Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice.

PubMed

Hatta, Yasuko; Hershberger, Karen; Shinya, Kyoko; Proll, Sean C; Dubielzig, Richard R; Hatta, Masato; Katze, Michael G; Kawaoka, Yoshihiro; Suresh, M

2010-10-07

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.

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.

Two-amino acids change in the nsp4 of SARS coronavirus abolishes viral replication.

PubMed

Sakai, Yusuke; Kawachi, Kengo; Terada, Yutaka; Omori, Hiroko; Matsuura, Yoshiharu; Kamitani, Wataru

2017-10-01

Infection with coronavirus rearranges the host cell membrane to assemble a replication/transcription complex in which replication of the viral genome and transcription of viral mRNA occur. Although coexistence of nsp3 and nsp4 is known to cause membrane rearrangement, the mechanisms underlying the interaction of these two proteins remain unclear. We demonstrated that binding of nsp4 with nsp3 is essential for membrane rearrangement and identified amino acid residues in nsp4 responsible for the interaction with nsp3. In addition, we revealed that the nsp3-nsp4 interaction is not sufficient to induce membrane rearrangement, suggesting the participation of other factors such as host proteins. Finally, we showed that loss of the nsp3-nsp4 interaction eliminated viral replication by using an infectious cDNA clone and replicon system of SARS-CoV. These findings provide clues to the mechanism of the replication/transcription complex assembly of SARS-CoV and could reveal an antiviral target for the treatment of betacoronavirus infection. Copyright © 2017 Elsevier Inc. All rights reserved.

In vitro replication of poliovirus

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

Lubinski, J.M.

1986-01-01

Poliovirus is a member of the Picornaviridae whose genome is a single stranded RNA molecule of positive polarity surrounded by a proteinaceous capsid. Replication of poliovirus occurs via negative strand intermediates in infected cells using a virally encoded RNA-dependent RNA polymerase and host cell proteins. The authors have exploited the fact that complete cDNA copies of the viral genome when transfected onto susceptible cells generate virus. Utilizing the bacteriophage SP6 DNA dependent RNA polymerase system to synthesize negative strands in vitro and using these in an in vitro reaction the authors have generated full length infectious plus strands. Mutagenesis ofmore » the 5' and 3' ends of the negative and positive strands demonstrated that replication could occur either de novo or be extensions of the templates from their 3' ends or from nicks occurring during replication. The appearance of dimeric RNA molecules generated in these reactions was not dependent upon the same protein required for de novo initiation. Full length dimeric RNA molecules using a 5' /sup 32/P end-labelled oligo uridylic acid primer and positive strand template were demonstrated in vitro containing only the 35,000 Mr host protein and the viral RNA-dependent RNA polymerase. A model for generating positive strands without protein priming by cleavage of dimeric RNA molecules was proposed.« less

The IMPORTance of the Nucleus during Flavivirus Replication

PubMed Central

Lopez-Denman, Adam J.; Mackenzie, Jason M.

2017-01-01

Flaviviruses are a large group of arboviruses of significant medical concern worldwide. With outbreaks a common occurrence, the need for efficient viral control is required more than ever. It is well understood that flaviviruses modulate the composition and structure of membranes in the cytoplasm that are crucial for efficient replication and evading immune detection. As the flavivirus genome consists of positive sense RNA, replication can occur wholly within the cytoplasm. What is becoming more evident is that some viral proteins also have the ability to translocate to the nucleus, with potential roles in replication and immune system perturbation. In this review, we discuss the current understanding of flavivirus nuclear localisation, and the function it has during flavivirus infection. We also describe—while closely related—the functional differences between similar viral proteins in their nuclear translocation. PMID:28106839

AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system.

PubMed

Schuck, Jana; Gursinsky, Torsten; Pantaleo, Vitantonio; Burgyán, Jozsef; Behrens, Sven-Erik

2013-05-01

AGO/RISC-mediated antiviral RNA silencing, an important component of the plant's immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (-)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing.

AGO/RISC-mediated antiviral RNA silencing in a plant in vitro system

PubMed Central

Schuck, Jana; Gursinsky, Torsten; Pantaleo, Vitantonio; Burgyán, Jozsef; Behrens, Sven-Erik

2013-01-01

AGO/RISC-mediated antiviral RNA silencing, an important component of the plant’s immune response against RNA virus infections, was recapitulated in vitro. Cytoplasmic extracts of tobacco protoplasts were applied that supported Tombusvirus RNA replication, as well as the formation of RNA-induced silencing complexes (RISC) that could be functionally reconstituted with various plant ARGONAUTE (AGO) proteins. For example, when RISC containing AGO1, 2, 3 or 5 were programmed with exogenous siRNAs that specifically targeted the viral RNA, endonucleolytic cleavages occurred and viral replication was inhibited. Antiviral RNA silencing was disabled by the viral silencing suppressor p19 when this was present early during RISC formation. Notably, with replicating viral RNA, only (+)RNA molecules were accessible to RISC, whereas (−)RNA replication intermediates were not. The vulnerability of viral RNAs to RISC activity also depended on the RNA structure of the target sequence. This was most evident when we characterized viral siRNAs (vsiRNAs) that were particularly effective in silencing with AGO1- or AGO2/RISC. These vsiRNAs targeted similar sites, suggesting that accessible parts of the viral (+)RNA may be collectively attacked by different AGO/RISC. The in vitro system was, hence, established as a valuable tool to define and characterize individual molecular determinants of antiviral RNA silencing. PMID:23535144

Early intranuclear replication of African swine fever virus genome modifies the landscape of the host cell nucleus.

PubMed

Simões, Margarida; Martins, Carlos; Ferreira, Fernando

2015-12-02

Although African swine fever virus (ASFV) replicates in viral cytoplasmic factories, the presence of viral DNA within the host cell nucleus has been previously reported to be essential for productive infection. Herein, we described, for the first time, the intranuclear distribution patterns of viral DNA replication events, preceding those that occur in the cytoplasmic compartment. Using BrdU pulse-labelling experiments, newly synthesized ASFV genomes were exclusively detected inside the host cell nucleus at the early phase of infection, both in swine monocyte-derived macrophages (MDMs) and Vero cells. From 8hpi onwards, BrdU labelling was only observed in ASFV cytoplasmic factories. Our results also show that ASFV specifically activates the Ataxia Telangiectasia Mutated Rad-3 related (ATR) pathway in ASFV-infected swine MDMs from the early phase of infection, most probably because ASFV genome is recognized as foreign DNA. Morphological changes of promyelocytic leukaemia nuclear bodies (PML-NBs), nuclear speckles and Cajal bodies were also found in ASFV-infected swine MDMs, strongly suggesting the viral modulation of cellular antiviral responses and cellular transcription, respectively. As described for other viral infections, the nuclear reorganization that takes place during ASFV infection may also provide an environment that favours its intranuclear replication events. Altogether, our results contribute for a better understanding of ASFV replication strategies, starting with an essential intranuclear DNA replication phase which induces host nucleus changes towards a successful viral infection. Copyright © 2015 Elsevier B.V. All rights reserved.

[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.

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter.

PubMed

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald; Zheng, Zhi-Ming

2017-05-30

The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P 811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis -acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. IMPORTANCE It has been known for decades that the activity of viral late promoters is associated with viral DNA replication among almost all DNA viruses. However, the mechanism of how DNA replication activates the viral late promoter and what components of the replication machinery are involved remain largely unknown. In this study, we characterized the P 811 promoter region of HPV18 and demonstrated that its activation depends on the orientation of DNA replication. Using single-stranded oligonucleotides targeting the replication fork on either leading or lagging strands, we showed that viral lagging-strand replication activates the promoter. We also identified a transcriptional repressor element located upstream of the promoter transcription start site which interacts with cellular proteins hnRNP D0B and hnRNP A/B and modulates the late promoter activity. This is the first report on how DNA replication activates a viral late promoter. Copyright © 2017 Wang et al.

Hsp70 Isoforms Are Essential for the Formation of Kaposi’s Sarcoma-Associated Herpesvirus Replication and Transcription Compartments

PubMed Central

Baquero-Pérez, Belinda; Whitehouse, Adrian

2015-01-01

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with various AIDS-related malignancies. Like other herpesviruses, multiple processes required for KSHV lytic replication, including viral transcription, viral DNA synthesis and capsid assembly occur in virus-induced intranuclear structures, termed replication and transcription compartments (RTCs). Here we utilised a novel methodology, combining subcellular fractionation and quantitative proteomics, to identify cellular proteins which are recruited to KSHV-induced RTCs and thus play a key role in KSHV lytic replication. We show that several isoforms of the HSP70 chaperone family, Hsc70 and iHsp70, are redistributed from the cytoplasm into the nucleus coinciding with the initial formation of KSHV-induced RTCs. We demonstrate that nuclear chaperone foci are dynamic, initially forming adjacent to newly formed KSHV RTCs, however during later time points the chaperones move within KSHV RTCs and completely co-localise with actively replicating viral DNA. The functional significance of Hsp70 isoforms recruitment into KSHV RTCs was also examined using the specific Hsp70 isoform small molecule inhibitor, VER-155008. Intriguingly, results highlight an essential role of Hsp70 isoforms in the KSHV replication cycle independent of protein stability and maturation. Notably, inhibition of Hsp70 isoforms precluded KSHV RTC formation and RNA polymerase II (RNAPII) relocalisation to the viral genome leading to the abolishment of global KSHV transcription and subsequent viral protein synthesis and DNA replication. These new findings have revealed novel mechanisms that regulate KSHV lytic replication and highlight the potential of HSP70 inhibitors as novel antiviral agents. PMID:26587836

Evasion of superinfection exclusion and elimination of primary viral RNA by an adapted strain of hepatitis C virus.

PubMed

Webster, Brian; Ott, Melanie; Greene, Warner C

2013-12-01

Cells that are productively infected by hepatitis C virus (HCV) are refractory to a second infection by HCV via a block in viral replication known as superinfection exclusion. The block occurs at a postentry step and likely involves translation or replication of the secondary viral RNA, but the mechanism is largely unknown. To characterize HCV superinfection exclusion, we selected for an HCV variant that could overcome the block. We produced a high-titer HC-J6/JFH1 (Jc1) viral genome with a fluorescent reporter inserted between NS5A and NS5B and used it to infect Huh7.5 cells containing a Jc1 replicon. With multiple passages of these infected cells, we isolated an HCV variant that can superinfect cells at high levels. Notably, the superinfectious virus rapidly cleared the primary replicon from superinfected cells. Viral competition experiments, using a novel strategy of sequence-barcoding viral strains, as well as superinfection of replicon cells demonstrated that mutations in E1, p7, NS5A, and the poly(U/UC) tract of the 3' untranslated region were important for superinfection. Furthermore, these mutations dramatically increased the infectivity of the virus in naive cells. Interestingly, viruses with a shorter poly(U/UC) and an NS5A domain II mutation were most effective in overcoming the postentry block. Neither of these changes affected viral RNA translation, indicating that the major barrier to postentry exclusion occurs at viral RNA replication. The evolution of the ability to superinfect after less than a month in culture and the concomitant exclusion of the primary replicon suggest that superinfection exclusion dramatically affects viral fitness and dynamics in vivo.

Macrophages in Progressive Human Immunodeficiency Virus/Simian Immunodeficiency Virus Infections

PubMed Central

DiNapoli, Sarah R.; Hirsch, Vanessa M.

2016-01-01

The cells that are targeted by primate lentiviruses (HIV and simian immunodeficiency virus [SIV]) are of intense interest given the renewed effort to identify potential cures for HIV. These viruses have been reported to infect multiple cell lineages of hematopoietic origin, including all phenotypic and functional CD4 T cell subsets. The two most commonly reported cell types that become infected in vivo are memory CD4 T cells and tissue-resident macrophages. Though viral infection of CD4 T cells is routinely detected in both HIV-infected humans and SIV-infected Asian macaques, significant viral infection of macrophages is only routinely observed in animal models wherein CD4 T cells are almost entirely depleted. Here we review the roles of macrophages in lentiviral disease progression, the evidence that macrophages support viral replication in vivo, the animal models where macrophage-mediated replication of SIV is thought to occur, how the virus can interact with macrophages in vivo, pathologies thought to be attributed to viral replication within macrophages, how viral replication in macrophages might contribute to the asymptomatic phase of HIV/SIV infection, and whether macrophages represent a long-lived reservoir for the virus. PMID:27307568

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

PubMed Central

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

2016-01-01

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

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

PubMed

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

2016-05-01

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

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes.

PubMed

Chotiwan, Nunya; Andre, Barbara G; Sanchez-Vargas, Irma; Islam, M Nurul; Grabowski, Jeffrey M; Hopf-Jannasch, Amber; Gough, Erik; Nakayasu, Ernesto; Blair, Carol D; Belisle, John T; Hill, Catherine A; Kuhn, Richard J; Perera, Rushika

2018-02-01

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes.

Dynamic remodeling of lipids coincides with dengue virus replication in the midgut of Aedes aegypti mosquitoes

PubMed Central

Chotiwan, Nunya; Andre, Barbara G.; Sanchez-Vargas, Irma; Islam, M. Nurul; Grabowski, Jeffrey M.; Hopf-Jannasch, Amber; Gough, Erik; Nakayasu, Ernesto; Blair, Carol D.; Hill, Catherine A.; Kuhn, Richard J.

2018-01-01

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes. PMID:29447265

In vitro models of viral-induced congenital deafness.

PubMed

Davis, G L

1981-10-01

Cytomegalovirus (CMV) infects 1 to 2 percent of liveborn infants in the United States and causes varying degrees of perceptive hearing loss. There are eight reported pathologic studies of temporal bones in CMV-infected neonates. Viral replication occurs in nonsensory endolabyrinthine epithelium, but viral antigen is also found in the organ of Corti and spiral ganglion neurons, and CMV has been cultured from perilymph. Further clinicopathologic correlation is frustrated, since the inner ear cannot be biopsied during life, and the number of temporal bones available for study is limited, owing to the decrease in the number of autopsies being performed. Inoculation of CMV into newborn mice, and extracorporeal preparations of mouse and guinea pig fetal inner ears, either in organ culture or as grafts on chick chorioallantoic membranes, yields viral perilabyrinthitis. The different ultrastructural appearances of CMV replicating in epithelial and mesenchymal cells show that animal CMV replicates in mesenchymal cells and human CMV replicates in epithelial cells of the inner ear. These different ultrastructural patterns indicate that the chromophobe (transitional) cells of the stria vascularis of the guinea pig are of mesenchymal origin.

Involvement of rainbow trout leucocytes in the pathogenesis of infectious hematopoietic necrosis

USGS Publications Warehouse

Chilmonczyk, S.; Winton, J.R.

1994-01-01

Rainbow trout Oncorhynchus myluss leucocytes were tested for their ability to support replication of infectious hematopoietic necrosis virus (IHNV). Viral replication occurred in vitro uslng leucocytes cultured from peripheral blood, kidney, and thymus where viral titers peaked at 2 to 4 d post-inoculation. Leucocytes collected from trout following waterborne challenge with IHNV were cocultured on EPC cell monolayers. These assays detected IHNV in leucocytes infected in vivo as early as 6 h post-exposure before the challenge virus had undergone replication. These data showed that leucocyte populations could serve as target cells in the initial phase of IHNV infection.

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

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.

Restricted Replication of Xenotropic Murine Leukemia Virus-Related Virus in Pigtailed Macaques

PubMed Central

Del Prete, Gregory Q.; Kearney, Mary F.; Spindler, Jon; Wiegand, Ann; Chertova, Elena; Roser, James D.; Estes, Jacob D.; Hao, Xing Pei; Trubey, Charles M.; Lara, Abigail; Lee, KyeongEun; Chaipan, Chawaree; Bess, Julian W.; Nagashima, Kunio; Keele, Brandon F.; Macallister, Rhonda; Smedley, Jeremy; Pathak, Vinay K.; KewalRamani, Vineet N.; Coffin, John M.

2012-01-01

Although xenotropic murine leukemia virus-related virus (XMRV) has been previously linked to prostate cancer and myalgic encephalomyelitis/chronic fatigue syndrome, recent data indicate that results interpreted as evidence of human XMRV infection reflect laboratory contamination rather than authentic in vivo infection. Nevertheless, XMRV is a retrovirus of undefined pathogenic potential that is able to replicate in human cells. Here we describe a comprehensive analysis of two male pigtailed macaques (Macaca nemestrina) experimentally infected with XMRV. Following intravenous inoculation with >1010 RNA copy equivalents of XMRV, viral replication was limited and transient, peaking at ≤2,200 viral RNA (vRNA) copies/ml plasma and becoming undetectable by 4 weeks postinfection, though viral DNA (vDNA) in peripheral blood mononuclear cells remained detectable through 119 days of follow-up. Similarly, vRNA was not detectable in lymph nodes by in situ hybridization despite detectable vDNA. Sequencing of cell-associated vDNA revealed extensive G-to-A hypermutation, suggestive of APOBEC-mediated viral restriction. Consistent with limited viral replication, we found transient upregulation of type I interferon responses that returned to baseline by 2 weeks postinfection, no detectable cellular immune responses, and limited or no spread to prostate tissue. Antibody responses, including neutralizing antibodies, however, were detectable by 2 weeks postinfection and maintained throughout the study. Both animals were healthy for the duration of follow-up. These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro, human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread. PMID:22238316

Recruitment of DNA Replication and Damage Response Proteins to Viral Replication Centers during Infection with NS2 Mutants of Minute Virus of Mice (MVM)

PubMed Central

Ruiz, Zandra; Mihaylov, Ivailo S.; Cotmore, Susan F.; Tattersall, Peter

2010-01-01

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA32, which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins. PMID:21193212

Recruitment of DNA replication and damage response proteins to viral replication centers during infection with NS2 mutants of Minute Virus of Mice (MVM).

PubMed

Ruiz, Zandra; Mihaylov, Ivailo S; Cotmore, Susan F; Tattersall, Peter

2011-02-20

MVM NS2 is essential for viral DNA amplification, but its mechanism of action is unknown. A classification scheme for autonomous parvovirus-associated replication (APAR) center development, based on NS1 distribution, was used to characterize abnormal APAR body maturation in NS2null mutant infections, and their organization examined for defects in host protein recruitment. Since acquisition of known replication factors appeared normal, we looked for differences in invoked DNA damage responses. We observed widespread association of H2AX/MDC1 damage response foci with viral replication centers, and sequestration and complex hyperphosphorylation of RPA(32), which occurred in wildtype and mutant infections. Quantifying these responses by western transfer indicated that both wildtype and NS2 mutant MVM elicited ATM activation, while phosphorylation of ATR, already basally activated in asynchronous A9 cells, was downregulated. We conclude that MVM infection invokes multiple damage responses that influence the APAR environment, but that NS2 does not modify the recruitment of cellular proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

Membrane dynamics associated with viral infection.

PubMed

de Armas-Rillo, Laura; Valera, María-Soledad; Marrero-Hernández, Sara; Valenzuela-Fernández, Agustín

2016-05-01

Viral replication and spreading are fundamental events in the viral life cycle, accounting for the assembly and egression of nascent virions, events that are directly associated with viral pathogenesis in target hosts. These processes occur in cellular compartments that are modified by specialized viral proteins, causing a rearrangement of different cell membranes in infected cells and affecting the ER, mitochondria, Golgi apparatus, vesicles and endosomes, as well as processes such as autophagic membrane flux. In fact, the activation or inhibition of membrane trafficking and other related activities are fundamental to ensure the adequate replication and spreading of certain viruses. In this review, data will be presented that support the key role of membrane dynamics in the viral cycle, especially in terms of the assembly, egression and infection processes. By defining how viruses orchestrate these events it will be possible to understand how they successfully complete their route of infection, establishing viral pathogenesis and provoking disease. © 2015 The Authors Reviews in Medical Virology Published by John Wiley & Sons, Ltd.

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

PubMed Central

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

2011-01-01

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

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.

Initiation and Reinitiation of DNA Synthesis during Replication of Bacteriophage T7*

PubMed Central

Dressler, David; Wolfson, John; Magazin, Marilyn

1972-01-01

In its first round of replication, the T7 chromosome follows a simple pattern, as viewed in the electron microscope. The iniation of DNA synthesis occurs about 17% from the genetic left end of the viral DNA rod. Bidirectional DNA synthesis from this origin then generates a replicating intermediate that we call an “eye form.” In the eye form, when synthesis in the leftward direction reaches the left end of the viral chromosome, the molecule is converted into a Y-shaped replicating rod. The remaining growing point continues synthesis rightward, until presumably it runs off the right end of the DNA rod, thus terminating replication. Numerous T7 chromosomes were found in which a second round of replication had begun before the first round had finished. Analysis of these reinitiated DNA molecules showed that the second round of replication, like the first, began 17% from the end of the chromosome and involved bidirectional DNA synthesis. Images PMID:4554539

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

Complex Dynamic Development of Poliovirus Membranous Replication Complexes

PubMed Central

Nair, Vinod; Hansen, Bryan T.; Hoyt, Forrest H.; Fischer, Elizabeth R.; Ehrenfeld, Ellie

2012-01-01

Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as “vesicles” are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses. PMID:22072780

Involvement of the Reparative DNA Polymerase Pol X of African Swine Fever Virus in the Maintenance of Viral Genome Stability In Vivo

PubMed Central

Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Suárez, Cristina; Salas, José

2013-01-01

The function of the African swine fever virus (ASFV) reparative DNA polymerase, Pol X, was investigated in the context of virus infection. Pol X is a late structural protein that localizes at cytoplasmic viral factories during DNA replication. Using an ASFV deletion mutant lacking the Pol X gene, we have shown that Pol X is not required for virus growth in Vero cells or swine macrophages under one-step growth conditions. However, at a low multiplicity of infection, when multiple rounds of replication occur, the growth of the mutant virus is impaired in swine macrophages but not in Vero cells, suggesting that Pol X is needed to repair the accumulated DNA damage. The replication of the mutant virus in Vero cells presents sensitivity to oxidative damage, and mutational analysis of viral DNA shows that deletion of Pol X results in an increase in the mutation frequency in macrophages. Therefore, our data reveal a biological role for ASFV Pol X in the context of the infected cell in the preservation of viral genetic information. PMID:23824796

Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms.

