus particle assembly. NS3 is a multifunctional enzyme that participates in different steps of the viral life cycle. Using reporter systems to dissect different viral processes, we identified a novel N-terminal unstructured region of the NS3 protein as crucial for production of viral particles. Based on our findings, we propose new ideas that include NS3 as a possible scaffold for the viral assembly process. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Protein Interactions during the Flavivirus and Hepacivirus Life Cycle.

PubMed

Gerold, Gisa; Bruening, Janina; Weigel, Bettina; Pietschmann, Thomas

2017-04-01

Protein-protein interactions govern biological functions in cells, in the extracellular milieu, and at the border between cells and extracellular space. Viruses are small intracellular parasites and thus rely on protein interactions to produce progeny inside host cells and to spread from cell to cell. Usage of host proteins by viruses can have severe consequences e.g. apoptosis, metabolic disequilibria, or altered cell proliferation and mobility. Understanding protein interactions during virus infection can thus educate us on viral infection and pathogenesis mechanisms. Moreover, it has led to important clinical translations, including the development of new therapeutic and vaccination strategies. Here, we will discuss protein interactions of members of the Flaviviridae family, which are small enveloped RNA viruses. Dengue virus, Zika virus and hepatitis C virus belong to the most prominent human pathogenic Flaviviridae With a genome of roughly ten kilobases encoding only ten viral proteins, Flaviviridae display intricate mechanisms to engage the host cell machinery for their purpose. In this review, we will highlight how dengue virus, hepatitis C virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, yellow fever virus, and Zika virus proteins engage host proteins and how this knowledge helps elucidate Flaviviridae infection. We will specifically address the protein composition of the virus particle as well as the protein interactions during virus entry, replication, particle assembly, and release from the host cell. Finally, we will give a perspective on future challenges in Flaviviridae interaction proteomics and why we believe these challenges should be met. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Natural polymorphisms in human APOBEC3H and HIV-1 Vif combine in primary T lymphocytes to affect viral G-to-A mutation levels and infectivity.

PubMed

Refsland, Eric W; Hultquist, Judd F; Luengas, Elizabeth M; Ikeda, Terumasa; Shaban, Nadine M; Law, Emily K; Brown, William L; Reilly, Cavan; Emerman, Michael; Harris, Reuben S

2014-11-01

The Vif protein of HIV-1 allows virus replication by degrading several members of the host-encoded APOBEC3 family of DNA cytosine deaminases. Polymorphisms in both host APOBEC3 genes and the viral vif gene have the potential to impact the extent of virus replication among individuals. The most genetically diverse of the seven human APOBEC3 genes is APOBEC3H with seven known haplotypes. Overexpression studies have shown that a subset of these variants express stable and active proteins, whereas the others encode proteins with a short half-life and little, if any, antiviral activity. We demonstrate that these stable/unstable phenotypes are an intrinsic property of endogenous APOBEC3H proteins in primary CD4+ T lymphocytes and confer differential resistance to HIV-1 infection in a manner that depends on natural variation in the Vif protein of the infecting virus. HIV-1 with a Vif protein hypo-functional for APOBEC3H degradation, yet fully able to counteract APOBEC3D, APOBEC3F, and APOBEC3G, was susceptible to restriction and hypermutation in stable APOBEC3H expressing lymphocytes, but not in unstable APOBEC3H expressing lymphocytes. In contrast, HIV-1 with hyper-functional Vif counteracted stable APOBEC3H proteins as well as all other endogenous APOBEC3s and replicated to high levels. We also found that APOBEC3H protein levels are induced over 10-fold by infection. Finally, we found that the global distribution of stable/unstable APOBEC3H haplotypes correlates with the distribution a critical hyper/hypo-functional Vif amino acid residue. These data combine to strongly suggest that stable APOBEC3H haplotypes present as in vivo barriers to HIV-1 replication, that Vif is capable of adapting to these restrictive pressures, and that an evolutionary equilibrium has yet to be reached.

Hepatitis C virus (HCV) induces formation of stress granules whose proteins regulate HCV RNA replication and virus assembly and egress.

PubMed

Garaigorta, Urtzi; Heim, Markus H; Boyd, Bryan; Wieland, Stefan; Chisari, Francis V

2012-10-01

Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

PubMed

Jin, H; Elliott, R M

1993-03-01

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities.

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

PubMed Central

Jin, H; Elliott, R M

1993-01-01

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities. Images PMID:8437222

The Acyclic Retinoid Peretinoin Inhibits Hepatitis C Virus Replication and Infectious Virus Release in Vitro

NASA Astrophysics Data System (ADS)

Shimakami, Tetsuro; Honda, Masao; Shirasaki, Takayoshi; Takabatake, Riuta; Liu, Fanwei; Murai, Kazuhisa; Shiomoto, Takayuki; Funaki, Masaya; Yamane, Daisuke; Murakami, Seishi; Lemon, Stanley M.; Kaneko, Shuichi

2014-04-01

Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.

Cucumber Necrosis Virus Recruits Cellular Heat Shock Protein 70 Homologs at Several Stages of Infection

PubMed Central

Alam, Syed Benazir

2015-01-01

ABSTRACT RNA viruses often depend on host factors for multiplication inside cells due to the constraints of their small genome size and limited coding capacity. One such factor that has been exploited by several plant and animal viruses is heat shock protein 70 (HSP70) family homologs which have been shown to play roles for different viruses in viral RNA replication, viral assembly, disassembly, and cell-to-cell movement. Using next generation sequence analysis, we reveal that several isoforms of Hsp70 and Hsc70 transcripts are induced to very high levels during cucumber necrosis virus (CNV) infection of Nicotiana benthamiana and that HSP70 proteins are also induced by at least 10-fold. We show that HSP70 family protein homologs are co-opted by CNV at several stages of infection. We have found that overexpression of Hsp70 or Hsc70 leads to enhanced CNV genomic RNA, coat protein (CP), and virion accumulation, whereas downregulation leads to a corresponding decrease. Hsc70-2 was found to increase solubility of CNV CP in vitro and to increase accumulation of CNV CP independently of viral RNA replication during coagroinfiltration in N. benthamiana. In addition, virus particle assembly into virus-like particles in CP agroinfiltrated plants was increased in the presence of Hsc70-2. HSP70 was found to increase the targeting of CNV CP to chloroplasts during infection, reinforcing the role of HSP70 in chloroplast targeting of host proteins. Hence, our findings have led to the discovery of a highly induced host factor that has been co-opted to play multiple roles during several stages of the CNV infection cycle. IMPORTANCE Because of the small size of its RNA genome, CNV is dependent on interaction with host cellular components to successfully complete its multiplication cycle. We have found that CNV induces HSP70 family homologs to a high level during infection, possibly as a result of the host response to the high levels of CNV proteins that accumulate during infection. Moreover, we have found that CNV co-opts HSP70 family homologs to facilitate several aspects of the infection process such as viral RNA, coat protein and virus accumulation. Chloroplast targeting of the CNV CP is also facilitated, which may aid in CNV suppression of host defense responses. Several viruses have been shown to induce HSP70 during infection and others to utilize HSP70 for specific aspects of infection such as replication, assembly, and disassembly. We speculate that HSP70 may play multiple roles in the infection processes of many viruses. PMID:26719261