PubMed Central

Ermak, G; Paszkowski, U; Wohlmuth, M; Scheid, O M; Paszkowski, J

1993-01-01

Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibition due to altered interaction between the replication complex and methylated viral DNA. Our results suggest that methylation-mediated reduction of viral replication is due to both repression mechanisms and that this provides two independent selection pressures for the maintenance of methylation-free replicons in infected cells. Images PMID:7688453

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

Insight into HIV-2 latency may disclose strategies for a cure for HIV-1 infection.

PubMed

Saleh, Suha; Vranckx, Lenard; Gijsbers, Rik; Christ, Frauke; Debyser, Zeger

2017-01-01

HIV-1 and HIV-2 originate from two distinct zoonotic transmissions of simian immunodeficiency viruses from primate to human. Although both share similar modes of transmission and can result in the development of AIDS with similar clinical manifestations, HIV-2 infection is generally milder and less likely to progress to AIDS. HIV is currently incurable due to the presence of HIV provirus integrated into the host DNA of long-lived memory cells of the immune system without active replication. As such, the latent virus is immunologically inert and remains insensitive to the administered antiviral drugs targeting active viral replication steps. Recent evidence suggests that persistent HIV replication may occur in anatomical sanctuaries such as the lymphoid tissue due to low drug penetration. At present, different strategies are being evaluated either to completely eradicate the virus from the patient (sterilising cure) or to allow treatment interruption without viral rebound (functional cure). Because HIV-2 is naturally less pathogenic and displays a more latent phenotype than HIV-1, it may represent a valuable model that provides elementary information to cure HIV-1 infection. Insight into the viral and cellular determinants of HIV-2 replication may therefore pave the way for alternative strategies to eradicate HIV-1 or promote viral remission.

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.

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.

BiP: Master Regulator of the Unfolded Protein Response and Crucial Factor in Flavivirus Biology


PubMed Central

Lewy, Tyler G.; Grabowski, Jeffrey M.; Bloom, Marshall E.

2017-01-01

Flaviviruses have an intimate relationship with their host cells, utilizing host proteins during replication. Much of viral genome replication and virion assembly occurs on and within the endoplasmic reticulum (ER). As a cellular protein folding hub, the ER provides an ideal environment for flaviviruses to replicate. Flaviviruses can interact with several ER processes, including the unfolded protein response (UPR), a cellular stress mechanism responsible for managing unfolded protein accumulation and ER stress. The UPR can alter the ER environment in several ways, including increasing ER volume and quantity of available chaperones, both of which can favor viral replication. BiP, a chaperone and master regulator of the UPR, has been demonstrated to play a key role in several flavivirus infections. Here we describe what is known in regard to BiP, its implicated role with flavivirus infection, and what remains to be discovered. PMID:28656015

BiP: Master Regulator of the Unfolded Protein Response and Crucial Factor in Flavivirus Biology
.

PubMed

Lewy, Tyler G; Grabowski, Jeffrey M; Bloom, Marshall E

2017-06-01

Flaviviruses have an intimate relationship with their host cells, utilizing host proteins during replication. Much of viral genome replication and virion assembly occurs on and within the endoplasmic reticulum (ER). As a cellular protein folding hub, the ER provides an ideal environment for flaviviruses to replicate. Flaviviruses can interact with several ER processes, including the unfolded protein response (UPR), a cellular stress mechanism responsible for managing unfolded protein accumulation and ER stress. The UPR can alter the ER environment in several ways, including increasing ER volume and quantity of available chaperones, both of which can favor viral replication. BiP, a chaperone and master regulator of the UPR, has been demonstrated to play a key role in several flavivirus infections. Here we describe what is known in regard to BiP, its implicated role with flavivirus infection, and what remains to be discovered.

Characterization of Reemerging Chikungunya Virus

PubMed Central

Sourisseau, Marion; Schilte, Clémentine; Casartelli, Nicoletta; Trouillet, Céline; Guivel-Benhassine, Florence; Rudnicka, Dominika; Sol-Foulon, Nathalie; Roux, Karin Le; Prevost, Marie-Christine; Fsihi, Hafida; Frenkiel, Marie-Pascale; Blanchet, Fabien; Afonso, Philippe V; Ceccaldi, Pierre-Emmanuel; Ozden, Simona; Gessain, Antoine; Schuffenecker, Isabelle; Verhasselt, Bruno; Zamborlini, Alessia; Saïb, Ali; Rey, Felix A; Arenzana-Seisdedos, Fernando; Desprès, Philippe; Michault, Alain; Albert, Matthew L; Schwartz, Olivier

2007-01-01

An unprecedented epidemic of chikungunya virus (CHIKV) infection recently started in countries of the Indian Ocean area, causing an acute and painful syndrome with strong fever, asthenia, skin rash, polyarthritis, and lethal cases of encephalitis. The basis for chikungunya disease and the tropism of CHIKV remain unknown. Here, we describe the replication characteristics of recent clinical CHIKV strains. Human epithelial and endothelial cells, primary fibroblasts and, to a lesser extent, monocyte-derived macrophages, were susceptible to infection and allowed viral production. In contrast, CHIKV did not replicate in lymphoid and monocytoid cell lines, primary lymphocytes and monocytes, or monocyte-derived dendritic cells. CHIKV replication was cytopathic and associated with an induction of apoptosis in infected cells. Chloroquine, bafilomycin-A1, and short hairpin RNAs against dynamin-2 inhibited viral production, indicating that viral entry occurs through pH-dependent endocytosis. CHIKV was highly sensitive to the antiviral activity of type I and II interferons. These results provide a general insight into the interaction between CHIKV and its mammalian host. PMID:17604450

A Re-Examination of Global Suppression of RNA Interference by HIV-1

PubMed Central

Sanghvi, Viraj R.; Steel, Laura F.

2011-01-01

The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing. PMID:21386885

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

Kaposi's Sarcoma-Associated Herpesvirus mRNA Accumulation in Nuclear Foci Is Influenced by Viral DNA Replication and Viral Noncoding Polyadenylated Nuclear RNA.

PubMed

Vallery, Tenaya K; Withers, Johanna B; Andoh, Joana A; Steitz, Joan A

2018-07-01

Kaposi's sarcoma-associated herpesvirus (KSHV), like other herpesviruses, replicates within the nuclei of its human cell host and hijacks host machinery for expression of its genes. The activities that culminate in viral DNA synthesis and assembly of viral proteins into capsids physically concentrate in nuclear areas termed viral replication compartments. We sought to better understand the spatiotemporal regulation of viral RNAs during the KSHV lytic phase by examining and quantifying the subcellular localization of select viral transcripts. We found that viral mRNAs, as expected, localized to the cytoplasm throughout the lytic phase. However, dependent on active viral DNA replication, viral transcripts also accumulated in the nucleus, often in foci in and around replication compartments, independent of the host shutoff effect. Our data point to involvement of the viral long noncoding polyadenylated nuclear (PAN) RNA in the localization of an early, intronless viral mRNA encoding ORF59-58 to nuclear foci that are associated with replication compartments. IMPORTANCE Late in the lytic phase, mRNAs from Kaposi's sarcoma-associated herpesvirus accumulate in the host cell nucleus near viral replication compartments, centers of viral DNA synthesis and virion production. This work contributes spatiotemporal data on herpesviral mRNAs within the lytic host cell and suggests a mechanism for viral RNA accumulation. Our findings indicate that the mechanism is independent of the host shutoff effect and splicing but dependent on active viral DNA synthesis and in part on the viral noncoding RNA, PAN RNA. PAN RNA is essential for the viral life cycle, and its contribution to the nuclear accumulation of viral messages may facilitate propagation of the virus. Copyright © 2018 American Society for Microbiology.

Three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated RNA.

PubMed

Miorin, Lisa; Romero-Brey, Inés; Maiuri, Paolo; Hoppe, Simone; Krijnse-Locker, Jacomine; Bartenschlager, Ralf; Marcello, Alessandro

2013-06-01

Flavivirus replication is accompanied by the rearrangement of cellular membranes that may facilitate viral genome replication and protect viral components from host cell responses. The topological organization of viral replication sites and the fate of replicated viral RNA are not fully understood. We exploited electron microscopy to map the organization of tick-borne encephalitis virus (TBEV) replication compartments in infected cells and in cells transfected with a replicon. Under both conditions, 80-nm vesicles were seen within the lumen of the endoplasmic reticulum (ER) that in infected cells also contained virions. By electron tomography, the vesicles appeared as invaginations of the ER membrane, displaying a pore that could enable release of newly synthesized viral RNA into the cytoplasm. To track the fate of TBEV RNA, we took advantage of our recently developed method of viral RNA fluorescent tagging for live-cell imaging combined with bleaching techniques. TBEV RNA was found outside virus-induced vesicles either associated to ER membranes or free to move within a defined area of juxtaposed ER cisternae. From our results, we propose a biologically relevant model of the possible topological organization of flavivirus replication compartments composed of replication vesicles and a confined extravesicular space where replicated viral RNA is retained. Hence, TBEV modifies the ER membrane architecture to provide a protected environment for viral replication and for the maintenance of newly replicated RNA available for subsequent steps of the virus life cycle.

The cellular Mre11 protein interferes with adenovirus E4 mutant DNA replication.

PubMed

Mathew, Shomita S; Bridge, Eileen

2007-09-01

Adenovirus type 5 (Ad5) relocalizes and degrades the host DNA repair protein Mre11, and efficiently initiates viral DNA replication. Mre11 associates with Ad E4 mutant DNA replication centers and is important for concatenating viral genomes. We have investigated the role of Mre11 in the E4 mutant DNA replication defect. RNAi-mediated knockdown of Mre11 dramatically rescues E4 mutant DNA replication in cells that do or do not concatenate viral genomes, suggesting that Mre11 inhibits DNA replication independent of genome concatenation. The mediator of DNA damage checkpoint 1 (Mdc1) protein is involved in recruiting and sustaining Mre11 at sites of DNA damage following ionizing radiation. We observe foci formation by Mdc1 in response to viral infection, indicating that this damage response protein is activated. However, knockdown of Mdc1 does not prevent Mre11 from localizing at viral DNA replication foci or rescue E4 mutant DNA replication. Our results are consistent with a model in which Mre11 interferes with DNA replication when it is localized at viral DNA replication foci.

A Host Susceptibility Gene, DR1, Facilitates Influenza A Virus Replication by Suppressing Host Innate Immunity and Enhancing Viral RNA Replication

PubMed Central

Hsu, Shih-Feng; Su, Wen-Chi; Jeng, King-Song

2015-01-01

ABSTRACT Influenza A virus (IAV) depends on cellular factors to complete its replication cycle; thus, investigation of the factors utilized by IAV may facilitate antiviral drug development. To this end, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNA interference (RNAi) screen. Knockdown (KD) of DR1 resulted in reductions of viral RNA and protein production, demonstrating that DR1 acts as a positive host factor in IAV replication. Genome-wide transcriptomic analysis showed that there was a strong induction of interferon-stimulated gene (ISG) expression after prolonged DR1 KD. We found that beta interferon (IFN-β) was induced by DR1 KD, thereby activating the JAK-STAT pathway to turn on ISG expression, which led to a strong inhibition of IAV replication. This result suggests that DR1 in normal cells suppresses IFN induction, probably to prevent undesired cytokine production, but that this suppression may create a milieu that favors IAV replication once cells are infected. Furthermore, biochemical assays of viral RNA replication showed that DR1 KD suppressed viral RNA replication. We also showed that DR1 associated with all three subunits of the viral RNA-dependent RNA polymerase (RdRp) complex, indicating that DR1 may interact with individual components of the viral RdRp complex to enhance viral RNA replication. Thus, DR1 may be considered a novel host susceptibility gene for IAV replication via a dual mechanism, not only suppressing the host defense to indirectly favor IAV replication but also directly facilitating viral RNA replication. IMPORTANCE Investigations of virus-host interactions involved in influenza A virus (IAV) replication are important for understanding viral pathogenesis and host defenses, which may manipulate influenza virus infection or prevent the emergence of drug resistance caused by a high error rate during viral RNA replication. For this purpose, a cellular transcriptional repressor, DR1, was identified from a genome-wide RNAi screen as a positive regulator in IAV replication. In the current studies, we showed that DR1 suppressed the gene expression of a large set of host innate immunity genes, which indirectly facilitated IAV replication in the event of IAV infection. Besides this scenario, DR1 also directly enhanced the viral RdRp activity, likely through associating with individual components of the viral RdRp complex. Thus, DR1 represents a novel host susceptibility gene for IAV replication via multiple functions, not only suppressing the host defense but also enhancing viral RNA replication. DR1 may be a potential target for drug development against influenza virus infection. PMID:25589657

Structural studies demonstrating a bacteriophage-like replication cycle of the eukaryote-infecting Paramecium bursaria chlorella virus-1

PubMed Central

Shimoni, Eyal; Dadosh, Tali; Rechav, Katya; Unger, Tamar

2017-01-01

A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA Paramecium bursaria chlorella virus-1 (PBCV-1). Our studies reveal that early infection stages of this eukaryotic-infecting virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle. PMID:28850602

Distinct temporal roles for the promyelocytic leukaemia (PML) protein in the sequential regulation of intracellular host immunity to HSV-1 infection

PubMed Central

Alandijany, Thamir; Conn, Kristen L.; McFarlane, Steven; Orr, Anne

2018-01-01

Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2’-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication. PMID:29309427

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

PubMed Central

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

1996-01-01

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

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

TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

PubMed Central

Kitamura, Kouichi; Wang, Zhe; Chowdhury, Sajeda; Monjurul, Ahasan Md; Wakae, Kousho; Koura, Miki; Shimadu, Miyuki; Kinoshita, Kazuo; Muramatsu, Masamichi

2015-01-01

Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner. PMID:25836330

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

PubMed

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

2016-06-24

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

Inclusion Bodies Are a Site of Ebolavirus Replication

PubMed Central

Hoenen, Thomas; Shabman, Reed S.; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F.; Becker, Stephan

2012-01-01

Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place. PMID:22915810

Inclusion bodies are a site of ebolavirus replication.

PubMed

Hoenen, Thomas; Shabman, Reed S; Groseth, Allison; Herwig, Astrid; Weber, Michaela; Schudt, Gordian; Dolnik, Olga; Basler, Christopher F; Becker, Stephan; Feldmann, Heinz

2012-11-01

Inclusion bodies are a characteristic feature of ebolavirus infections in cells. They contain large numbers of preformed nucleocapsids, but their biological significance has been debated, and they have been suggested to be aggregates of viral proteins without any further biological function. However, recent data for other viruses that produce similar structures have suggested that inclusion bodies might be involved in genome replication and transcription. In order to study filovirus inclusion bodies, we fused mCherry to the ebolavirus polymerase L, which is found in inclusion bodies. The resulting L-mCherry fusion protein was functional in minigenome assays and incorporated into virus-like particles. Importantly, L-mCherry fluorescence in transfected cells was readily detectable and distributed in a punctate pattern characteristic for inclusion bodies. A recombinant ebolavirus encoding L-mCherry instead of L was rescued and showed virtually identical growth kinetics and endpoint titers to those for wild-type virus. Using this virus, we showed that the onset of inclusion body formation corresponds to the onset of viral genome replication, but that viral transcription occurs prior to inclusion body formation. Live-cell imaging further showed that inclusion bodies are highly dynamic structures and that they can undergo dramatic reorganization during cell division. Finally, by labeling nascent RNAs using click technology we showed that inclusion bodies are indeed the site of viral RNA synthesis. Based on these data we conclude that, rather than being inert aggregates of nucleocapsids, ebolavirus inclusion bodies are in fact complex and dynamic structures and an important site at which viral RNA replication takes place.

Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis.

PubMed Central

Winnacker, E L

1975-01-01

Isolated nuclei from adenovirus type 2-infected HeLa cells catalyze the incorporation of all four deoxyribonucleoside triphosphates into viral DNA. The observed DNA synthesis occurs via a transient formation of DNA fragments with a sedimentation coefficient of 10S. The fragments are precursors to unit-length viral DNA, they are self-complementary to an extent of at least 70%, and they are distributed along most of the viral chromosome. In addition, accumulation of 10S DNA fragments is observed either in intact, virus-infected HeLa cells under conditions where viral DNA synthesis is inhibited by hydroxyurea or in isolated nuclei from virus-infected HeLa cells at low concentrations of deoxyribonucleotides. Under these suboptimal conditions for DNA synthesis in isolated nuclei, ribonucleoside triphosphates determine the size distribution of DNA intermediates. The evidence presented suggests that a ribonucleoside-dependent initiation step as well at two DNA polymerase catalyzed reactions are involved in the discontinuous replication of adenovirus type 2 DNA. PMID:1117487

ADARs, Viruses and Innate Immunity

PubMed Central

Samuel, Charles E.

2013-01-01

Double-stranded RNA (dsRNA) functions both as a substrate of ADARs and also as a molecular trigger of innate immune responses. ADARs, adenosine deaminases that act on RNA, catalyze the deamination of adenosine (A) to produce inosine (I) in dsRNA. ADARs thereby can destablize RNA structures, because the generated I:U mismatch pairs are less stable than A:U base pairs. Additionally, I is read as G instead of A by ribosomes during translation and by viral RNA-dependent RNA polymerases during RNA replication. Members of several virus families have the capacity to produce dsRNA during viral genome transcription and replication. Sequence changes (A-to-G, and U-to-C) characteristic of A-to-I editing occur during virus growth and persistence. Foreign viral dsRNA also mediates both the induction and the action of interferons. In this chapter our current understanding of the role and significance of ADARs in the context of innate immunity, and as determinants of the outcome of viral infection, will be considered. PMID:21809195

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome

PubMed Central

Hartley, Carol A.; Vaz, Paola K.; Diaz-Méndez, Andrés; García, Maricarmen; Spatz, Stephen; Devlin, Joanne M.

2017-01-01

ABSTRACT Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome. IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease. PMID:28939604

MicroRNA reduction of neuronal West Nile virus replication attenuates and affords a protective immune response in mice

PubMed Central

Brostoff, Terza; Pesavento, Patricia A.; Barker, Christopher M.; Kenney, Joan L.; Dietrich, Elizabeth A.; Duggal, Nisha K.; Bosco-Lauth, Angela M.; Brault, Aaron C.

2017-01-01

West Nile virus (WNV) is an important agent of human encephalitis that has quickly become endemic across much of the United States since its identification in North America in 1999. While the majority (~75%) of infections are subclinical, neurologic disease can occur in a subset of cases, with outcomes including permanent neurologic damage and death. Currently, there are no WNV vaccines approved for use in humans. This study introduces a novel vaccine platform for WNV to reduce viral replication in the central nervous system while maintaining peripheral replication to elicit strong neutralizing antibody titers. Vaccine candidates were engineered to incorporate microRNA (miRNA) target sequences for a cognate miRNA expressed only in neurons, allowing the host miRNAs to target viral transcription through endogenous RNA silencing. To maintain stability, these targets were incorporated in multiple locations within the 3′-untranslated region, flanking sequences essential for viral replication without affecting the viral open reading frame. All candidates replicated comparably to wild type WNV in vitro within cells that did not express the cognate miRNA. Insertional control viruses were also capable of neuroinvasion and neurovirulence in vivo in CD-1 mice. Vaccine viruses were safe at all doses tested and did not demonstrate mutations associated with a reversion to virulence when serially passaged in mice. All vaccine constructs were protective from lethal challenge in mice, producing 93–100% protection at the highest dose tested. Overall, this is a safe and effective attenuation strategy with broad potential application for vaccine development. PMID:27637937

MicroRNA reduction of neuronal West Nile virus replication attenuates and affords a protective immune response in mice.

PubMed

Brostoff, Terza; Pesavento, Patricia A; Barker, Christopher M; Kenney, Joan L; Dietrich, Elizabeth A; Duggal, Nisha K; Bosco-Lauth, Angela M; Brault, Aaron C

2016-10-17

West Nile virus (WNV) is an important agent of human encephalitis that has quickly become endemic across much of the United States since its identification in North America in 1999. While the majority (∼75%) of infections are subclinical, neurologic disease can occur in a subset of cases, with outcomes including permanent neurologic damage and death. Currently, there are no WNV vaccines approved for use in humans. This study introduces a novel vaccine platform for WNV to reduce viral replication in the central nervous system while maintaining peripheral replication to elicit strong neutralizing antibody titers. Vaccine candidates were engineered to incorporate microRNA (miRNA) target sequences for a cognate miRNA expressed only in neurons, allowing the host miRNAs to target viral transcription through endogenous RNA silencing. To maintain stability, these targets were incorporated in multiple locations within the 3'-untranslated region, flanking sequences essential for viral replication without affecting the viral open reading frame. All candidates replicated comparably to wild type WNV in vitro within cells that did not express the cognate miRNA. Insertional control viruses were also capable of neuroinvasion and neurovirulence in vivo in CD-1 mice. Vaccine viruses were safe at all doses tested and did not demonstrate mutations associated with a reversion to virulence when serially passaged in mice. All vaccine constructs were protective from lethal challenge in mice, producing 93-100% protection at the highest dose tested. Overall, this is a safe and effective attenuation strategy with broad potential application for vaccine development. Published by Elsevier Ltd.

Structural Insights into the Coupling of Virion Assembly and Rotavirus Replication

PubMed Central

Trask, Shane D.; McDonald, Sarah M.; Patton, John T.

2013-01-01

Preface Viral replication is rapid and robust, but it is far from a chaotic process. Instead, successful production of infectious progeny requires that events occur in the correct place and at the correct time. Rotavirus, a segmented double-stranded RNA virus of the Reoviridae family, seems to govern its replication through ordered disassembly and assembly of a triple-layered icosahedral capsid. In recent years, high-resolution structural data have provided unprecedented insight into these events. In this Review, we explore the current understanding of rotavirus replication and how it compares to other Reoviridae family members. PMID:22266782

Viral persistence, latent reservoir, and blips: a review on HIV-1 dynamics and modeling during HAART and related treatment implications

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

Rong, Libin; Perelson, Alan

2008-01-01

HIV-1 eradication from infected individuals has not been achieved with the use of highly active antiretroviral therapy (HAART) for a prolonged period of time. The cellular reservoir for HIV-1 in resting memory CD4{sup +} T cells remains a major obstacle to viral elimination. The reservoir does not decay significantly over long periods of time as is able to release replication competent HIV-1 upon cell activation. Residual ongoing viral replication may likely occur in many patients because low levels of virus can be detected in plasma by sensitive assays and transient episodes of viremia, or HIV-1 blips, are often observed inmore » patients even with successful viral suppression for many years. Here we review our current knowledge of the factors contributing to viral persistence, the latent reservoir, and blips, and mathematical models developed to explore them and their relationships. We show how mathematical modeling can help improve our understanding of HIV-1 dynamics in patients on HAART and the quantitative events underlying HIV-1 latency, reservoir stability, low-level viremic persistence, and emergence of intermittent viral blips. We also discuss treatment implications related to these studies.« less

The role of virulence for in vivo superinfection fitness of a vertebrate RNA virus

USGS Publications Warehouse

Kell, Alison M.; Wargo, Andrew R.; Kurath, Gael

2013-01-01

We have developed a novel, in vivo superinfection fitness assay to examine superinfection dynamics and the role of virulence in superinfection fitness. This assay involves controlled, sequential infections of a natural, vertebrate host, Oncorhynchus mykiss (rainbow trout), with variants of a co-evolved viral pathogen, infectious hematopoietic necrosis virus (IHNV). Intervals between infections ranged from 12 hours to 7 days, and both frequency of superinfection and viral replication levels were examined. Using virus genotype pairs of equal and unequal virulence, we observed that superinfection generally occurred with decreasing frequency as the interval between exposures to each genotype increased. For both the equal virulence and unequal virulence genotype pairs, the frequency of superinfection was the same regardless of which genotype was used in the primary exposure. However, the ability to replicate in the context of superinfection did not differ between the genotypes of equal or unequal virulence tested here. For both genotype pairs, the mean viral load of the secondary virus was significantly reduced in superinfection, while primary virus replication was unaffected. Our results demonstrate, for the two genotype pairs examined, that superinfection restriction does occur for IHNV, and that higher virulence did not correlate with a significant difference in superinfection fitness. To our knowledge, this is the first assay to examine the role of virulence of an RNA virus in determining superinfection fitness dynamics within a natural, vertebrate host.

Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.

PubMed

Dembowski, Jill A; DeLuca, Neal A

2015-05-01

Much of the HSV-1 life cycle is carried out in the cell nucleus, including the expression, replication, repair, and packaging of viral genomes. Viral proteins, as well as cellular factors, play essential roles in these processes. Isolation of proteins on nascent DNA (iPOND) was developed to label and purify cellular replication forks. We adapted aspects of this method to label viral genomes to both image, and purify replicating HSV-1 genomes for the identification of associated proteins. Many viral and cellular factors were enriched on viral genomes, including factors that mediate DNA replication, repair, chromatin remodeling, transcription, and RNA processing. As infection proceeded, packaging and structural components were enriched to a greater extent. Among the more abundant proteins that copurified with genomes were the viral transcription factor ICP4 and the replication protein ICP8. Furthermore, all seven viral replication proteins were enriched on viral genomes, along with cellular PCNA and topoisomerases, while other cellular replication proteins were not detected. The chromatin-remodeling complexes present on viral genomes included the INO80, SWI/SNF, NURD, and FACT complexes, which may prevent chromatinization of the genome. Consistent with this conclusion, histones were not readily recovered with purified viral genomes, and imaging studies revealed an underrepresentation of histones on viral genomes. RNA polymerase II, the mediator complex, TFIID, TFIIH, and several other transcriptional activators and repressors were also affinity purified with viral DNA. The presence of INO80, NURD, SWI/SNF, mediator, TFIID, and TFIIH components is consistent with previous studies in which these complexes copurified with ICP4. Therefore, ICP4 is likely involved in the recruitment of these key cellular chromatin remodeling and transcription factors to viral genomes. Taken together, iPOND is a valuable method for the study of viral genome dynamics during infection and provides a comprehensive view of how HSV-1 selectively utilizes cellular resources.

SV40 Utilizes ATM Kinase Activity to Prevent Non-homologous End Joining of Broken Viral DNA Replication Products

PubMed Central

Sowd, Gregory A.; Mody, Dviti; Eggold, Joshua; Cortez, David; Friedman, Katherine L.; Fanning, Ellen

2014-01-01

Simian virus 40 (SV40) and cellular DNA replication rely on host ATM and ATR DNA damage signaling kinases to facilitate DNA repair and elicit cell cycle arrest following DNA damage. During SV40 DNA replication, ATM kinase activity prevents concatemerization of the viral genome whereas ATR activity prevents accumulation of aberrant genomes resulting from breakage of a moving replication fork as it converges with a stalled fork. However, the repair pathways that ATM and ATR orchestrate to prevent these aberrant SV40 DNA replication products are unclear. Using two-dimensional gel electrophoresis and Southern blotting, we show that ATR kinase activity, but not DNA-PKcs kinase activity, facilitates some aspects of double strand break (DSB) repair when ATM is inhibited during SV40 infection. To clarify which repair factors associate with viral DNA replication centers, we examined the localization of DSB repair proteins in response to SV40 infection. Under normal conditions, viral replication centers exclusively associate with homology-directed repair (HDR) and do not colocalize with non-homologous end joining (NHEJ) factors. Following ATM inhibition, but not ATR inhibition, activated DNA-PKcs and KU70/80 accumulate at the viral replication centers while CtIP and BLM, proteins that initiate 5′ to 3′ end resection during HDR, become undetectable. Similar to what has been observed during cellular DSB repair in S phase, these data suggest that ATM kinase influences DSB repair pathway choice by preventing the recruitment of NHEJ factors to replicating viral DNA. These data may explain how ATM prevents concatemerization of the viral genome and promotes viral propagation. We suggest that inhibitors of DNA damage signaling and DNA repair could be used during infection to disrupt productive viral DNA replication. PMID:25474690

DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication.

PubMed

Bristol, Molly L; Wang, Xu; Smith, Nathan W; Son, Minkyeong P; Evans, Michael R; Morgan, Iain M

2016-06-22

Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.

Requirement of multiple cis-acting elements in the human cytomegalovirus major immediate-early distal enhancer for viral gene expression and replication.

PubMed

Meier, Jeffery L; Keller, Michael J; McCoy, James J

2002-01-01

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer's orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at -300 or -345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication.

Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility

PubMed Central

Webel, Rike; Hakki, Morgan; Prichard, Mark N.; Rawlinson, William D.; Marschall, Manfred

2014-01-01

ABSTRACT The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of maribavir. IMPORTANCE The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why maribavir appears to have variable antiviral activity in cell culture and in vivo. PMID:24522923

TIA-1 and TIAR interact with 5'-UTR of enterovirus 71 genome and facilitate viral replication.

PubMed

Wang, Xiaohui; Wang, Huanru; Li, Yixuan; Jin, Yu; Chu, Ying; Su, Airong; Wu, Zhiwei

2015-10-16

Enterovirus 71 is one of the major causative pathogens of HFMD in children. Upon infection, the viral RNA is translated in an IRES-dependent manner and requires several host factors for effective replication. Here, we found that T-cell-restricted intracellular antigen 1 (TIA-1), and TIA-1 related protein (TIAR) were translocated from nucleus to cytoplasm after EV71 infection and localized to the sites of viral replication. We found that TIA-1 and TIAR can facilitate EV71 replication by enhancing the viral genome synthesis in host cells. We demonstrated that both proteins bound to the stem-loop I of 5'-UTR of viral genome and improved the stability of viral genomic RNA. Our results suggest that TIA-1 and TIAR are two new host factors that interact with 5-UTR of EV71 genome and positively regulate viral replication. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

A noda-like virus isolated from the sweetpotato pest spodoptera eridania (Cramer) (Lep.; noctuidae)

PubMed

Zeddam; Rodriguez; Ravallec; Lagnaoui

1999-11-01

A small isometric virus has been isolated from larvae of the sweetpotato pest Spodoptera eridania (Cramer) collected near Pariacoto, Ancash province, Peru. It is designated the Pariacoto virus (PaV). In addition to its high pathogenicity on its natural host Spodoptera eridania, PaV was found to replicate in Spodoptera ochrea (Hampson) larvae but not in Spodoptera frugiperda (Smith) larvae. The size of the viral particle was estimated to be about 30 nm in diameter. Polyacrylamide gel electrophoresis showed a protein of approximately 40.5 kDa. After agarose gel electrophoresis, the viral genome appeared to be bipartite RNA. Gel immunodiffusion tests showed no serological relationship between PaV and Nodamura virus, the type species for insect nodaviruses. Electron microscopy confirmed that viral replication occurs in the cytoplasm. These properties are similar to those of other members of family Nodaviridae, to which the virus is currently assigned. Copyright 1999 Academic Press.

Decoupling activation and exhaustion of B cells in spontaneous controllers of HIV infection

PubMed Central

Sciaranghella, Gaia; Tong, Neath; Mahan, Alison E.; Suscovich, Todd J.; Alter, Galit

2013-01-01

Objective To define the impact of chronic viremia and associated immune activation on B-cell exhaustion in HIV infection. Design Progressive HIV infection is marked by B-cell anergy and exhaustion coupled with dramatic hypergammaglobulinemia. Although both upregulation of CD95 and loss of CD21 have been used as markers of infection-associated B-cell dysfunction, little is known regarding the specific profiles of dysfunctional B cells and whether persistent viral replication and its associated immune activation play a central role in driving B-cell dysfunction. Methods Multiparameter flow cytometry was used to define the profile of dysfunctional B cells. The changes in the expression of CD21 and CD95 were tracked on B-cell subpopulations in patients with differential control of viral replication. Results Although the emergence of exhausted, CD21low tissue-like memory B cells followed similar patterns in both progressors and controllers, the frequency of CD21low activated memory B cells was lower in spontaneous controllers. Conclusion Our results suggest that the loss of CD21 and the upregulation of CD95 occur as separate events during the development of B-cell dysfunction. The loss of CD21 is a marker of B-cell exhaustion induced in the absence of appreciable viral replication, whereas the upregulation of CD95 is tightly linked to persistent viral replication and its associated immune activation. Thus, these dysfunctional profiles potentially represent two functionally distinct states within the B-cell compartment. PMID:23135171

Construction and characterization of poliovirus subgenomic replicons

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

Kaplan, G.; Racaniello, V.R.

1988-05-01

Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3,064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blotmore » (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of ({sup 35}S-labeled) viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2A{sup pro}, 2C, and 3D{sup pol} and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs.« less

Epstein-Barr Viral Productive Amplification Reprograms Nuclear Architecture, DNA Replication and Histone Deposition

PubMed Central

Chiu, Ya-Fang; Sugden, Arthur U.; Sugden, Bill

2014-01-01

Summary The spontaneous transition of Epstein-Barr Virus (EBV) from latency to productive infection is infrequent, making its analysis in the resulting mixed cell populations difficult. We engineered cells to support this transition efficiently and developed EBV DNA variants that could be visualized and measured as fluorescent signals over multiple cell cycles. This approach revealed that EBV’s productive replication began synchronously for viral DNAs within a cell but asynchronously between cells. EBV DNA amplification was delayed until early S-phase and occurred in factories characterized by the absence of cellular DNA and histones, by a sequential redistribution of PCNA, and by localization away from the nuclear periphery. The earliest amplified DNAs lacked histones accompanying a decline in four histone chaperones. Thus, EBV transitions from being dependent on the cellular replication machinery during latency to commandeering both that machinery and nuclear structure for its own reproductive needs. PMID:24331459

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

PubMed

Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

2017-05-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

Sp100 colocalizes with HPV replication foci and restricts the productive stage of the infectious cycle

PubMed Central

Khurana, Simran; Warburton, Alix

2017-01-01

We have shown previously that Sp100 (a component of the ND10 nuclear body) represses transcription, replication and establishment of incoming human papillomavirus (HPV) DNA in the early stages of infection. In this follow up study, we show that Sp100 does not substantially regulate viral infection in the maintenance phase, however at late stages of infection Sp100 interacts with amplifying viral genomes to repress viral processes. We find that Sp100 localizes to HPV16 replication foci generated in primary keratinocytes, to HPV31 replication foci that form in differentiated cells, and to HPV16 replication foci in CIN 1 cervical biopsies. To analyze this further, Sp100 was down regulated by siRNA treatment of differentiating HPV31 containing cells and levels of viral transcription and replication were assessed. This revealed that Sp100 represses viral transcription and replication in differentiated cells. Analysis of Sp100 binding to viral chromatin showed that Sp100 bound across the viral genome, and that binding increased at late stages of infection. Therefore, Sp100 represses the HPV life cycle at both early and late stages of infection. PMID:28968443

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

The Canonical Immediate Early 3 Gene Product pIE611 of Mouse Cytomegalovirus Is Dispensable for Viral Replication but Mediates Transcriptional and Posttranscriptional Regulation of Viral Gene Products.

PubMed

Rattay, Stephanie; Trilling, Mirko; Megger, Dominik A; Sitek, Barbara; Meyer, Helmut E; Hengel, Hartmut; Le-Trilling, Vu Thuy Khanh

2015-08-01

Transcription of mouse cytomegalovirus (MCMV) immediate early ie1 and ie3 is controlled by the major immediate early promoter/enhancer (MIEP) and requires differential splicing. Based on complete loss of genome replication of an MCMV mutant carrying a deletion of the ie3-specific exon 5, the multifunctional IE3 protein (611 amino acids; pIE611) is considered essential for viral replication. Our analysis of ie3 transcription resulted in the identification of novel ie3 isoforms derived from alternatively spliced ie3 transcripts. Construction of an IE3-hemagglutinin (IE3-HA) virus by insertion of an in-frame HA epitope sequence allowed detection of the IE3 isoforms in infected cells, verifying that the newly identified transcripts code for proteins. This prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication. To determine the role of pIE611 for viral gene expression during MCMV infection in an unbiased global approach, we used label-free quantitative mass spectrometry to delineate pIE611-dependent changes of the MCMV proteome. Interestingly, further analysis revealed transcriptional as well as posttranscriptional regulation of MCMV gene products by pIE611. Cytomegaloviruses are pathogenic betaherpesviruses persisting in a lifelong latency from which reactivation can occur under conditions of immunosuppression, immunoimmaturity, or inflammation. The switch from latency to reactivation requires expression of immediate early genes. Therefore, understanding of immediate early gene regulation might add insights into viral pathogenesis. The mouse cytomegalovirus (MCMV) immediate early 3 protein (611 amino acids; pIE611) is considered essential for viral replication. The identification of novel protein isoforms derived from alternatively spliced ie3 transcripts prompted the construction of an MCMV mutant lacking ie611 but retaining the coding capacity for the newly identified isoforms ie453 and ie310. Using Δie611 MCMV, we demonstrated the dispensability of the canonical ie3 gene product pIE611 for viral replication and delineated pIE611-dependent changes of the MCMV proteome. Our findings have fundamental implications for the interpretation of earlier studies on pIE3 functions and highlight the complex orchestration of MCMV gene regulation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Requirement of Multiple cis-Acting Elements in the Human Cytomegalovirus Major Immediate-Early Distal Enhancer for Viral Gene Expression and Replication

PubMed Central

Meier, Jeffery L.; Keller, Michael J.; McCoy, James J.

2002-01-01

We have shown previously that the human cytomegalovirus (HCMV) major immediate-early (MIE) distal enhancer is needed for MIE promoter-dependent transcription and viral replication at low multiplicities of infection (MOI). To understand how this region works, we constructed and analyzed a series of HCMVs with various distal enhancer mutations. We show that the distal enhancer is composed of at least two parts that function independently to coordinately activate MIE promoter-dependent transcription and viral replication. One such part is contained in a 47-bp segment that has consensus binding sites for CREB/ATF, SP1, and YY1. At low MOI, these working parts likely function in cis to directly activate MIE gene expression, thus allowing viral replication to ensue. Three findings support the view that these working parts are likely cis-acting elements. (i) Deletion of either part of a bisegmented distal enhancer only slightly alters MIE gene transcription and viral replication. (ii) Reversing the distal enhancer’s orientation largely preserves MIE gene transcription and viral replication. (iii) Placement of stop codons at −300 or −345 in all reading frames does not impair MIE gene transcription and viral replication. Lastly, we show that these working parts are dispensable at high MOI, partly because of compensatory stimulation of MIE promoter activity and viral replication that is induced by a virion-associated component(s) present at a high viral particle/cell ratio. We conclude that the distal enhancer is a complex multicomponent cis-acting region that is required to augment both MIE promoter-dependent transcription and HCMV replication. PMID:11739696

Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

PubMed

Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

2018-03-01

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication. Copyright © 2018 American Society for Microbiology.

pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth.

PubMed

Slayton, Mark; Hossain, Tanvir; Biegalke, Bonita J

2018-05-01

The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Copyright © 2018 Elsevier Inc. All rights reserved.

An Adenovirus DNA Replication Factor, but Not Incoming Genome Complexes, Targets PML Nuclear Bodies.

PubMed

Komatsu, Tetsuro; Nagata, Kyosuke; Wodrich, Harald

2016-02-01

Promyelocytic leukemia protein nuclear bodies (PML-NBs) are subnuclear domains implicated in cellular antiviral responses. Despite the antiviral activity, several nuclear replicating DNA viruses use the domains as deposition sites for the incoming viral genomes and/or as sites for viral DNA replication, suggesting that PML-NBs are functionally relevant during early viral infection to establish productive replication. Although PML-NBs and their components have also been implicated in the adenoviral life cycle, it remains unclear whether incoming adenoviral genome complexes target PML-NBs. Here we show using immunofluorescence and live-cell imaging analyses that incoming adenovirus genome complexes neither localize at nor recruit components of PML-NBs during early phases of infection. We further show that the viral DNA binding protein (DBP), an early expressed viral gene and essential DNA replication factor, independently targets PML-NBs. We show that DBP oligomerization is required to selectively recruit the PML-NB components Sp100 and USP7. Depletion experiments suggest that the absence of one PML-NB component might not affect the recruitment of other components toward DBP oligomers. Thus, our findings suggest a model in which an adenoviral DNA replication factor, but not incoming viral genome complexes, targets and modulates PML-NBs to support a conducive state for viral DNA replication and argue against a generalized concept that PML-NBs target incoming viral genomes. The immediate fate upon nuclear delivery of genomes of incoming DNA viruses is largely unclear. Early reports suggested that incoming genomes of herpesviruses are targeted and repressed by PML-NBs immediately upon nuclear import. Genome localization and/or viral DNA replication has also been observed at PML-NBs for other DNA viruses. Thus, it was suggested that PML-NBs may immediately sense and target nuclear viral genomes and hence serve as sites for deposition of incoming viral genomes and/or subsequent viral DNA replication. Here we performed a detailed analyses of the spatiotemporal distribution of incoming adenoviral genome complexes and found, in contrast to the expectation, that an adenoviral DNA replication factor, but not incoming genomes, targets PML-NBs. Thus, our findings may explain why adenoviral genomes could be observed at PML-NBs in earlier reports but argue against a generalized role for PML-NBs in targeting invading viral genomes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

The Human Cytomegalovirus UL51 Protein Is Essential for Viral Genome Cleavage-Packaging and Interacts with the Terminase Subunits pUL56 and pUL89

PubMed Central

Borst, Eva Maria; Kleine-Albers, Jennifer; Gabaev, Ildar; Babić, Marina; Wagner, Karen; Binz, Anne; Degenhardt, Inga; Kalesse, Markus; Jonjić, Stipan; Bauerfeind, Rudolf

2013-01-01

Cleavage of human cytomegalovirus (HCMV) genomes as well as their packaging into capsids is an enzymatic process mediated by viral proteins and therefore a promising target for antiviral therapy. The HCMV proteins pUL56 and pUL89 form the terminase and play a central role in cleavage-packaging, but several additional viral proteins, including pUL51, had been suggested to contribute to this process, although they remain largely uncharacterized. To study the function of pUL51 in infected cells, we constructed HCMV mutants encoding epitope-tagged versions of pUL51 and used a conditionally replicating virus (HCMV-UL51-ddFKBP), in which pUL51 levels could be regulated by a synthetic ligand. In cells infected with HCMV-UL51-ddFKBP, viral DNA replication was not affected when pUL51 was knocked down. However, no unit-length genomes and no DNA-filled C capsids were found, indicating that cleavage of concatemeric HCMV DNA and genome packaging into capsids did not occur in the absence of pUL51. pUL51 was expressed mainly with late kinetics and was targeted to nuclear replication compartments, where it colocalized with pUL56 and pUL89. Upon pUL51 knockdown, pUL56 and pUL89 were no longer detectable in replication compartments, suggesting that pUL51 is needed for their correct subnuclear localization. Moreover, pUL51 was found in a complex with the terminase subunits pUL56 and pUL89. Our data provide evidence that pUL51 is crucial for HCMV genome cleavage-packaging and may represent a third component of the viral terminase complex. Interference with the interactions between the terminase subunits by antiviral drugs could be a strategy to disrupt the HCMV replication cycle. PMID:23175377

Herpes Simplex Virus DNA Packaging without Measurable DNA Synthesis

PubMed Central

Church, Geoffrey A.; Dasgupta, Anindya; Wilson, Duncan W.

1998-01-01

Herpes simplex virus (HSV) type 1 DNA synthesis and packaging occur within the nuclei of infected cells; however, the extent to which the two processes are coupled remains unclear. Correct packaging is thought to be dependent upon DNA debranching or other repair processes, and such events commonly involve new DNA synthesis. Furthermore, the HSV UL15 gene product, essential for packaging, nevertheless localizes to sites of active DNA replication and may link the two events. It has previously been difficult to determine whether packaging requires concomitant DNA synthesis due to the complexity of these processes and of the viral life cycle; however, we have recently described a model system which simplifies the study of HSV assembly. Cells infected with HSV strain tsProt.A accumulate unpackaged capsids at the nonpermissive temperature of 39°C. Following release of the temperature block, these capsids proceed to package viral DNA in a single, synchronous wave. Here we report that, when DNA replication was inhibited prior to release of the temperature block, DNA packaging and later events in viral assembly nevertheless occurred at near-normal levels. We conclude that, under our conditions, HSV DNA packaging does not require detectable levels of DNA synthesis. PMID:9525593

Evidence supporting a role for TopBP1 and Brd4 in the initiation but not continuation of human papillomavirus 16 E1/E2-mediated DNA replication.

PubMed

Gauson, Elaine J; Donaldson, Mary M; Dornan, Edward S; Wang, Xu; Bristol, Molly; Bodily, Jason M; Morgan, Iain M

2015-05-01

To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evidence Supporting a Role for TopBP1 and Brd4 in the Initiation but Not Continuation of Human Papillomavirus 16 E1/E2-Mediated DNA Replication

PubMed Central

Gauson, Elaine J.; Donaldson, Mary M.; Dornan, Edward S.; Wang, Xu; Bristol, Molly; Bodily, Jason M.

2015-01-01

ABSTRACT To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. IMPORTANCE Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted. PMID:25694599

DDB1 Stimulates Viral Transcription of Hepatitis B Virus via HBx-Independent Mechanisms.

PubMed

Kim, Woohyun; Lee, Sooyoung; Son, Yeongnam; Ko, Chunkyu; Ryu, Wang-Shick

2016-11-01

HBx, a small regulatory protein of hepatitis B virus (HBV), augments viral DNA replication by stimulating viral transcription. Among numerous reported HBx-binding proteins, DDB1 has drawn attention, because DDB1 acts as a substrate receptor of the Cul4-DDB1 ubiquitin E3 ligase. Previous work reported that the DDB1-HBx interaction is indispensable for HBx-stimulated viral DNA replication, suggesting that the Cul4-DDB1 ubiquitin E3 ligase might target cellular restriction factors for ubiquitination and proteasomal degradation. To gain further insight into the DDB1-HBx interaction, we generated HBx mutants deficient for DDB1 binding (i.e., R96A, L98A, and G99A) and examined whether they support HBx-stimulated viral DNA replication. In contrast to data from previous reports, our results showed that the HBx mutants deficient for DDB1 binding supported viral DNA replication to nearly wild-type levels, revealing that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, we found that DDB1 directly stimulates viral transcription regardless of HBx expression. Through an HBV infection study, importantly, we demonstrated that DDB1 stimulates viral transcription from covalently closed circular DNA, a physiological template for viral transcription. Overall, we concluded that DDB1 stimulates viral transcription via a mechanism that does not involve an interaction with HBx. DDB1 constitutes a cullin-based ubiquitin E3 ligase, where DDB1 serves as an adaptor linking the cullin scaffold to the substrate receptor. Previous findings that the DDB1-binding ability of HBx is essential for HBx-stimulated viral DNA replication led to the hypothesis that HBx could downregulate host restriction factors that limit HBV replication through the cullin ubiquitin E3 ligase that requires the DDB1-HBx interaction. Consistent with this hypothesis, recent work identified Smc5/6 as a host restriction factor that is regulated by the viral cullin ubiquitin E3 ligase. In contrast, here we found that the DDB1-HBx interaction is largely dispensable for HBx-stimulated viral DNA replication. Instead, our results clearly showed that DDB1, regardless of HBx expression, enhances viral transcription. Overall, besides its role in the viral cullin ubiquitin E3 ligase, DDB1 itself stimulates viral transcription via HBx-independent mechanisms. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Replication of a chronic hepatitis B virus genotype F1b construct.

PubMed

Hernández, Sergio; Jiménez, Gustavo; Alarcón, Valentina; Prieto, Cristian; Muñoz, Francisca; Riquelme, Constanza; Venegas, Mauricio; Brahm, Javier; Loyola, Alejandra; Villanueva, Rodrigo A

2016-03-01

Genotype F is one of the less-studied genotypes of human hepatitis B virus, although it is widely distributed in regions of Central and South American. Our previous studies have shown that HBV genotype F is prevalent in Chile, and phylogenetic analysis of its full-length sequence amplified from the sera of chronically infected patients identified it as HBV subgenotype F1b. We have previously reported the full-length sequence of a HBV molecular clone obtained from a patient chronically infected with genotype F1b. In this report, we established a system to study HBV replication based on hepatoma cell lines transfected with full-length monomers of the HBV genome. Culture supernatants were analyzed after transfection and found to contain both HBsAg and HBeAg viral antigens. Consistently, fractionated cell extracts revealed the presence of viral replication, with both cytoplasmic and nuclear DNA intermediates. Analysis of HBV-transfected cells by indirect immunofluorescence or immunoelectron microscopy revealed the expression of viral antigens and cytoplasmic viral particles, respectively. To test the functionality of the ongoing viral replication further at the level of chromatinized cccDNA, transfected cells were treated with a histone deacetylase inhibitor, and this resulted in increased viral replication. This correlated with changes posttranslational modifications of histones at viral promoters. Thus, the development of this viral replication system for HBV genotype F will facilitate studies on the regulation of viral replication and the identification of new antiviral drugs.

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex

PubMed Central

Ganaie, Safder S.; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve

2017-01-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases. PMID:28459842

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

Murine Gammaherpesvirus 68 LANA and SOX Homologs Counteract ATM-Driven p53 Activity during Lytic Viral Replication

PubMed Central

Sifford, Jeffrey M.; Stahl, James A.; Salinas, Eduardo

2015-01-01

ABSTRACT Tumor suppressor p53 is activated in response to numerous cellular stresses, including viral infection. However, whether murine gammaherpesvirus 68 (MHV68) provokes p53 during the lytic replication cycle has not been extensively evaluated. Here, we demonstrate that MHV68 lytic infection induces p53 phosphorylation and stabilization in a manner that is dependent on the DNA damage response (DDR) kinase ataxia telangiectasia mutated (ATM). The induction of p53 during MHV68 infection occurred in multiple cell types, including splenocytes of infected mice. ATM and p53 activation required early viral gene expression but occurred independently of viral DNA replication. At early time points during infection, p53-responsive cellular genes were induced, coinciding with p53 stabilization and phosphorylation. However, p53-related gene expression subsided as infection progressed, even though p53 remained stable and phosphorylated. Infected cells also failed to initiate p53-dependent gene expression and undergo apoptosis in response to treatment with exogenous p53 agonists. The inhibition of p53 responses during infection required the expression of the MHV68 homologs of the shutoff and exonuclease protein (muSOX) and latency-associated nuclear antigen (mLANA). Interestingly, mLANA, but not muSOX, was necessary to prevent p53-mediated death in MHV68-infected cells under the conditions tested. This suggests that muSOX and mLANA are differentially required for inhibiting p53 in specific settings. These data reveal that DDR responses triggered by MHV68 infection promote p53 activation. However, MHV68 encodes at least two proteins capable of limiting the potential consequences of p53 function. IMPORTANCE Gammaherpesviruses are oncogenic herpesviruses that establish lifelong chronic infections. Defining how gammaherpesviruses overcome host responses to infection is important for understanding how these viruses infect and cause disease. Here, we establish that murine gammaherpesvirus 68 induces the activation of tumor suppressor p53. p53 activation was dependent on the DNA damage response kinase ataxia telangiectasia mutated. Although active early after infection, p53 became dominantly inhibited as the infection cycle progressed. Viral inhibition of p53 was mediated by the murine gammaherpesvirus 68 homologs of muSOX and mLANA. The inhibition of the p53 pathway enabled infected cells to evade p53-mediated cell death responses. These data demonstrate that a gammaherpesvirus encodes multiple proteins to limit p53-mediated responses to productive viral infection, which likely benefits acute viral replication and the establishment of chronic infection. PMID:26676792

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells.

PubMed

Strang, Blair L

2015-02-01

In human cytomegalovirus (HCMV)-infected cells, a dramatic remodelling of the nuclear architecture is linked to the creation, utilization and manipulation of subnuclear structures. This review outlines the involvement of several viral and cellular subnuclear structures in areas of HCMV replication and virus-host interaction that include viral transcription, viral DNA synthesis and the production of DNA-filled viral capsids. The structures discussed include those that promote or impede HCMV replication (such as viral replication compartments and promyelocytic leukaemia nuclear bodies, respectively) and those whose role in the infected cell is unclear (for example, nucleoli and nuclear speckles). Viral and cellular proteins associated with subnuclear structures are also discussed. The data reviewed here highlight advances in our understanding of HCMV biology and emphasize the complexity of HCMV replication and virus-host interactions in the nucleus. © 2015 The Authors.

Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing.

PubMed

Bierle, Craig J; Anderholm, Kaitlyn M; Wang, Jian Ben; McVoy, Michael A; Schleiss, Mark R

2016-08-01

The cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome in Escherichia coli and successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates. The cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infection in vivo Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Oligomerization transforms human APOBEC3G from an efficient enzyme to a slowly dissociating nucleic acid-binding protein

NASA Astrophysics Data System (ADS)

Chaurasiya, Kathy R.; McCauley, Micah J.; Wang, Wei; Qualley, Dominic F.; Wu, Tiyun; Kitamura, Shingo; Geertsema, Hylkje; Chan, Denise S. B.; Hertz, Amber; Iwatani, Yasumasa; Levin, Judith G.; Musier-Forsyth, Karin; Rouzina, Ioulia; Williams, Mark C.

2014-01-01

The human APOBEC3 proteins are a family of DNA-editing enzymes that play an important role in the innate immune response against retroviruses and retrotransposons. APOBEC3G is a member of this family that inhibits HIV-1 replication in the absence of the viral infectivity factor Vif. Inhibition of HIV replication occurs by both deamination of viral single-stranded DNA and a deamination-independent mechanism. Efficient deamination requires rapid binding to and dissociation from ssDNA. However, a relatively slow dissociation rate is required for the proposed deaminase-independent roadblock mechanism in which APOBEC3G binds the viral template strand and blocks reverse transcriptase-catalysed DNA elongation. Here, we show that APOBEC3G initially binds ssDNA with rapid on-off rates and subsequently converts to a slowly dissociating mode. In contrast, an oligomerization-deficient APOBEC3G mutant did not exhibit a slow off rate. We propose that catalytically active monomers or dimers slowly oligomerize on the viral genome and inhibit reverse transcription.

Inhibition of host protein synthesis by Sindbis virus: correlation with viral RNA replication and release of nuclear proteins to the cytoplasm.

PubMed

Sanz, Miguel A; García-Moreno, Manuel; Carrasco, Luis

2015-04-01

Infection of mammalian cells by Sindbis virus (SINV) profoundly blocks cellular mRNA translation. Experimental evidence points to viral non-structural proteins (nsPs), in particular nsP2, as the mediator of this inhibition. However, individual expression of nsP1, nsP2, nsP3 or nsP1-4 does not block cellular protein synthesis in BHK cells. Trans-complementation of a defective SINV replicon lacking most of the coding region for nsPs by the co-expression of nsP1-4 propitiates viral RNA replication at low levels, and inhibition of cellular translation is not observed. Exit of nuclear proteins including T-cell intracellular antigen and polypyrimidine tract-binding protein is clearly detected in SINV-infected cells, but not upon the expression of nsPs, even when the defective replicon was complemented. Analysis of a SINV variant with a point mutation in nsP2, exhibiting defects in the shut-off of host protein synthesis, indicates that both viral RNA replication and the release of nuclear proteins to the cytoplasm are greatly inhibited. Furthermore, nucleoside analogues that inhibit cellular and viral RNA synthesis impede the blockade of host mRNA translation, in addition to the release of nuclear proteins. Prevention of the shut-off of host mRNA translation by nucleoside analogues is not due to the inhibition of eIF2α phosphorylation, as this prevention is also observed in PKR(-/-) mouse embryonic fibroblasts that do not phosphorylate eIF2α after SINV infection. Collectively, our observations are consistent with the concept that for the inhibition of cellular protein synthesis to occur, viral RNA replication must take place at control levels, leading to the release of nuclear proteins to the cytoplasm. © 2014 John Wiley & Sons Ltd.

Genetic Diversity of Infectious Laryngotracheitis Virus during In Vivo Coinfection Parallels Viral Replication and Arises from Recombination Hot Spots within the Genome.

PubMed

Loncoman, Carlos A; Hartley, Carol A; Coppo, Mauricio J C; Vaz, Paola K; Diaz-Méndez, Andrés; Browning, Glenn F; García, Maricarmen; Spatz, Stephen; Devlin, Joanne M

2017-12-01

Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1 ) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome. IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease. Copyright © 2017 American Society for Microbiology.

Process of infection with bacteriophage phi chi 174. XL. Viral DNA replication of phi chi 174 mutants blocked in progeny single-stranded DNA synthesis.

PubMed Central

Fukuda, A; Sinsheimer, R L

1976-01-01

Mutation in several different cistrons of bacteriophage phi chi 174 blocks net progeny single-stranded DNA synthesis at the late period of infection (15). For the study of the functions of these cistrons in single-stranded DNA synthesis, asymmetric replication of replicative form DNA was examined at the late period of infection with amber mutants of these cistrons. While the normal, rapid process of asymmetric single-stranded viral DNA synthesis is blocked at the late period of these mutant infections, an asymmetric synthesis of the viral strand of replicative-form DNA is observed in this period, though at a reduced level, together with degradation of prelabeled viral strand. Some intermediate replicative-form molecules were also detected. Asymmetric synthesis of the viral strand of replicative-form DNA at the late period of phi chi infection is completely inhibited in the presence of a low concentration (35mug/ml) of chloramphenicol (which also blocks net single-stranded viral DNA synthesis). These results are discussed in terms of the possible role of the specific viral proteins for normal single-stranded DNA synthesis. PMID:1255871

Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region

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

Bienz, K.; Egger, D.; Troxler, M.

1990-03-01

Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but didmore » not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed.« less

Metabolism Goes Viral

PubMed Central

Miyake-Stoner, Shigeki J.; O’Shea, Clodagh C.

2014-01-01

Viral and cellular oncogenes converge in targeting critical protein interaction networks to reprogram the cellular DNA and protein replication machinery for pathological replication. In this issue, Thai et al. (2014) show that adenovirus E4ORF1 activates MYC glycolytic targets to induce a Warburg-like effect that converts glucose into nucleotides for viral replication. PMID:24703688

Detection of human cytomegalovirus DNA replication in non-permissive Vero and 293 cells.

PubMed

Ellsmore, Victoria; Reid, G Gordon; Stow, Nigel D

2003-03-01

Human cytomegalovirus (HCMV) displays an exceptionally restricted host range in tissue culture with human fibroblasts being the principal fully permissive system. Nevertheless, immediate early (IE) proteins are expressed following infection of many non-permissive cell types of human, simian and murine origin, and viral origin-dependent DNA synthesis has been reconstituted by transfection of plasmids into Vero cells, a non-permissive line from African green monkey. We have examined the accumulation of HCMV strain AD169 DNA, and the replication of transfected HCMV origin-containing plasmids, in infected Vero and human embryonic kidney 293 cells, which were previously reported to express the major IE protein in a small proportion of infected cells but to be non-permissive for viral DNA synthesis. In Vero cells accumulation of origin-containing plasmid but not viral DNA occurred, whilst in 293 cells both DNAs accumulated. Immunofluorescence experiments indicated that following infection with 3 p.f.u. per cell, a small fraction of both cell types expressed the UL44 DNA replication protein. Neither cell line, however, supported the generation of infectious progeny virus. These results suggest that IE proteins expressed in Vero and 293 cells can induce the synthesis of early proteins capable of functioning in viral DNA replication, but there is a failure in later events on the pathway to infectious virus production. This provides further support for transfected Vero cells being a valid system in which to study HCMV DNA synthesis, and suggests that 293 cells may also prove useful in similar experiments.

Trial watch

PubMed Central

Vacchelli, Erika; Eggermont, Alexander; Sautès-Fridman, Catherine; Galon, Jérôme; Zitvogel, Laurence; Kroemer, Guido; Galluzzi, Lorenzo

2013-01-01

Oncolytic virotherapy is emerging as a promising approach for the treatment of several neoplasms. The term “oncolytic viruses” is generally employed to indicate naturally occurring or genetically engineered attenuated viral particles that cause the demise of malignant cells while sparing their non-transformed counterparts. From a conceptual standpoint, oncolytic viruses differ from so-called “oncotropic viruses” in that only the former are able to kill cancer cells, even though both display a preferential tropism for malignant tissues. Of note, such a specificity can originate at several different steps of the viral cycle, including the entry of virions (transductional specificity) as well as their intracellular survival and replication (post-transcriptional and transcriptional specificity). During the past two decades, a large array of replication-competent and replication-incompetent oncolytic viruses has been developed and engineered to express gene products that would specifically promote the death of infected (cancer) cells. However, contrarily to long-standing beliefs, the antineoplastic activity of oncolytic viruses is not a mere consequence of the cytopathic effect, i.e., the lethal outcome of an intense, productive viral infection, but rather involves the elicitation of an antitumor immune response. In line with this notion, oncolytic viruses genetically modified to drive the local production of immunostimulatory cytokines exert more robust therapeutic effects than their non-engineered counterparts. Moreover, the efficacy of oncolytic virotherapy is significantly improved by some extent of initial immunosuppression (facilitating viral replication and spread) followed by the administration of immunostimulatory molecules (boosting antitumor immune responses). In this Trial Watch, we will discuss the results of recent clinical trials that have evaluated/are evaluating the safety and antineoplastic potential of oncolytic virotherapy. PMID:23894720

Role of hydrogen sulfide in paramyxovirus infections.

PubMed

Li, Hui; Ma, Yinghong; Escaffre, Oliver; Ivanciuc, Teodora; Komaravelli, Narayana; Kelley, John P; Coletta, Ciro; Szabo, Csaba; Rockx, Barry; Garofalo, Roberto P; Casola, Antonella

2015-05-01

Hydrogen sulfide (H2S) is an endogenous gaseous mediator that has gained increasing recognition as an important player in modulating acute and chronic inflammatory diseases. However, its role in virus-induced lung inflammation is currently unknown. Respiratory syncytial virus (RSV) is a major cause of upper and lower respiratory tract infections in children for which no vaccine or effective treatment is available. Using the slow-releasing H2S donor GYY4137 and propargylglycin (PAG), an inhibitor of cystathionine-γ-lyase (CSE), a key enzyme that produces intracellular H2S, we found that RSV infection led to a reduced ability to generate and maintain intracellular H2S levels in airway epithelial cells (AECs). Inhibition of CSE with PAG resulted in increased viral replication and chemokine secretion. On the other hand, treatment of AECs with the H2S donor GYY4137 reduced proinflammatory mediator production and significantly reduced viral replication, even when administered several hours after viral absorption. GYY4137 also significantly reduced replication and inflammatory chemokine production induced by human metapneumovirus (hMPV) and Nipah virus (NiV), suggesting a broad inhibitory effect of H2S on paramyxovirus infections. GYY4137 treatment had no effect on RSV genome replication or viral mRNA and protein synthesis, but it inhibited syncytium formation and virus assembly/release. GYY4137 inhibition of proinflammatory gene expression occurred by modulation of the activation of the key transcription factors nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF-3) at a step subsequent to their nuclear translocation. H2S antiviral and immunoregulatory properties could represent a novel treatment strategy for paramyxovirus infections. RSV is a global health concern, causing significant morbidity and economic losses as well as mortality in developing countries. After decades of intensive research, no vaccine or effective treatment, with the exception of immunoprophylaxis, is available for this infection as well as for other important respiratory mucosal viruses. This study identifies hydrogen sulfide as a novel cellular mediator that can modulate viral replication and proinflammatory gene expression, both important determinants of lung injury in respiratory viral infections, with potential for rapid translation of such findings into novel therapeutic approaches for viral bronchiolitis and pneumonia. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Productive replication of human papillomavirus 31 requires DNA repair factor Nbs1.

PubMed

Anacker, Daniel C; Gautam, Dipendra; Gillespie, Kenric A; Chappell, William H; Moody, Cary A

2014-08-01

Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

PubMed Central

Anacker, Daniel C.; Gautam, Dipendra; Gillespie, Kenric A.; Chappell, William H.

2014-01-01

ABSTRACT Activation of the ATM (ataxia telangiectasia-mutated kinase)-dependent DNA damage response (DDR) is necessary for productive replication of human papillomavirus 31 (HPV31). We previously found that DNA repair and homologous recombination (HR) factors localize to sites of HPV replication, suggesting that ATM activity is required to recruit factors to viral genomes that can productively replicate viral DNA in a recombination-dependent manner. The Mre11-Rad50-Nbs1 (MRN) complex is an essential component of the DDR that is necessary for ATM-mediated HR repair and localizes to HPV DNA foci. In this study, we demonstrate that the HPV E7 protein is sufficient to increase levels of the MRN complex and also interacts with MRN components. We have found that Nbs1 depletion blocks productive viral replication and results in decreased localization of Mre11, Rad50, and the principal HR factor Rad51 to HPV DNA foci upon differentiation. Nbs1 contributes to the DDR by acting as an upstream activator of ATM in response to double-strand DNA breaks (DSBs) and as a downstream effector of ATM activity in the intra-S-phase checkpoint. We have found that phosphorylation of ATM and its downstream target Chk2, as well as SMC1 (structural maintenance of chromosome 1), is maintained upon Nbs1 knockdown in differentiating cells. Given that ATM and Chk2 are required for productive replication, our results suggest that Nbs1 contributes to viral replication outside its role as an ATM activator, potentially through ensuring localization of DNA repair factors to viral genomes that are necessary for efficient productive replication. IMPORTANCE The mechanisms that regulate human papillomavirus (HPV) replication during the viral life cycle are not well understood. Our finding that Nbs1 is necessary for productive replication even in the presence of ATM (ataxia telangiectasia-mutated kinase) and Chk2 phosphorylation offers evidence that Nbs1 contributes to viral replication downstream of facilitating ATM activation. Nbs1 is required for the recruitment of Mre11 and Rad50 to viral genomes, suggesting that the MRN complex plays a direct role in facilitating productive viral replication, potentially through the processing of substrates that are recognized by the key homologous recombination (HR) factor Rad51. The discovery that E7 increases levels of MRN components, and MRN complex formation, identifies a novel role for E7 in facilitating productive replication. Our study not only identifies DNA repair factors necessary for HPV replication but also provides a deeper understanding of how HPV utilizes the DNA damage response to regulate viral replication. PMID:24850735

Construction and characterization of poliovirus subgenomic replicons.

PubMed

Kaplan, G; Racaniello, V R

1988-05-01

Poliovirus RNAs containing in-frame deletions within the capsid-coding region were produced by in vitro transcription of altered poliovirus type 1 cDNA by using bacteriophage T7 RNA polymerase. Three RNAs were transcribed that contained deletions of 2,317 nucleotides (bases 747 to 3064), 1,781 nucleotides (bases 1,175 to 2,956), and 1,295 nucleotides (bases 1,175 to 2,470). All three subgenomic RNAs replicated after transfection into HeLa cells, demonstrating that sequences encoding the capsid polypeptides are not essential for viral RNA replication in vivo. Viral RNA containing the largest deletion (R1) replicated approximately three times better than full-length RNA produced in vitro. Northern blot (RNA blot) hybridization analysis of total cellular RNA from HeLa cells at different times after transfection with R1 demonstrated the presence of increasing amounts of the expected 5.1-kilobase subgenomic RNA. Analysis by immunoprecipitation of viral proteins induced after transfection of R1 RNA into HeLa cells revealed the presence of proteins 2Apro, 2C, and 3Dpol and its precursors, suggesting that the polyprotein cleavages are similar to those occurring in virus-infected cells. Replication of P2/Lansing virion RNA was inhibited by cotransfection with the R1 replicon, as demonstrated by hybridization analysis with a serotype-specific oligonucleotide probe. A higher level of inhibition of RNA replication was observed when P2/Lansing RNA was cotransfected into HeLa cells with truncated R1 transcripts (R1-PvuII) that were missing 395 3' nucleotides and a poly(A) tail. These internally and terminally deleted RNAs inhibited the replication of subgenomic replicons R1, R2, and R3 and caused a reduction in plaque size when cotransfected with P1/Mahoney or P2/Lansing viral RNA, suggesting that individual cells had received both RNAs. No inhibition of plaque size was observed when replicon RNAs were used that were missing 1,384 or 1,839 3' nucleotides or contained plasmid-derived sequences downstream of the 3' poly(A). The trans-acting inhibitory effect of R1-PvuII on the replication of poliovirus P2/Lansing RNA did not involve entry of RNA into cells and appeared to reduce viral translation and RNA synthesis late in the infection cycle.

Metabolism goes viral.

PubMed

Miyake-Stoner, Shigeki J; O'Shea, Clodagh C

2014-04-01

Viral and cellular oncogenes converge in targeting critical protein interaction networks to reprogram the cellular DNA and protein replication machinery for pathological replication. In this issue, Thai et al. (2014) show that adenovirus E4ORF1 activates MYC glycolytic targets to induce a Warburg-like effect that converts glucose into nucleotides for viral replication. Copyright © 2014 Elsevier Inc. All rights reserved.

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

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

Filling Knowledge Gaps for Mimivirus Entry, Uncoating, and Morphogenesis

PubMed Central

Andrade, Ana Cláudia dos Santos Pereira; Rodrigues, Rodrigo Araújo Lima; Oliveira, Graziele Pereira; Andrade, Kétyllen Reis; Bonjardim, Cláudio Antônio; La Scola, Bernard; Kroon, Erna Geessien

2017-01-01

ABSTRACT Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assay results, shed light on unclear points concerning the mimivirus replication cycle. We found that treatment with cytochalasin, a phagocytosis inhibitor, negatively impacted the incorporation of mimivirus particles by Acanthamoeba castellanii, causing a negative effect on viral growth in amoeba monolayers. Treatment of amoebas with bafilomicin significantly impacted mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In-depth analyses of particle morphogenesis suggest that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals from previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory where particles acquire the surface fibrils. Furthermore, we reinforce the concept that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimivirus. IMPORTANCE Investigating the viral life cycle is essential to a better understanding of virus biology. The combination of biological assays and microscopic images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many knowledge gaps remain to be filled. In this study, we conducted a new examination of the replication cycle of mimivirus and provide new evidence concerning some stages of the cycle which were previously unclear, mainly entry, uncoating, and morphogenesis. Furthermore, we demonstrate that atypical virion morphologies can occur even in the absence of virophages. Our results, along with previous data, allow us to present an ultimate model for the mimivirus replication cycle. PMID:28878069

Filling Knowledge Gaps for Mimivirus Entry, Uncoating, and Morphogenesis.

PubMed

Andrade, Ana Cláudia Dos Santos Pereira; Rodrigues, Rodrigo Araújo Lima; Oliveira, Graziele Pereira; Andrade, Kétyllen Reis; Bonjardim, Cláudio Antônio; La Scola, Bernard; Kroon, Erna Geessien; Abrahão, Jônatas Santos

2017-11-15

Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assay results, shed light on unclear points concerning the mimivirus replication cycle. We found that treatment with cytochalasin, a phagocytosis inhibitor, negatively impacted the incorporation of mimivirus particles by Acanthamoeba castellanii , causing a negative effect on viral growth in amoeba monolayers. Treatment of amoebas with bafilomicin significantly impacted mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In-depth analyses of particle morphogenesis suggest that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals from previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory where particles acquire the surface fibrils. Furthermore, we reinforce the concept that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimivirus. IMPORTANCE Investigating the viral life cycle is essential to a better understanding of virus biology. The combination of biological assays and microscopic images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many knowledge gaps remain to be filled. In this study, we conducted a new examination of the replication cycle of mimivirus and provide new evidence concerning some stages of the cycle which were previously unclear, mainly entry, uncoating, and morphogenesis. Furthermore, we demonstrate that atypical virion morphologies can occur even in the absence of virophages. Our results, along with previous data, allow us to present an ultimate model for the mimivirus replication cycle. Copyright © 2017 American Society for Microbiology.

Reovirus Nonstructural Protein σNS Acts as an RNA-Stability Factor Promoting Viral Genome Replication.

PubMed

Zamora, Paula F; Hu, Liya; Knowlton, Jonathan J; Lahr, Roni M; Moreno, Rodolfo A; Berman, Andrea J; Prasad, B V Venkataram; Dermody, Terence S

2018-05-16

Viral nonstructural proteins, which are not packaged into virions, are essential for replication of most viruses. Reovirus, a nonenveloped, double-stranded RNA (dsRNA) virus, encodes three nonstructural proteins that are required for viral replication and dissemination in the host. Reovirus nonstructural protein σNS is a single-stranded RNA (ssRNA)-binding protein that must be expressed in infected cells for production of viral progeny. However, activities of σNS during individual steps of the reovirus replication cycle are poorly understood. We explored the function of σNS by disrupting its expression during infection using cells expressing a small interfering RNA (siRNA) targeting the σNS-encoding S3 gene and found that σNS is required for viral genome replication. Using complementary biochemical assays, we determined that σNS forms complexes with viral and nonviral RNAs. We also discovered that σNS increases RNA half-life using in vitro and cell-based RNA degradation experiments. Cryo-electron microscopy revealed that σNS and ssRNAs organize into long, filamentous structures. Collectively, our findings indicate that σNS functions as an RNA-binding protein that increases viral RNA half-life. These results suggest that σNS forms RNA-protein complexes in preparation for genome replication. IMPORTANCE Following infection, viruses synthesize nonstructural proteins that mediate viral replication and promote dissemination. Viruses from the Reoviridae family encode nonstructural proteins that are required for the formation of progeny viruses. Although nonstructural proteins of different Reoviridae family viruses are diverged in primary sequence, these proteins are functionally homologous and appear to facilitate conserved mechanisms of dsRNA virus replication. Using in vitro and cell-culture approaches, we found that the mammalian reovirus nonstructural protein σNS binds and stabilizes viral RNA and is required for genome synthesis. This work contributes new knowledge about basic mechanisms of dsRNA virus replication and provides a foundation for future studies to determine how viruses in the Reoviridae family assort and replicate their genomes. Copyright © 2018 American Society for Microbiology.

Multiplex CRISPR/Cas9 system impairs HCMV replication by excising an essential viral gene.

PubMed

Gergen, Janina; Coulon, Flora; Creneguy, Alison; Elain-Duret, Nathan; Gutierrez, Alejandra; Pinkenburg, Olaf; Verhoeyen, Els; Anegon, Ignacio; Nguyen, Tuan Huy; Halary, Franck Albert; Haspot, Fabienne

2018-01-01

Anti-HCMV treatments used in immunosuppressed patients reduce viral replication, but resistant viral strains can emerge. Moreover, these drugs do not target latently infected cells. We designed two anti-viral CRISPR/Cas9 strategies to target the UL122/123 gene, a key regulator of lytic replication and reactivation from latency. The singleplex strategy contains one gRNA to target the start codon. The multiplex strategy contains three gRNAs to excise the complete UL122/123 gene. Primary fibroblasts and U-251 MG cells were transduced with lentiviral vectors encoding Cas9 and one or three gRNAs. Both strategies induced mutations in the target gene and a concomitant reduction of immediate early (IE) protein expression in primary fibroblasts. Further detailed analysis in U-251 MG cells showed that the singleplex strategy induced 50% of indels in the viral genome, leading to a reduction in IE protein expression. The multiplex strategy excised the IE gene in 90% of all viral genomes and thus led to the inhibition of IE protein expression. Consequently, viral genome replication and late protein expression were reduced by 90%. Finally, the production of new viral particles was nearly abrogated. In conclusion, the multiplex anti-UL122/123 CRISPR/Cas9 system can target the viral genome efficiently enough to significantly prevent viral replication.

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

Noise-induced bistability in the quasi-neutral coexistence of viral RNAs under different replication modes.

PubMed

Sardanyés, Josep; Arderiu, Andreu; Elena, Santiago F; Alarcón, Tomás

2018-05-01

Evolutionary and dynamical investigations into real viral populations indicate that RNA replication can range between the two extremes represented by so-called 'stamping machine replication' (SMR) and 'geometric replication' (GR). The impact of asymmetries in replication for single-stranded (+) sense RNA viruses has been mainly studied with deterministic models. However, viral replication should be better described by including stochasticity, as the cell infection process is typically initiated with a very small number of RNA macromolecules, and thus largely influenced by intrinsic noise. Under appropriate conditions, deterministic theoretical descriptions of viral RNA replication predict a quasi-neutral coexistence scenario, with a line of fixed points involving different strands' equilibrium ratios depending on the initial conditions. Recent research into the quasi-neutral coexistence in two competing populations reveals that stochastic fluctuations fundamentally alter the mean-field scenario, and one of the two species outcompetes the other. In this article, we study this phenomenon for viral RNA replication modes by means of stochastic simulations and a diffusion approximation. Our results reveal that noise has a strong impact on the amplification of viral RNAs, also causing the emergence of noise-induced bistability. We provide analytical criteria for the dominance of (+) sense strands depending on the initial populations on the line of equilibria, which are in agreement with direct stochastic simulation results. The biological implications of this noise-driven mechanism are discussed within the framework of the evolutionary dynamics of RNA viruses with different modes of replication. © 2018 The Author(s).

Enterovirus 3A Facilitates Viral Replication by Promoting Phosphatidylinositol 4-Kinase IIIβ–ACBD3 Interaction

PubMed Central

Xiao, Xia; Lei, Xiaobo; Zhang, Zhenzhen; Ma, Yijie; Qi, Jianli; Wu, Chao; Xiao, Yan; Li, Li

2017-01-01

ABSTRACT Like other enteroviruses, enterovirus 71 (EV71) relies on phosphatidylinositol 4-kinase IIIβ (PI4KB) for genome RNA replication. However, how PI4KB is recruited to the genome replication sites of EV71 remains elusive. Recently, we reported that a host factor, ACBD3, is needed for EV71 replication by interacting with viral 3A protein. Here, we show that ACBD3 is required for the recruitment of PI4KB to RNA replication sites. Overexpression of viral 3A or EV71 infection stimulates the interaction of PI4KB and ACBD3. Consistently, EV71 infection induces the production of phosphatidylinositol-4-phosphate (PI4P). Furthermore, PI4KB, ACBD3, and 3A are all localized to the viral-RNA replication sites. Accordingly, PI4KB or ACBD3 depletion by small interfering RNA (siRNA) leads to a reduction in PI4P production after EV71 infection. I44A or H54Y substitution in 3A interrupts the stimulation of PI4KB and ACBD3. Further analysis suggests that stimulation of ACBD3-PI4KB interaction is also important for the replication of enterovirus 68 but disadvantageous to human rhinovirus 16. These results reveal a mechanism of enterovirus replication that involves a selective strategy for recruitment of PI4KB to the RNA replication sites. IMPORTANCE Enterovirus 71, like other human enteroviruses, replicates its genome within host cells, where viral proteins efficiently utilize cellular machineries. While multiple factors are involved, it is largely unclear how viral replication is controlled. We show that the 3A protein of enterovirus 71 recruits an enzyme, phosphatidylinositol 4-kinase IIIβ, by interacting with ACBD3, which alters cellular membranes through the production of a lipid, PI4P. Consequently, the viral and host proteins form a large complex that is necessary for RNA synthesis at replication sites. Notably, PI4KB-ACBD3 interaction also differentially mediates the replication of enterovirus 68 and rhinovirus 16. These results provide new insight into the molecular network of enterovirus replication. PMID:28701404

The anti-obesity drug orlistat reveals anti-viral activity.

PubMed

Ammer, Elisabeth; Nietzsche, Sandor; Rien, Christian; Kühnl, Alexander; Mader, Theresa; Heller, Regine; Sauerbrei, Andreas; Henke, Andreas

2015-12-01

The administration of drugs to inhibit metabolic pathways not only reduces the risk of obesity-induced diseases in humans but may also hamper the replication of different viral pathogens. In order to investigate the value of the US Food and Drug Administration-approved anti-obesity drug orlistat in view of its anti-viral activity against different human-pathogenic viruses, several anti-viral studies, electron microscopy analyses as well as fatty acid uptake experiments were performed. The results indicate that administrations of non-cytotoxic concentrations of orlistat reduced the replication of coxsackievirus B3 (CVB3) in different cell types significantly. Moreover, orlistat revealed cell protective effects and modified the formation of multi-layered structures in CVB3-infected cells, which are necessary for viral replication. Lowering fatty acid uptake from the extracellular environment by phloretin administrations had only marginal impact on CVB3 replication. Finally, orlistat reduced also the replication of varicella-zoster virus moderately but had no significant influence on the replication of influenza A viruses. The data support further experiments into the value of orlistat as an inhibitor of the fatty acid synthase to develop new anti-viral compounds, which are based on the modulation of cellular metabolic pathways.

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication

PubMed Central

2013-01-01

Background Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. Methods To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. Results In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Conclusions Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication. PMID:23800076

The eukaryotic translation initiation factor 3 subunit L protein interacts with Flavivirus NS5 and may modulate yellow fever virus replication.

PubMed

Morais, Ana Ts; Terzian, Ana Cb; Duarte, Danilo Vb; Bronzoni, Roberta Vm; Madrid, Maria Cfs; Gavioli, Arieli F; Gil, Laura Hvg; Oliveira, Amanda G; Zanelli, Cleslei F; Valentini, Sandro R; Rahal, Paula; Nogueira, Mauricio L

2013-06-22

Yellow fever virus (YFV) belongs to the Flavivirus genus and causes an important disease. An alarming resurgence of viral circulation and the expansion of YFV-endemic zones have been detected in Africa and South America in recent years. NS5 is a viral protein that contains methyltransferase and RNA-dependent RNA polymerase (RdRp) domains, which are essential for viral replication, and the interactions between NS5 and cellular proteins have been studied to better understand viral replication. The aim of this study was to characterize the interaction of the NS5 protein with eukaryotic translation initiation factor 3 subunit L (eIF3L) and to evaluate the role of eIF3L in yellow fever replication. To identify interactions of YFV NS5 with cellular proteins, we performed a two-hybrid screen using the YFV NS5 RdRp domain as bait with a human cDNA library, and RNApol deletion mutants were generated and analyzed using the two-hybrid system for mapping the interactions. The RNApol region involved was segmented into three fragments and analyzed using an eIF3L-expressing yeast strain. To map the NS5 residues that are critical for the interactions, we performed site-direct mutagenesis in segment 3 of the interaction domain (ID) and confirmed the interaction using in vitro assays and in vivo coimmunoprecipitation. The significance of eIF3L for YFV replication was investigated using eIF3L overexpression and RNA interference. In this work, we describe and characterize the interaction of NS5 with the translation factor eIF3L. The interaction between NS5 and eIF3L was confirmed using in vitro binding and in vivo coimmunoprecipitation assays. This interaction occurs at a region (the interaction domain of the RNApol domain) that is conserved in several flaviviruses and that is, therefore, likely to be relevant to the genus. eIF3L overexpression and plaque reduction assays showed a slight effect on YFV replication, indicating that the interaction of eIF3L with YFV NS5 may play a role in YFV replication. Although the precise function of eIF3L on interactions with viral proteins is not entirely understood, these results indicate an interaction of eIF3L with YF NS5 and that eIF3L overexpression facilitates translation, which has potential implications for virus replication.

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

A postdoctoral position is available in the Viral Recombination Section (VRS), HIV Dynamics and Replication Program, CCR.  The VRS studies retroviral replication using human immunodeficiency viruses and other retroviruses, with a particular emphasis on the mechanisms of viral RNA biology, specific RNA packaging, virus assembly, and HIV replication.  Molecular tools and advanced imaging approaches are used to dissect various aspects of viral replication mechanisms.  A more complete description of the projects can be found at http://home.ncifcrf.gov/hivdrp/Hu_res.html.

SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

PubMed Central

Luo, Yong; Deng, Xuefeng; Cheng, Fang; Li, Yi

2013-01-01

Activation of a host DNA damage response (DDR) is essential for DNA replication of minute virus of canines (MVC), a member of the genus Bocavirus of the Parvoviridae family; however, the mechanism by which DDR contributes to viral DNA replication is unknown. In the current study, we demonstrate that MVC infection triggers the intra-S-phase arrest to slow down host cellular DNA replication and to recruit cellular DNA replication factors for viral DNA replication. The intra-S-phase arrest is regulated by ATM (ataxia telangiectasia-mutated kinase) signaling in a p53-independent manner. Moreover, we demonstrate that SMC1 (structural maintenance of chromosomes 1) is the key regulator of the intra-S-phase arrest induced during infection. Either knockdown of SMC1 or complementation with a dominant negative SMC1 mutant blocks both the intra-S-phase arrest and viral DNA replication. Finally, we show that the intra-S-phase arrest induced during MVC infection was caused neither by damaged host cellular DNA nor by viral proteins but by replicating viral genomes physically associated with the DNA damage sensor, the Mre11-Rad50-Nbs1 (MRN) complex. In conclusion, the feedback loop between MVC DNA replication and the intra-S-phase arrest is mediated by ATM-SMC1 signaling and plays a critical role in MVC DNA replication. Thus, our findings unravel the mechanism underlying DDR signaling-facilitated MVC DNA replication and demonstrate a novel strategy of DNA virus-host interaction. PMID:23365434

Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types.

PubMed

Selth, Luke A; Randles, John W; Rezaian, M Ali

2002-04-10

We have previously shown that the soil-borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA. However, the reproducibility of this observation with other geminiviruses has been questioned. Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium. Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication-competent by changing their configuration so that two copies of the viral ori were present. Furthermore, we report that low-level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid. These findings were reinforced by expression studies using beta-glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E. coli.

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

PubMed

Muñoz-Espín, Daniel; Daniel, Richard; Kawai, Yoshikazu; Carballido-López, Rut; Castilla-Llorente, Virginia; Errington, Jeff; Meijer, Wilfried J J; Salas, Margarita

2009-08-11

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

Peripheral and central immune cell reservoirs in tissues from asymptomatic cats chronically infected with feline immunodeficiency virus.

PubMed

Eckstrand, C D; Sparger, E E; Pitt, K A; Murphy, B G

2017-01-01

Feline immunodeficiency virus (FIV) infection in cats results in life-long viral persistence and progressive immunopathology. We have previously described a cohort of experimentally infected cats demonstrating a progressive decline of peripheral blood CD4+ T-cell over six years in the face of apparent peripheral viral latency. More recently we reported findings from this same cohort that revealed popliteal lymph node tissue as sites for ongoing viral replication suggesting that tissue reservoirs are important in FIV immunopathogenesis during the late asymptomatic phase of infection. Results reported herein characterize important tissue reservoirs of active viral replication during the late asymptomatic phase by examining biopsied specimens of spleen, mesenteric lymph node (MLN), and intestine from FIV-infected and uninfected control cats. Peripheral blood collected coincident with harvest of tissues demonstrated severe CD4+ T-cell depletion, undetectable plasma viral gag RNA and rarely detectable peripheral blood mononuclear cell (PBMC)-associated viral RNA (vRNA) by real-time PCR. However, vRNA was detectable in all three tissue sites from three of four FIV-infected cats despite the absence of detectable vRNA in plasma. A novel in situ hybridization assay identified B cell lymphoid follicular domains as microanatomical foci of ongoing FIV replication. Additionally, we demonstrated that CD4+ leukocyte depletion in tissues, and CD4+ and CD21+ leukocytes as important cellular reservoirs of ongoing replication. These findings revealed that tissue reservoirs support foci of ongoing viral replication, in spite of highly restricted viral replication in blood. Lentiviral eradication strategies will need address tissue viral reservoirs.

Peripheral and central immune cell reservoirs in tissues from asymptomatic cats chronically infected with feline immunodeficiency virus

PubMed Central

Sparger, E. E.; Pitt, K. A.

2017-01-01

Feline immunodeficiency virus (FIV) infection in cats results in life-long viral persistence and progressive immunopathology. We have previously described a cohort of experimentally infected cats demonstrating a progressive decline of peripheral blood CD4+ T-cell over six years in the face of apparent peripheral viral latency. More recently we reported findings from this same cohort that revealed popliteal lymph node tissue as sites for ongoing viral replication suggesting that tissue reservoirs are important in FIV immunopathogenesis during the late asymptomatic phase of infection. Results reported herein characterize important tissue reservoirs of active viral replication during the late asymptomatic phase by examining biopsied specimens of spleen, mesenteric lymph node (MLN), and intestine from FIV-infected and uninfected control cats. Peripheral blood collected coincident with harvest of tissues demonstrated severe CD4+ T-cell depletion, undetectable plasma viral gag RNA and rarely detectable peripheral blood mononuclear cell (PBMC)-associated viral RNA (vRNA) by real-time PCR. However, vRNA was detectable in all three tissue sites from three of four FIV-infected cats despite the absence of detectable vRNA in plasma. A novel in situ hybridization assay identified B cell lymphoid follicular domains as microanatomical foci of ongoing FIV replication. Additionally, we demonstrated that CD4+ leukocyte depletion in tissues, and CD4+ and CD21+ leukocytes as important cellular reservoirs of ongoing replication. These findings revealed that tissue reservoirs support foci of ongoing viral replication, in spite of highly restricted viral replication in blood. Lentiviral eradication strategies will need address tissue viral reservoirs. PMID:28384338

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

PubMed

Ustav, M; Stenlund, A

1991-02-01

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.

Hematopoietic Cancer Cell Lines Can Support Replication of Sabin Poliovirus Type 1

PubMed Central

van Eikenhorst, Gerco; de Gruijl, Tanja D.; van der Pol, Leo A.; Bakker, Wilfried A. M.

2015-01-01

Viral vaccines can be produced in adherent or in suspension cells. The objective of this work was to screen human suspension cell lines for the capacity to support viral replication. As the first step, it was investigated whether poliovirus can replicate in such cell lines. Sabin poliovirus type 1 was serially passaged on five human cell lines, HL60, K562, KG1, THP-1, and U937. Sabin type 1 was capable of efficiently replicating in three cell lines (K562, KG1, and U937), yielding high viral titers after replication. Expression of CD155, the poliovirus receptor, did not explain susceptibility to replication, since all cell lines expressed CD155. Furthermore, we showed that passaged virus replicated more efficiently than parental virus in KG1 cells, yielding higher virus titers in the supernatant early after infection. Infection of cell lines at an MOI of 0.01 resulted in high viral titers in the supernatant at day 4. Infection of K562 with passaged Sabin type 1 in a bioreactor system yielded high viral titers in the supernatant. Altogether, these data suggest that K562, KG1, and U937 cell lines are useful for propagation of poliovirus. PMID:25815312

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.

Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells*

PubMed Central

Narayanan, Aarthi; Kehn-Hall, Kylene; Senina, Svetlana; Lundberg, Lindsay; Van Duyne, Rachel; Guendel, Irene; Das, Ravi; Baer, Alan; Bethel, Laura; Turell, Michael; Hartman, Amy Lynn; Das, Bhaskar; Bailey, Charles; Kashanchi, Fatah

2012-01-01

Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets. PMID:22847000

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 Damage Signaling Is Induced in the Absence of Epstein-Barr Virus (EBV) Lytic DNA Replication and in Response to Expression of ZEBRA.

PubMed

Wang'ondu, Ruth; Teal, Stuart; Park, Richard; Heston, Lee; Delecluse, Henri; Miller, George

2015-01-01

Epstein Barr virus (EBV), like other oncogenic viruses, modulates the activity of cellular DNA damage responses (DDR) during its life cycle. Our aim was to characterize the role of early lytic proteins and viral lytic DNA replication in activation of DNA damage signaling during the EBV lytic cycle. Our data challenge the prevalent hypothesis that activation of DDR pathways during the EBV lytic cycle occurs solely in response to large amounts of exogenous double stranded DNA products generated during lytic viral DNA replication. In immunofluorescence or immunoblot assays, DDR activation markers, specifically phosphorylated ATM (pATM), H2AX (γH2AX), or 53BP1 (p53BP1), were induced in the presence or absence of viral DNA amplification or replication compartments during the EBV lytic cycle. In assays with an ATM inhibitor and DNA damaging reagents in Burkitt lymphoma cell lines, γH2AX induction was necessary for optimal expression of early EBV genes, but not sufficient for lytic reactivation. Studies in lytically reactivated EBV-positive cells in which early EBV proteins, BGLF4, BGLF5, or BALF2, were not expressed showed that these proteins were not necessary for DDR activation during the EBV lytic cycle. Expression of ZEBRA, a viral protein that is necessary for EBV entry into the lytic phase, induced pATM foci and γH2AX independent of other EBV gene products. ZEBRA mutants deficient in DNA binding, Z(R183E) and Z(S186E), did not induce foci of pATM. ZEBRA co-localized with HP1β, a heterochromatin associated protein involved in DNA damage signaling. We propose a model of DDR activation during the EBV lytic cycle in which ZEBRA induces ATM kinase phosphorylation, in a DNA binding dependent manner, to modulate gene expression. ATM and H2AX phosphorylation induced prior to EBV replication may be critical for creating a microenvironment of viral and cellular gene expression that enables lytic cycle progression.

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.

Different Pathogenesis of CCR5-Using Primary HIV-1 Isolates from Non-Switch and Switch Virus Patients in Human Lymphoid Tissue Ex Vivo

NASA Technical Reports Server (NTRS)

Iarlsson, Ingrid; Grivel, Jean-Charles; Chen. Silvia; Karlsson, Anders; Albert, Jan; Fenyol, Eva Maria; Margolis, Leonid B.

2005-01-01

CCR5-utilizing HIV-1 variants (R5) typically transmit infection and dominate its early stages, whereas emergence of CXCR4-using (X4 or R5X4) HIV-1 is often associated with disease progression. However, such a switch in co-receptor usage can only be detected in approximately onehalf of HIV-infected patients (switch virus patients), and progression to immunodeficiency may also occur in patients without detectable switch in co-receptor usage (non-switch virus patients). Here, we used a system of ex vivo-infected tonsillar tissue to compare the pathogenesis of sequential primary R5 HIV-1 isolates from the switch and non-switch patients. Inoculation of ex vivo tissue with these R5 isolates resulted in viral replication and CCR5(+)CD4(+) T cell depletion. The levels of such depletion by HIV-1 isolated from non-switch virus patients were significantly higher than those by R5 HIV-1 isolates from switch virus patients. T cell depletion seemed to be controlled by viral factors and did not significantly vary between tissues from different donors. In contrast, viral replication did not correlate with the switch status of the patients; in tissues fiom different donors it varied 30-fold and seemed to be controlled by a combination of viral and tissue factors. Nevertheless, replication-level hierarchy among sequential isolates remained constant in tissues from various donors. Viral load in vivo was higher in switch virus patients compared to non-switch virus patients. The high cytopathogenicity of CCR5(+)CD4(+) T cells by R5 HIV-1 isolates from non-switch virus patients may explain the steady decline of CD4(+) T cells in the absence of CXCR4 using virus; elimination of target cells by these isolates may limit their own replication in vivo.

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.

Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells

PubMed Central

Lin, C.; Agnes, J. T.; Behrens, N.; Tagawa, Y.; Gershwin, L. J.; Corbeil, L. B.

2016-01-01

Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2—RSAD2) and ISG15 (IFN-stimulated gene 15—ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo. PMID:26859677

Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells.

PubMed

Lin, C; Agnes, J T; Behrens, N; Shao, M; Tagawa, Y; Gershwin, L J; Corbeil, L B

2016-01-01

Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2--RSAD2) and ISG15 (IFN-stimulated gene 15--ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.

Single-Cell Analysis of the Impact of Host Cell Heterogeneity on Infection with Foot-and-Mouth Disease Virus.

PubMed

Xin, Xiu; Wang, Hailong; Han, Lingling; Wang, Mingzhen; Fang, Hui; Hao, Yao; Li, Jiadai; Zhang, Hu; Zheng, Congyi; Shen, Chao

2018-05-01

Viral infection and replication are affected by host cell heterogeneity, but the mechanisms underlying the effects remain unclear. Using single-cell analysis, we investigated the effects of host cell heterogeneity, including cell size, inclusion, and cell cycle, on foot-and-mouth disease virus (FMDV) infection (acute and persistent infections) and replication. We detected various viral genome replication levels in FMDV-infected cells. Large cells and cells with a high number of inclusions generated more viral RNA copies and viral protein and a higher proportion of infectious cells than other cells. Additionally, we found that the viral titer was 10- to 100-fold higher in cells in G 2 /M than those in other cell cycle phases and identified a strong correlation between cell size, inclusion, and cell cycle heterogeneity, which all affected the infection and replication of FMDV. Furthermore, we demonstrated that host cell heterogeneity influenced the adsorption of FMDV due to differences in the levels of FMDV integrin receptors expression. Collectively, these results further our understanding of the evolution of a virus in a single host cell. IMPORTANCE It is important to understand how host cell heterogeneity affects viral infection and replication. Using single-cell analysis, we found that viral genome replication levels exhibited dramatic variability in foot-and-mouth disease virus (FMDV)-infected cells. We also found a strong correlation between heterogeneity in cell size, inclusion number, and cell cycle status and that all of these characteristics affect the infection and replication of FMDV. Moreover, we found that host cell heterogeneity influenced the viral adsorption as differences in the levels of FMDV integrin receptors' expression. This study provided new ideas for the studies of correlation between FMDV infection mechanisms and host cells. Copyright © 2018 American Society for Microbiology.

Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

NASA Astrophysics Data System (ADS)

Laporta, Robert F.; Taichman, Lorne B.

1982-06-01

Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

Human CD134 (OX40) expressed on T cells plays a key role for human herpesvirus 6B replication after allogeneic hematopoietic stem cell transplantation.

PubMed

Nagamata, Satoshi; Nagasaka, Miwako; Kawabata, Akiko; Kishimoto, Kenji; Hasegawa, Daiichiro; Kosaka, Yoshiyuki; Mori, Takeshi; Morioka, Ichiro; Nishimura, Noriyuki; Iijima, Kazumoto; Yamada, Hideto; Kawamoto, Shinichiro; Yakushijin, Kimikazu; Matsuoka, Hiroshi; Mori, Yasuko

2018-05-01

CD134 (OX40), which is a cellular receptor for human herpesvirus-6B (HHV-6B) and expresses on activated T cells, may play a key role for HHV-6B replication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Therefore, we examined the CD134 expression on T cells and HHV-6B replication after allo-HSCT, and analyzed the correlation between them. Twenty-three patients after allo-HSCT were enrolled. The percentages of CD134-positive cells within the CD4 + and CD8 + cell populations were measured by flow cytometry, and the viral copy number of HHV-6B was simultaneously quantified by real-time PCR. The correlation between CD134 and HHV-6B viral load was then statistically analyzed. HHV-6B reactivation occurred in 11 of 23 patients (47.8%). CD134 expression was seen on T cells and was coincident with the time of peak viral load. The percentage of CD134-positive cells decreased significantly when HHV-6B DNA disappeared (p = .005 in CD4 + T cells, p = .02 in CD8 + T cells). In the 4 patients who underwent umbilical cord blood transplantation (UCBT), the viral load varied with the percentage of CD134-positive cells. In the comparison between the HHV-6B reactivation group and non-reactivation group, maximum percentages of CD134-positive cells among CD4 + T cells in reactivation group were significantly higher than those in non-reactivation group (p = .04). This is the first study to show that a correlation of CD134 expression on T cells with HHV-6B replication after allo-HSCT, especially in UCBT. The results possibly indicate that CD134 on T cells plays a key role for HHV-6B replication after allo-HSCT. Copyright © 2018 Elsevier B.V. All rights reserved.

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter

PubMed Central

Wang, Xiaohong; Liu, Haibin; Ge, Hui; Ajiro, Masahiko; Sharma, Nishi R.; Meyers, Craig; Morozov, Pavel; Tuschl, Thomas; Klar, Amar; Court, Donald

2017-01-01

ABSTRACT The life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis-acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors. PMID:28559488

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly.

PubMed

Romero-Brey, Inés; Bartenschlager, Ralf

2016-06-07

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles.

Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly

PubMed Central

Romero-Brey, Inés; Bartenschlager, Ralf

2016-01-01

The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles. PMID:27338443

Increased inflammation in sanctuary sites may explain viral blips in HIV infection

DOE PAGES

Piovoso, Michael J.; Cardozo, E. Fabian; Zurakowski, Ryan

2016-08-01

Here, combined antiretroviral therapy (cART) suppress HIV-1 viral replication, such that viral load in plasma remains below the limit of detection in standard assays. However, intermittent episodes of transient viremia (blips) occur in a set of HIV-patients. Given that follicular hyperplasia occurs during lymphoid inflammation as a normal response to infection, it is hypothesised that when the diameter of the lymph node follicle (LNF) increases and crosses a critical size, a viral blip occurs due to cryptic viremia. To study this hypothesis, a theoretical analysis of a mathematical model is performed to find the conditions for virus suppression in allmore » compartments and different scenarios of LNF size changes are simulated. According to the analysis, blips with duration of around 30 days arise when the diameter rise rate is between 0.02 and 0.03 days –1. Moreover, the final diameter of the site is directly related to the steady states of the virus load after the occurrence of a blip. When the value of R 0 is around 2.1, to have a steady-state below the limit of detection after the viral blip, the maximum final diameters should be greater than 0.7 mm so that there is a relative loss of connection between compartments.« less

Increased inflammation in sanctuary sites may explain viral blips in HIV infection.

PubMed

Cardozo, E Fabian; Piovoso, Michael J; Zurakowski, Ryan

2016-08-01

Combined antiretroviral therapy (cART) suppress HIV-1 viral replication, such that viral load in plasma remains below the limit of detection in standard assays. However, intermittent episodes of transient viremia (blips) occur in a set of HIV-patients. Given that follicular hyperplasia occurs during lymphoid inflammation as a normal response to infection, it is hypothesised that when the diameter of the lymph node follicle (LNF) increases and crosses a critical size, a viral blip occurs due to cryptic viremia. To study this hypothesis, a theoretical analysis of a mathematical model is performed to find the conditions for virus suppression in all compartments and different scenarios of LNF size changes are simulated. According to the analysis, blips with duration of around 30 days arise when the diameter rise rate is between 0.02 and 0.03 days(-1). Moreover, the final diameter of the site is directly related to the steady states of the virus load after the occurrence of a blip. When the value of R0 is around 2.1, to have a steady-state below the limit of detection after the viral blip, the maximum final diameters should be greater than 0.7 mm so that there is a relative loss of connection between compartments.

Increased inflammation in sanctuary sites may explain viral blips in HIV infection

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

Piovoso, Michael J.; Cardozo, E. Fabian; Zurakowski, Ryan

Here, combined antiretroviral therapy (cART) suppress HIV-1 viral replication, such that viral load in plasma remains below the limit of detection in standard assays. However, intermittent episodes of transient viremia (blips) occur in a set of HIV-patients. Given that follicular hyperplasia occurs during lymphoid inflammation as a normal response to infection, it is hypothesised that when the diameter of the lymph node follicle (LNF) increases and crosses a critical size, a viral blip occurs due to cryptic viremia. To study this hypothesis, a theoretical analysis of a mathematical model is performed to find the conditions for virus suppression in allmore » compartments and different scenarios of LNF size changes are simulated. According to the analysis, blips with duration of around 30 days arise when the diameter rise rate is between 0.02 and 0.03 days –1. Moreover, the final diameter of the site is directly related to the steady states of the virus load after the occurrence of a blip. When the value of R 0 is around 2.1, to have a steady-state below the limit of detection after the viral blip, the maximum final diameters should be greater than 0.7 mm so that there is a relative loss of connection between compartments.« less

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

PubMed Central

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

2014-01-01

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

Ebola virus vaccines: an overview of current approaches

PubMed Central

Marzi, Andrea; Feldmann, Heinz

2016-01-01

Ebola hemorrhagic fever is one of the most fatal viral diseases worldwide affecting humans and nonhuman primates. Although infections only occur frequently in Central Africa, the virus has the potential to spread globally and is classified as a category A pathogen that could be misused as a bioterrorism agent. As of today there is no vaccine or treatment licensed to counteract Ebola virus infections. DNA, subunit and several viral vector approaches, replicating and non-replicating, have been tested as potential vaccine platforms and their protective efficacy has been evaluated in nonhuman primate models for Ebola virus infections, which closely resemble disease progression in humans. Though these vaccine platforms seem to confer protection through different mechanisms, several of them are efficacious against lethal disease in nonhuman primates attesting that vaccination against Ebola virus infections is feasible. PMID:24575870

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.

Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames.

PubMed Central

Ustav, M; Stenlund, A

1991-01-01

Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells. Images PMID:1846806

H2AX phosphorylation and DNA damage kinase activity are dispensable for herpes simplex virus replication.

PubMed

Botting, Carolyn; Lu, Xu; Triezenberg, Steven J

2016-01-27

Herpes simplex virus type 1 (HSV-1) can establish both lytic and latent infections in humans. The phosphorylation of histone H2AX, a common marker of DNA damage, during lytic infection by HSV-1 is well established. However, the role(s) of H2AX phosphorylation in lytic infection remain unclear. Following infection of human foreskin fibroblasts by HSV-1 or HSV-2, we assayed the phosphorylation of H2AX in the presence of inhibitors of transcription, translation, or viral DNA replication, or in the presence of inhibitors of ATM and ATR kinases (KU-55933 and VE-821, respectively). We also assayed viral replication in fibroblasts in the presence of the kinase inhibitors or siRNAs specific for ATM and ATR, as well as in cell lines deficient for either ATR or ATM. The expression of viral immediate-early and early proteins (including the viral DNA polymerase), but not viral DNA replication or late protein expression, were required for H2AX phosphorylation following HSV-1 infection. Inhibition of ATM kinase activity prevented HSV-stimulated H2AX phosphorylation but had only a minor effect on DNA replication and virus yield in HFF cells. These results differ from previous reports of a dramatic reduction in viral yield following chemical inhibition of ATM in oral keratinocytes or following infection of ATM(-/-) cells. Inhibition of the closely related kinase ATR (whether by chemical inhibitor or siRNA disruption) had no effect on H2AX phosphorylation and reduced viral DNA replication only moderately. During infection by HSV-2, H2AX phosphorylation was similarly dispensable but was dependent on both ATM activity and viral DNA replication. H2AX phosphorylation represents a cell type-specific and virus type-specific host response to HSV infection with little impact on viral infection.

Hili Inhibits HIV Replication in Activated T Cells.

PubMed

Peterlin, B Matija; Liu, Pingyang; Wang, Xiaoyun; Cary, Daniele; Shao, Wei; Leoz, Marie; Hong, Tian; Pan, Tao; Fujinaga, Koh

2017-06-01

P-element-induced wimpy-like (Piwil) proteins restrict the replication of mobile genetic elements in the germ line. They are also expressed in many transformed cell lines. In this study, we discovered that the human Piwil 2 (Hili) protein can also inhibit HIV replication, especially in activated CD4 + T cells that are the preferred target cells for this virus in the infected host. Although resting cells did not express Hili, its expression was rapidly induced following T cell activation. In these cells and transformed cell lines, depletion of Hili increased levels of viral proteins and new viral particles. Further studies revealed that Hili binds to tRNA. Some of the tRNAs represent rare tRNA species, whose codons are overrepresented in the viral genome. Targeting tRNA Arg (UCU) with an antisense oligonucleotide replicated effects of Hili and also inhibited HIV replication. Finally, Hili also inhibited the retrotransposition of the endogenous intracysternal A particle (IAP) by a similar mechanism. Thus, Hili joins a list of host proteins that inhibit the replication of HIV and other mobile genetic elements. IMPORTANCE Piwil proteins inhibit the movement of mobile genetic elements in the germ line. In their absence, sperm does not form and male mice are sterile. This inhibition is thought to occur via small Piwi-interacting RNAs (piRNAs). However, in some species and in human somatic cells, Piwil proteins bind primarily to tRNA. In this report, we demonstrate that human Piwil proteins, especially Hili, not only bind to select tRNA species, including rare tRNAs, but also inhibit HIV replication. Importantly, T cell activation induces the expression of Hili in CD4 + T cells. Since Hili also inhibited the movement of an endogenous retrovirus (IAP), our finding shed new light on this intracellular resistance to exogenous and endogenous retroviruses as well as other mobile genetic elements. Copyright © 2017 American Society for Microbiology.

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.

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

The emerging role of nuclear viral DNA sensors.

PubMed

Diner, Benjamin A; Lum, Krystal K; Cristea, Ileana M

2015-10-30

Detecting pathogenic DNA by intracellular receptors termed "sensors" is critical toward galvanizing host immune responses and eliminating microbial infections. Emerging evidence has challenged the dogma that sensing of viral DNA occurs exclusively in sub-cellular compartments normally devoid of cellular DNA. The interferon-inducible protein IFI16 was shown to bind nuclear viral DNA and initiate immune signaling, culminating in antiviral cytokine secretion. Here, we review the newly characterized nucleus-originating immune signaling pathways, their links to other crucial host defenses, and unique mechanisms by which viruses suppress their functions. We frame these findings in the context of human pathologies associated with nuclear replicating DNA viruses. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficiency in Complexity: Composition and Dynamic Nature of Mimivirus Replication Factories

PubMed Central

Milrot, Elad; Mutsafi, Yael; Ben-Dor, Shifra; Levin, Yishai; Savidor, Alon; Kartvelishvily, Elena

2016-01-01

ABSTRACT The recent discovery of multiple giant double-stranded DNA (dsDNA) viruses blurred the consensual distinction between viruses and cells due to their size, as well as to their structural and genetic complexity. A dramatic feature revealed by these viruses as well as by many positive-strand RNA viruses is their ability to rapidly form elaborate intracellular organelles, termed “viral factories,” where viral progeny are continuously generated. Here we report the first isolation of viral factories at progressive postinfection time points. The isolated factories were subjected to mass spectrometry-based proteomics, bioinformatics, and imaging analyses. These analyses revealed that numerous viral proteins are present in the factories but not in mature virions, thus implying that multiple and diverse proteins are required to promote the efficiency of viral factories as “production lines” of viral progeny. Moreover, our results highlight the dynamic and highly complex nature of viral factories, provide new and general insights into viral infection, and substantiate the intriguing notion that viral factories may represent the living state of viruses. IMPORTANCE Large dsDNA viruses such as vaccinia virus and the giant mimivirus, as well as many positive-strand RNA viruses, generate elaborate cytoplasmic organelles in which the multiple and diverse transactions required for viral replication and assembly occur. These organelles, which were termed “viral factories,” are attracting much interest due to the increasing realization that the rapid and continuous production of viral progeny is a direct outcome of the elaborate structure and composition of the factories, which act as efficient production lines. To get new insights into the nature and function of viral factories, we devised a method that allows, for the first time, the isolation of these organelles. Analyses of the isolated factories generated at different times postinfection by mass spectrometry-based proteomics provide new perceptions of their role and reveal the highly dynamic nature of these organelles. PMID:27581975

Cidofovir inhibits polyomavirus BK replication in human renal tubular cells downstream of viral early gene expression.

PubMed

Bernhoff, E; Gutteberg, T J; Sandvik, K; Hirsch, H H; Rinaldo, C H

2008-07-01

The human polyomavirus BK (BKV) causes nephropathy and hemorrhagic cystitis in kidney and bone marrow transplant patients, respectively. The anti-viral cidofovir (CDV) has been used in small case series but the effects on BKV replication are unclear, since polyomaviruses do not encode viral DNA polymerases. We investigated the effects of CDV on BKV(Dunlop) replication in primary human renal proximal tubule epithelial cells (RPTECs). CDV inhibited the generation of viral progeny in a dose-dependent manner yielding a 90% reduction at 40 microg/mL. Early steps such as receptor binding and entry seemed unaffected. Initial large T-antigen transcription and expression were also unaffected, but subsequent intra-cellular BKV DNA replication was reduced by >90%. Late viral mRNA and corresponding protein levels were also 90% reduced. In uninfected RPTECs, CDV 40 microg/mL reduced cellular DNA replication and metabolic activity by 7% and 11% in BrdU and WST-1 assays, respectively. BKV infection increased DNA replication to 142% and metabolic activity to 116%, respectively, which were reduced by CDV 40 microg/mL to levels of uninfected untreated RPTECs. Our results show that CDV inhibits BKV DNA replication downstream of large T-antigen expression and involves significant host cell toxicity. This should be considered in current treatment and drug development.

Persistence of an Oncogenic Papillomavirus Genome Requires cis Elements from the Viral Transcriptional Enhancer

PubMed Central

Van Doorslaer, Koenraad; Chen, Dan; Chapman, Sandra; Khan, Jameela

2017-01-01

ABSTRACT Human papillomavirus (HPV) genomes are replicated and maintained as extrachromosomal plasmids during persistent infection. The viral E2 proteins are thought to promote stable maintenance replication by tethering the viral DNA to host chromatin. However, this has been very difficult to prove genetically, as the E2 protein is involved in transcriptional regulation and initiation of replication, as well as its assumed role in genome maintenance. This makes mutational analysis of viral trans factors and cis elements in the background of the viral genome problematic and difficult to interpret. To circumvent this problem, we have developed a complementation assay in which the complete wild-type HPV18 genome is transfected into primary human keratinocytes along with subgenomic or mutated replicons that contain the minimal replication origin. The wild-type genome provides the E1 and E2 proteins in trans, allowing us to determine additional cis elements that are required for long-term replication and partitioning of the replicon. We found that, in addition to the core replication origin (and the three E2 binding sites located therein), additional sequences from the transcriptional enhancer portion of the URR (upstream regulatory region) are required in cis for long-term genome replication. PMID:29162712

Replicating viral vector platform exploits alarmin signals for potent CD8+ T cell-mediated tumour immunotherapy

PubMed Central

Kallert, Sandra M.; Darbre, Stephanie; Bonilla, Weldy V.; Kreutzfeldt, Mario; Page, Nicolas; Müller, Philipp; Kreuzaler, Matthias; Lu, Min; Favre, Stéphanie; Kreppel, Florian; Löhning, Max; Luther, Sanjiv A.; Zippelius, Alfred; Merkler, Doron; Pinschewer, Daniel D.

2017-01-01

Viral infections lead to alarmin release and elicit potent cytotoxic effector T lymphocyte (CTLeff) responses. Conversely, the induction of protective tumour-specific CTLeff and their recruitment into the tumour remain challenging tasks. Here we show that lymphocytic choriomeningitis virus (LCMV) can be engineered to serve as a replication competent, stably-attenuated immunotherapy vector (artLCMV). artLCMV delivers tumour-associated antigens to dendritic cells for efficient CTL priming. Unlike replication-deficient vectors, artLCMV targets also lymphoid tissue stroma cells expressing the alarmin interleukin-33. By triggering interleukin-33 signals, artLCMV elicits CTLeff responses of higher magnitude and functionality than those induced by replication-deficient vectors. Superior anti-tumour efficacy of artLCMV immunotherapy depends on interleukin-33 signalling, and a massive CTLeff influx triggers an inflammatory conversion of the tumour microenvironment. Our observations suggest that replicating viral delivery systems can release alarmins for improved anti-tumour efficacy. These mechanistic insights may outweigh safety concerns around replicating viral vectors in cancer immunotherapy. PMID:28548102

Vesicular stomatitis virus infects resident cells of the central nervous system and induces replication-dependent inflammatory responses

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

Chauhan, Vinita S.; Furr, Samantha R.; Sterka, David G.

2010-05-10

Vesicular stomatitis virus (VSV) infection of mice via intranasal administration results in a severe encephalitis with rapid activation and proliferation of microglia and astrocytes. We have recently shown that these glial cells express RIG-I and MDA5, cytosolic pattern recognition receptors for viral RNA. However, it is unclear whether VSV can replicate in glial cells or if such replication is required for their inflammatory responses. Here we demonstrate that primary microglia and astrocytes are permissive for VSV infection and limited productive replication. Importantly, we show that viral replication is required for robust inflammatory mediator production by these cells. Finally, we havemore » confirmed that in vivo VSV administration can result in viral infection of glial cells in situ. These results suggest that viral replication within resident glial cells might play an important role in CNS inflammation following infection with VSV and possibly other neurotropic nonsegmented negative-strand RNA viruses.« less

Structural insights into the rhabdovirus transcription/replication complex.

PubMed

Ivanov, Ivan; Yabukarski, Filip; Ruigrok, Rob W H; Jamin, Marc

2011-12-01

The rhabdoviruses have a non-segmented single stranded negative-sense RNA genome. Their multiplication in a host cell requires three viral proteins in addition to the viral RNA genome. The nucleoprotein (N) tightly encapsidates the viral RNA, and the N-RNA complex serves as the template for both transcription and replication. The viral RNA-dependent RNA polymerase is a two subunit complex that consists of a large subunit, L, and a non-catalytic cofactor, the phosphoprotein, P. P also acts as a chaperone of nascent RNA-free N by forming a N(0)-P complex that prevents N from binding to cellular RNAs and from polymerizing in the absence of RNA. Here, we discuss the recent molecular and structural studies of individual components and multi-molecular complexes that are involved in the transcription/replication complex of these viruses with regard to their implication in viral transcription and replication. Copyright © 2011 Elsevier B.V. All rights reserved.

Nordihydroguaiaretic acid (NDGA) inhibits replication and viral morphogenesis of dengue virus.

PubMed

Soto-Acosta, Rubén; Bautista-Carbajal, Patricia; Syed, Gulam H; Siddiqui, Aleem; Del Angel, Rosa M

2014-09-01

Dengue is the most common mosquito borne viral disease in humans. The infection with any of the 4 dengue virus serotypes (DENV) can either be asymptomatic or manifest in two clinical forms, the mild dengue fever or the more severe dengue hemorrhagic fever that may progress into dengue shock syndrome. A DENV replicative cycle relies on host lipid metabolism; specifically, DENV infection modulates cholesterol and fatty acid synthesis, generating a lipid-enriched cellular environment necessary for viral replication. Thus, the aim of this work was to evaluate the anti-DENV effect of the Nordihydroguaiaretic acid (NDGA), a hypolipidemic agent with antioxidant and anti-inflammatory properties. A dose-dependent inhibition in viral yield and NS1 secretion was observed in supernatants of infected cells treated for 24 and 48 h with different concentrations of NDGA. To evaluate the effect of NDGA in DENV replication, a DENV4 replicon transfected Vero cells were treated with different concentrations of NDGA. NDGA treatment significantly reduced DENV replication, reiterating the importance of lipids in viral replication. NDGA treatment also led to reduction in number of lipid droplets (LDs), the neutral lipid storage organelles involved in DENV morphogenesis that are known to increase in number during DENV infection. Furthermore, NDGA treatment resulted in dissociation of the C protein from LDs. Overall our results suggest that NDGA inhibits DENV infection by targeting genome replication and viral assembly. Copyright © 2014 Elsevier B.V. All rights reserved.

Root cause investigation of a viral contamination incident occurred during master cell bank (MCB) testing and characterization--a case study.

PubMed

Chen, Dayue; Nims, Raymond; Dusing, Sandra; Miller, Pamela; Luo, Wen; Quertinmont, Michelle; Parekh, Bhavin; Poorbaugh, Josh; Boose, Jeri Ann; Atkinson, E Morrey

2008-11-01

An adventitious agent contamination occurred during a routine 9 CFR bovine viral screening test at BioReliance for an Eli Lilly Chinese Hamster Ovary (CHO) cell-derived Master Cell Bank (MCB) intended for biological production. Scientists from the sponsor (Eli Lilly and Company) and the testing service company (BioReliance) jointly conducted a systematic investigation in an attempt to determine the root cause of the contamination. Our investigation resulted in the identification of the viral nature of the contaminant. Subsequent experiments indicated that the viral contaminant was a non-enveloped and non-hemadsorbing virus. Transmission electron microscopy (TEM) revealed that the viral contaminant was 25-30 nm in size and morphologically resembled viruses of the family Picornaviridae. The contaminant virus was readily inactivated when exposed to acidic pH, suggesting that the viral contaminant was a member of rhinoviruses. Although incapable of infecting CHO cells, the viral contaminant replicated efficiently in Vero cell with a life cycle of approximately 16 h. Our investigation provided compelling data demonstrating that the viral contaminant did not originate from the MCB. Instead, it was introduced into the process during cell passaging and a possible entry point was proposed. We identified the viral contaminant as an equine rhinitis A virus using molecular cloning and DNA sequencing. Finally, our investigation led us to conclude that the source of the viral contaminant was the equine serum added to the cell growth medium in the 9 CFR bovine virus test.

Thymic HIV-2 infection uncovers posttranscriptional control of viral replication in human thymocytes.

PubMed

Nunes-Cabaço, Helena; Matoso, Paula; Foxall, Russell B; Tendeiro, Rita; Pires, Ana R; Carvalho, Tânia; Pinheiro, Ana I; Soares, Rui S; Sousa, Ana E

2015-02-01

A unique HIV-host equilibrium exists in untreated HIV-2-infected individuals. This equilibrium is characterized by low to undetectable levels of viremia throughout the disease course, despite the establishment of disseminated HIV-2 reservoirs at levels comparable to those observed in untreated HIV-1 infection. Although the clinical spectrum is similar in the two infections, HIV-2 infection is associated with a much lower rate of CD4 T-cell decline and has a limited impact on the mortality of infected adults. Here we investigated HIV-2 infection of the human thymus, the primary organ for T-cell production. Human thymic tissue and suspensions of total or purified CD4 single-positive thymocytes were infected with HIV-2 or HIV-1 primary isolates using either CCR5 or CXCR4 coreceptors. We found that HIV-2 infected both thymic organ cultures and thymocyte suspensions, as attested to by the total HIV DNA and cell-associated viral mRNA levels. Nevertheless, thymocytes featured reduced levels of intracellular Gag viral protein, irrespective of HIV-2 coreceptor tropism and cell differentiation stage, in agreement with the low viral load in culture supernatants. Our data show that HIV-2 is able to infect the human thymus, but the HIV-2 replication cycle in thymocytes is impaired, providing a new model to identify therapeutic targets for viral replication control. HIV-1 infects the thymus, leading to a decrease in CD4 T-cell production that contributes to the characteristic CD4 T-cell loss. HIV-2 infection is associated with a very low rate of progression to AIDS and is therefore considered a unique naturally occurring model of attenuated HIV disease. HIV-2-infected individuals feature low to undetectable plasma viral loads, in spite of the numbers of circulating infected T cells being similar to those found in patients infected with HIV-1. We assessed, for the first time, the direct impact of HIV-2 infection on the human thymus. We show that HIV-2 is able to infect the thymus but that the HIV-2 replication cycle in thymocytes is impaired. We propose that this system will be important to devise immunotherapies that target viral production, aiding the design of future therapeutic strategies for HIV control. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cytomegalovirus: virological facts for clinicians.

PubMed

Bodaghi, B; Michelson, S

1999-12-01

Human cytomegalovirus (HCMV) is a complex DNA virus encoding more than 200 viral proteins. This highly adapted opportunist agent has developed several ways to evade the immune system. Among all clinical features due to HCMV, retinitis occurs especially in severely immunosuppressed patients, particularly during the end phase of HIV infection. Highly active antiretroviral therapy (HAART) has significantly reduced the incidence of this complication. However, in this HAART era, we observe the emergence of new clinical patterns in patients presenting with cicatricial HCMV retinitis. These patterns could be potentially related to immune mechanisms directed against viral antigens expressed at the surface of retinal cells that are still latently infected without any viral replication. We used a model of human retinal pigment epithelial (RPE) cells to evaluate virus-host interactions in the presence of different cytokines in the eye which play a major role in immunological or infectious conditions. Two different enzymatic pathways seem to be particularly involved during infection. Lack of tryptophan and production of nitric oxide seem to block HCMV replication in RPE cells. We propose a model to explain some of the mechanisms involved during severe immunosuppression and also after immune recovery.

Pathogenesis of occult chronic hepatitis B virus infection

PubMed Central

de la Fuente, Rocio Aller; Gutiérrez, María L; Garcia-Samaniego, Javier; Fernández-Rodriguez, Conrado; Lledó, Jose Luis; Castellano, Gregorio

2011-01-01

Occult hepatitis B infection (OBI) is characterized by hepatitis B virus (HBV) DNA in serum in the absence of hepatitis B surface antigen (HBsAg) presenting HBsAg-negative and anti-HBc positive serological patterns. Occult HBV status is associated in some cases with mutant viruses undetectable by HBsAg assays; but more frequently it is due to a strong suppression of viral replication and gene expression. OBI is an entity with world-wide diffusion. The failure to detect HBsAg, despite the persistence of the viral DNA, is due in most cases to the strong suppression of viral replication and gene expression that characterizes this “occult” HBV infection; although the mechanisms responsible for suppression of HBV are not well understood. The majority of OBI cases are secondary to overt HBV infection and represent a residual low viremia level suppressed by a strong immune response together with histological derangements which occurred during acute or chronic HBV infection. Much evidence suggests that it can favour the progression of liver fibrosis and the development of hepatocellular carcinoma. PMID:21472118

TAM Receptors Are Not Required for Zika Virus Infection in Mice.

PubMed

Hastings, Andrew K; Yockey, Laura J; Jagger, Brett W; Hwang, Jesse; Uraki, Ryuta; Gaitsch, Hallie F; Parnell, Lindsay A; Cao, Bin; Mysorekar, Indira U; Rothlin, Carla V; Fikrig, Erol; Diamond, Michael S; Iwasaki, Akiko

2017-04-18

Tyro3, Axl, and Mertk (TAM) receptors are candidate entry receptors for infection with the Zika virus (ZIKV), an emerging flavivirus of global public health concern. To investigate the requirement of TAM receptors for ZIKV infection, we used several routes of viral inoculation and compared viral replication in wild-type versus Axl -/- , Mertk -/- , Axl -/- Mertk -/- , and Axl -/- Tyro3 -/- mice in various organs. Pregnant and non-pregnant mice treated with interferon-α-receptor (IFNAR)-blocking (MAR1-5A3) antibody and infected subcutaneously with ZIKV showed no reliance on TAMs for infection. In the absence of IFNAR-blocking antibody, adult female mice challenged intravaginally with ZIKV showed no difference in mucosal viral titers. Similarly, in young mice that were infected with ZIKV intracranially or intraperitoneally, ZIKV replication occurred in the absence of TAM receptors, and no differences in cell tropism were observed. These findings indicate that, in mice, TAM receptors are not required for ZIKV entry and infection. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Analyses of a whole-genome inter-clade recombination map of hepatitis delta virus suggest a host polymerase-driven and viral RNA structure-promoted template-switching mechanism for viral RNA recombination

PubMed Central

Chao, Mei; Wang, Tzu-Chi; Lin, Chia-Chi; Yung-Liang Wang, Robert; Lin, Wen-Bin; Lee, Shang-En; Cheng, Ying-Yu; Yeh, Chau-Ting; Iang, Shan-Bei

2017-01-01

The genome of hepatitis delta virus (HDV) is a 1.7-kb single-stranded circular RNA that folds into an unbranched rod-like structure and has ribozyme activity. HDV redirects host RNA polymerase(s) (RNAP) to perform viral RNA-directed RNA transcription. RNA recombination is known to contribute to the genetic heterogeneity of HDV, but its molecular mechanism is poorly understood. Here, we established a whole-genome HDV-1/HDV-4 recombination map using two cloned sequences coexisting in cultured cells. Our functional analyses of the resulting chimeric delta antigens (the only viral-encoded protein) and recombinant genomes provide insights into how recombination promotes the genotypic and phenotypic diversity of HDV. Our examination of crossover distribution and subsequent mutagenesis analyses demonstrated that ribozyme activity on HDV genome, which is required for viral replication, also contributes to the generation of an inter-clade junction. These data provide circumstantial evidence supporting our contention that HDV RNA recombination occurs via a replication-dependent mechanism. Furthermore, we identify an intrinsic asymmetric bulge on the HDV genome, which appears to promote recombination events in the vicinity. We therefore propose a mammalian RNAP-driven and viral-RNA-structure-promoted template-switching mechanism for HDV genetic recombination. The present findings improve our understanding of the capacities of the host RNAP beyond typical DNA-directed transcription. PMID:28977829

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

The Human Cytomegalovirus IE2 and UL112-113 Proteins Accumulate in Viral DNA Replication Compartments That Initiate from the Periphery of Promyelocytic Leukemia Protein-Associated Nuclear Bodies (PODs or ND10)

PubMed Central

Ahn, Jin-Hyun; Jang, Won-Jong; Hayward, Gary S.

1999-01-01

During human cytomegalovirus (HCMV) infection, the periphery of promyelocytic leukemia protein (PML)-associated nuclear bodies (also known as PML oncogenic domains [PODs] or ND10) are sites for both input viral genome deposition and immediate-early (IE) gene transcription. At very early times after infection, the IE1 protein localizes to and subsequently disrupts PODs, whereas the IE2 protein localizes within or adjacent to PODs. This process appears to be required for efficient viral gene expression and DNA replication. We have investigated the initiation of viral DNA replication compartment formation by studying the localization of viral IE proteins, DNA replication proteins, and the PML protein during productive infection. Localization of IE2 adjacent to PODs between 2 and 6 h after infection was confirmed by confocal microscopy of human fibroblasts (HF cells) infected with both wild-type HCMV(Towne) and with an IE1-deletion mutant HCMV(CR208) that fails to disrupt PODs. In HCMV(Towne)-infected HF cells at 24 to 48 h, IE2 also accumulated in newly formed viral DNA replication compartments containing the polymerase processivity factor (UL44), the single-stranded DNA binding protein (SSB; UL57), the UL112-113 accessory protein, and newly incorporated bromodeoxyuridine (BrdU). Double labeling of the HCMV(CR208)-infected HF cells demonstrated that formation of viral DNA replication compartments initiates within granular structures that bud from the periphery of some of the PODs and subsequently coalesce into larger structures that are flanked by PODs. In transient DNA transfection assays, both the N terminus (codons 136 to 290) and the C terminus (codons 379 to 579) of IE2 exon 5, but not the central region between them, were found to be necessary for both the punctate distribution of IE2 and its association with PODs. Like IE2, the UL112-113 accessory replication protein was also distributed in a POD-associated pattern in both DNA-transfected and virus-infected cells beginning at 6 h. Furthermore, when all six replication core machinery proteins (polymerase complex, SSB, and helicase-primase complex) were expressed together in the presence of UL112-113, they also accumulated at POD-associated sites, suggesting that the UL112-113 protein (but not IE2) may play a role in recruitment of viral replication fork proteins into the periphery of PODs. These results show that (i) subsequent to accumulating at the periphery of PODs, IE2 is incorporated together with the core proteins into viral DNA replication compartments that initiate from the periphery of PODs and then grow to fill the space between groups of PODs, and (ii) the UL112-113 protein appears to have a key role in assembling and recruiting the core replication machinery proteins in the initial stages of viral replication compartment formation. PMID:10559364

The FANC pathway is activated by adenovirus infection and promotes viral replication-dependent recombination

PubMed Central

Cherubini, Gioia; Naim, Valeria; Caruso, Paola; Burla, Romina; Bogliolo, Massimo; Cundari, Enrico; Benihoud, Karim; Saggio, Isabella; Rosselli, Filippo

2011-01-01

Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination. PMID:21421559

Visualization and Measurement of ATP Levels in Living Cells Replicating Hepatitis C Virus Genome RNA

PubMed Central

Ando, Tomomi; Imamura, Hiromi; Suzuki, Ryosuke; Aizaki, Hideki; Watanabe, Toshiki; Wakita, Takaji; Suzuki, Tetsuro

2012-01-01

Adenosine 5′-triphosphate (ATP) is the primary energy currency of all living organisms and participates in a variety of cellular processes. Although ATP requirements during viral lifecycles have been examined in a number of studies, a method by which ATP production can be monitored in real-time, and by which ATP can be quantified in individual cells and subcellular compartments, is lacking, thereby hindering studies aimed at elucidating the precise mechanisms by which viral replication energized by ATP is controlled. In this study, we investigated the fluctuation and distribution of ATP in cells during RNA replication of the hepatitis C virus (HCV), a member of the Flaviviridae family. We demonstrated that cells involved in viral RNA replication actively consumed ATP, thereby reducing cytoplasmic ATP levels. Subsequently, a method to measure ATP levels at putative subcellular sites of HCV RNA replication in living cells was developed by introducing a recently-established Förster resonance energy transfer (FRET)-based ATP indicator, called ATeam, into the NS5A coding region of the HCV replicon. Using this method, we were able to observe the formation of ATP-enriched dot-like structures, which co-localize with non-structural viral proteins, within the cytoplasm of HCV-replicating cells but not in non-replicating cells. The obtained FRET signals allowed us to estimate ATP concentrations within HCV replicating cells as ∼5 mM at possible replicating sites and ∼1 mM at peripheral sites that did not appear to be involved in HCV replication. In contrast, cytoplasmic ATP levels in non-replicating Huh-7 cells were estimated as ∼2 mM. To our knowledge, this is the first study to demonstrate changes in ATP concentration within cells during replication of the HCV genome and increased ATP levels at distinct sites within replicating cells. ATeam may be a powerful tool for the study of energy metabolism during replication of the viral genome. PMID:22396648

Baculovirus LEF-11 nuclear localization signal is important for viral DNA replication.

PubMed

Chen, Tingting; Dong, Zhanqi; Hu, Nan; Hu, Zhigang; Dong, Feifan; Jiang, Yaming; Li, Jun; Chen, Peng; Lu, Cheng; Pan, Minhui

2017-06-15

Baculovirus LEF-11 is a small nuclear protein that is involved in viral late gene transcription and DNA replication. However, the characteristics of its nuclear localization signal and its impact on viral DNA replication are unknown. In the present study, systemic bioinformatics analysis showed that the baculovirus LEF-11 contains monopartite and bipartite classical nuclear localization signal sequences (cNLSs), which were also detected in a few alphabaculovirus species. Localization of representative LEF-11 proteins of four baculovirus genera indicated that the nuclear localization characteristics of baculovirus LEF-11 coincided with the predicted results. Moreover, Bombyx mori nucleopolyhedrovirus (BmNPV) LEF-11 could be transported into the nucleus during viral infection in the absence of a cNLSs. Further investigations demonstrated that the NLS of BmNPV LEF-11 is important for viral DNA replication. The findings of the present study indicate that the characteristics of the baculovirus LEF-11 protein and the NLS is essential to virus DNA replication and nuclear transport mechanisms. Copyright © 2017 Elsevier B.V. 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

Impact of radiation therapy on the oncolytic adenovirus dl520: implications on the treatment of glioblastoma.

PubMed

Bieler, Alexa; Mantwill, Klaus; Holzmüller, Regina; Jürchott, Karsten; Kaszubiak, Alexander; Stärk, Sybille; Glockzin, Gabriel; Lage, Hermann; Grosu, Anca-Ligia; Gansbacher, Bernd; Holm, Per Sonne

2008-03-01

Viral oncolytic therapy is emerging as a new form of anticancer therapy and has shown promising preclinical results, especially in combination with radio- and chemotherapy. We recently reported that nuclear localization of the human transcription factor YB-1 in multidrug-resistant cells facilitates E1-independent adenoviral replication. The aim of this study was to evaluate the combined treatment of the conditionally-replicating adenovirus dl520 and radiotherapy in glioma cell lines in vitro and in human tumor xenografts. Furthermore, the dependency of YB-1 on dl520 replication was verified by shRNA directed down regulation of YB-1. Localization of YB-1 was determined by immunostaining. Glioma cell lines LN-18, U373 and U87 were infected with dl520. Induction of cytopathic effect (CPE), viral replication, viral yield and viral release were determined after viral infection, radiation therapy and the combination of both treatment modalities. The capacity of treatments alone or combined to induce tumor growth inhibition of subcutaneous U373 tumors was tested also in nude mice. Quantitative real-time PCR demonstrated that the shRNA-mediated down regulation of YB-1 is leading to a dramatic decrease in adenoviral replication of dl520. Immunostaining analysis showed that the YB-1 protein was predominantly located in the cytoplasm in the perinuclear space and less abundant in the nucleus. After irradiation we found an increase of nuclear YB-1. The addition of radiotherapy increased the oncolytic effect of dl520 with enhanced viral replication, viral yield and viral release. The oncolytic activity of dl520 plus radiation inhibited the growth of subcutaneous U373 tumors in a xenograft mouse model. Radiation mediated increase of nuclear YB-1 in glioma cells enhanced the oncolytic potential of adenovirus dl520.

Vaccination of calves using the BRSV nucleocapsid protein in a DNA prime-protein boost strategy stimulates cell-mediated immunity and protects the lungs against BRSV replication and pathology.

PubMed

Letellier, Carine; Boxus, Mathieu; Rosar, Laurent; Toussaint, Jean-François; Walravens, Karl; Roels, Stefan; Meyer, Gilles; Letesson, Jean-Jacques; Kerkhofs, Pierre

2008-09-02

Respiratory syncytial virus (RSV) is a major cause of respiratory disease in both cattle and young children. Despite the development of vaccines against bovine (B)RSV, incomplete protection and exacerbation of subsequent RSV disease have occurred. In order to circumvent these problems, calves were vaccinated with the nucleocapsid protein, known to be a major target of CD8(+) T cells in cattle. This was performed according to a DNA prime-protein boost strategy. The results showed that DNA vaccination primed a specific T-cell-mediated response, as indicated by both a lymphoproliferative response and IFN-gamma production. These responses were enhanced after protein boost. After challenge, mock-vaccinated calves displayed gross pneumonic lesions and viral replication in the lungs. In contrast, calves vaccinated by successive administrations of plasmid DNA and protein exhibited protection against the development of pneumonic lesions and the viral replication in the BAL fluids and the lungs. The protection correlated to the cell-mediated immunity and not to the antibody response.

Copy number determination of genetically-modified hematopoietic stem cells.

PubMed

Schuesler, Todd; Reeves, Lilith; Kalle, Christof von; Grassman, Elke

2009-01-01

Human gene transfer with gammaretroviral, murine leukemia virus (MLV) based vectors has been shown to effectively insert and express transgene sequences at a level of therapeutic benefit. However, there are numerous reports of disruption of the normal cellular processes caused by the viral insertion, even of replication deficient gammaretroviral vectors. Current gammaretroviral and lentiviral vectors do not control the site of insertion into the genome, hence, the possibility of disruption of the target cell genome. Risk related to viral insertions is linked to the number of insertions of the transgene into the cellular DNA, as has been demonstrated for replication competent and replication deficient retroviruses in experiments. At high number of insertions per cell, cell transformation due to vector induced activation of proto-oncogenes is more likely to occur, in particular since more than one transforming event is needed for oncogenesis. Thus, determination of the vector copy number in bulk transduced populations, individual colony forming units, and tissue from the recipient of the transduced cells is an increasingly important safety assay and has become a standard, though not straightforward assay, since the inception of quantitative PCR.

Postdoctoral Fellow | Center for Cancer Research

Cancer.gov

A postdoctoral position is available in the Viral Recombination Section (VRS), HIV Dynamics and Replication Program, CCR.  The VRS studies retroviral replication using human immunodeficiency viruses and other retroviruses, with a particular emphasis on the mechanisms of viral RNA biology, specific RNA packaging, virus assembly, and HIV replication.  Molecular tools and

A therapeutic HIV-1 vaccine enhances anti-HIV-1 immune responses in patients under highly active antiretroviral therapy.

PubMed

Tung, Frank Y; Tung, Jack K; Pallikkuth, Suresh; Pahwa, Savita; Fischl, Margaret A

2016-04-27

HIV-1 specific cellular immunity plays an important role in controlling viral replication. In this first-in-human therapeutic vaccination study, a replication-defective HIV-1 vaccine (HIVAX) was tested in HIV-1 infected participants undergoing highly active antiretroviral therapy (HAART) to enhance anti-HIV immunity (Clinicaltrials.gov, identifier NCT01428596). A010 was a randomized, placebo-controlled trial to evaluate the safety and the immunogenicity of a replication defective HIV-1 vaccine (HIVAX) given as a subcutaneous injection to HIV-1 infected participants who were receiving HAART with HIV-1 viral load <50 copies/ml and CD4 cell count >500 cells/mm(3). HIV-1 specific immune responses were monitored by INF-γ enzyme linked immunospot (Elispot) and intracellular cytokine staining (ICS) assay after vaccination. Following the randomized placebo-controlled vaccination phase, subjects who received HIVAX vaccine and who met eligibility underwent a 12-week analytical antiretroviral treatment interruption (ATI). Viral load was monitored throughout the study. HIVAX was well tolerated in trial participants. Transient grade 1 to 2 (mild to moderate) injection site reactions occurred in 8 of 10 vaccinated participants. HIVAX was immunogenic in all vaccinated participants. The functionality of T cells was significantly enhanced after vaccination. Median viral load (3.45 log10 copies/ml, range of 96-12,830 copies/ml) at the end of the 12-week treatment interruption in HIVAX vaccinated group was significantly lower than the pre-treatment levels. Three vaccinated participants extended ATI for up to 2 years with stable CD4 cells and low viral loads. HIVAX vaccine is generally safe, elicits strong anti-HIV-1 immune responses, and may play an important role in controlling viral load during treatment interruption in HIV-1 infected participants. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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.

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.

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

Challenges in identifying and determining the impacts of infection with pestiviruses on the herd health of free ranging cervid populations

USDA-ARS?s Scientific Manuscript database

Although most commonly associated with the infection of domestic livestock, the replication of pestiviruses, in particular bovine viral diarrhea virus (BVDV), occurs in a wide range of free ranging cervids including white-tailed deer, mule deer, fallow deer, elk, red deer, roe deer, eland and moused...

Polymorphisms in the hemagglutinin gene influenced the viral shedding of pandemic 2009 influenza virus in swine

USDA-ARS?s Scientific Manuscript database

The contribution of influenza virus quasi-species for transmission efficiency and replication is poorly understood. In the present study we show that naturally occurring polymorphisms present in the hemagglutinin (HA) gene of two 2009 pandemic H1N1 isolates, A/California/04/2009 (Ca/09) and A/Mexico...

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

Intracellular coordination of potyviral RNA functions in infection

PubMed Central

Mäkinen, Kristiina; Hafrén, Anders

2014-01-01

Establishment of an infection cycle requires mechanisms to allocate the genomes of (+)-stranded RNA viruses in a balanced ratio to translation, replication, encapsidation, and movement, as well as mechanisms to prevent translocation of viral RNA (vRNA) to cellular RNA degradation pathways. The ratio of vRNA allocated to various functions is likely balanced by the availability of regulatory proteins or competition of the interaction sites within regulatory ribonucleoprotein complexes. Due to the transient nature of viral processes and the interdependency between vRNA pathways, it is technically demanding to work out the exact molecular mechanisms underlying vRNA regulation. A substantial number of viral and host proteins have been identified that facilitate the steps that lead to the assembly of a functional potyviral RNA replication complex and their fusion with chloroplasts. Simultaneously with on-going viral replication, part of the replicated potyviral RNA enters movement pathways. Although not much is known about the processes of potyviral RNA release from viral replication complexes, the molecular interactions involved in these processes determine the fate of the replicated vRNA. Some viral and host cell proteins have been described that direct replicated potyviral RNA to translation to enable potyviral gene expression and productive infection. The antiviral defense of the cell causes vRNA degradation by RNA silencing. We hypothesize that also plant pathways involved in mRNA decay may have a role in the coordination of potyviral RNA expression. In this review, we discuss the roles of different potyviral and host proteins in the coordination of various potyviral RNA functions. PMID:24723931

Intracellular coordination of potyviral RNA functions in infection.

PubMed

Mäkinen, Kristiina; Hafrén, Anders

2014-01-01

Establishment of an infection cycle requires mechanisms to allocate the genomes of (+)-stranded RNA viruses in a balanced ratio to translation, replication, encapsidation, and movement, as well as mechanisms to prevent translocation of viral RNA (vRNA) to cellular RNA degradation pathways. The ratio of vRNA allocated to various functions is likely balanced by the availability of regulatory proteins or competition of the interaction sites within regulatory ribonucleoprotein complexes. Due to the transient nature of viral processes and the interdependency between vRNA pathways, it is technically demanding to work out the exact molecular mechanisms underlying vRNA regulation. A substantial number of viral and host proteins have been identified that facilitate the steps that lead to the assembly of a functional potyviral RNA replication complex and their fusion with chloroplasts. Simultaneously with on-going viral replication, part of the replicated potyviral RNA enters movement pathways. Although not much is known about the processes of potyviral RNA release from viral replication complexes, the molecular interactions involved in these processes determine the fate of the replicated vRNA. Some viral and host cell proteins have been described that direct replicated potyviral RNA to translation to enable potyviral gene expression and productive infection. The antiviral defense of the cell causes vRNA degradation by RNA silencing. We hypothesize that also plant pathways involved in mRNA decay may have a role in the coordination of potyviral RNA expression. In this review, we discuss the roles of different potyviral and host proteins in the coordination of various potyviral RNA functions.

Gefitinib and pyrrolidine dithiocarbamate decrease viral replication and cytokine production in dengue virus infected human monocyte cultures.

PubMed

Duran, Anyelo; Valero, Nereida; Mosquera, Jesús; Fuenmayor, Edgard; Alvarez-Mon, Melchor

2017-12-15

The epidermal growth factor receptor (EGFR) and nucleotide-binding and oligomerization-domain containing 2 (NOD2) are important in cancer and in microbial recognition, respectively. These molecules trigger intracellular signaling pathways inducing the expression of inflammatory genes by NF-kB translocation. Gefitinib (GBTC) and pyrrolidine dithiocarbamate (PDTC) are capable of inhibiting EGFR/NOD2 and NF-kB, respectively. In earlier stages of dengue virus (DENV) infection, monocytes are capable of sustaining viral replication and increasing cytokine production, suggesting that monocyte/macrophages play an important role in early DENV replication. GBTC and PDTC have not been used to modify the pathogenesis of DENV in infected cells. This study was aimed to determine the effect of GBTC and PDTC on viral replication and cytokine production in DENV serotype 2 (DENV2)-infected human monocyte cultures. GBTC and PDTC were used to inhibit EGFR/NOD2 and NF-kB, respectively. Cytokine production was measured by ELISA and viral replication by plaque forming unit assay. Increased DENV2 replication and anti-viral cytokine production (IFN-α/β, TNF-α, IL-12 and IL-18) in infected cultures were found. These parameters were decreased after EGFR/NOD2 or NF-kB inhibitions. The inhibitory effects of GBTC and PDTC on viral replication and cytokine production can be beneficial in the treatment of patients infected by dengue and suggest a possible role of EGFR/NOD2 receptors and NF-kB in dengue pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition and recovery of the replication of depurinated parvovirus DNA in mouse fibroblasts

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

Vos, J.M.; Avalosse, B.; Su, Z.Z.

Apurinic sites were introduced in the single-stranded DNA of parvovirus minute-virus-of-mice (MVM) and their effect on viral DNA synthesis was measured in mouse fibroblasts. Approximately one apurinic site per viral genome, is sufficient to block its replication in untreated cells. The exposure of host cells to a sublethal dose of UV-light 15 hours prior to virus infection, enhances their ability to support the replication of depurinated MVM. Cell preirradiation induces the apparent overcome of 10-15% of viral DNA replication blocks. These results indicate that apurinic sites prevent mammalian cells from replicating single-stranded DNA unless a recovery process is activated bymore » cell UV-irradiation.« less

Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

PubMed

McCune, Broc T; Tang, Wei; Lu, Jia; Eaglesham, James B; Thorne, Lucy; Mayer, Anne E; Condiff, Emily; Nice, Timothy J; Goodfellow, Ian; Krezel, Andrzej M; Virgin, Herbert W

2017-07-11

The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication. IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the VAPA host protein. The NS1/2-VAPA interaction is conserved between murine and human noroviruses and was important for early steps in murine norovirus replication. Using structure-function analysis, we found that NS1/2 contains a short sequence that molecularly mimics the FFAT motif that is found in multiple host proteins that bind VAPA. This represents to our knowledge the first example of functionally important mimicry of a host FFAT motif by a microbial protein. Copyright © 2017 McCune et al.

Defining the roles for Vpr in HIV-1-associated neuropathogenesis

PubMed Central

James, Tony; Nonnemacher, Michael R.; Wigdahl, Brian; Krebs, Fred C.

2016-01-01

It is increasingly evident that the human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has a unique role in neuropathogenesis. Its ability to induce G2/M arrest coupled with its capacity to increase viral gene transcription gives it a unique role in sustaining viral replication and aiding in the establishment and maintenance of a systemic infection. The requirement of Vpr for HIV-1 infection and replication in cells of monocytic origin (a key lineage of cells involved in HIV-1 neuroinvasion) suggests an important role in establishing and sustaining infection in the central nervous system (CNS). Contributions of Vpr to neuropathogenesis can be expanded further through (i) naturally occurring HIV-1 sequence variation that results in functionally divergent Vpr variants; (ii) the dual activities of Vpr as a intracellular protein delivered and expressed during HIV-1 infection and as an extracellular protein that can act on neighboring, uninfected cells; (iii) cell type-dependent consequences of Vpr expression and exposure, including cell cycle arrest, metabolic dysregulation, and cytotoxicity; and (iv) the effects of Vpr on exosome-based intercellular communication in the CNS. Revealing the effects of this pleiotropic viral protein is an essential part of a greater understanding of HIV-1-associated pathogenesis and potential approaches to treating and preventing disease caused by HIV-1 infection. PMID:27056720

HIV–host interactome revealed directly from infected cells

PubMed Central

Luo, Yang; Jacobs, Erica Y.; Greco, Todd M.; Mohammed, Kevin D.; Tong, Tommy; Keegan, Sarah; Binley, James M.; Cristea, Ileana M.; Fenyö, David; Rout, Michael P.; Chait, Brian T.; Muesing, Mark A.

2016-01-01

Although genetically compact, HIV-1 commandeers vast arrays of cellular machinery to sustain and protect it during cycles of viral outgrowth. Transposon-mediated saturation linker scanning mutagenesis was used to isolate fully replication-competent viruses harbouring a potent foreign epitope tag. Using these viral isolates, we performed differential isotopic labelling and affinity-capture mass spectrometric analyses on samples obtained from cultures of human lymphocytes to classify the vicinal interactomes of the viral Env and Vif proteins as they occur during natural infection. Importantly, interacting proteins were recovered without bias, regardless of their potential for positive, negative or neutral impact on viral replication. We identified specific host associations made with trimerized Env during its biosynthesis, at virological synapses, with innate immune effectors (such as HLA-E) and with certain cellular signalling pathways (for example, Notch1). We also defined Vif associations with host proteins involved in the control of nuclear transcription and nucleoside biosynthesis as well as those interacting stably or transiently with the cytoplasmic protein degradation apparatus. Our approach is broadly applicable to elucidating pathogen–host interactomes, providing high-certainty identification of interactors by their direct access during cycling infection. Understanding the pathophysiological consequences of these associations is likely to provide strategic targets for antiviral intervention. PMID:27375898

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

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

Host phylogeny determines viral persistence and replication in novel hosts.

PubMed

Longdon, Ben; Hadfield, Jarrod D; Webster, Claire L; Obbard, Darren J; Jiggins, Francis M

2011-09-01

Pathogens switching to new hosts can result in the emergence of new infectious diseases, and determining which species are likely to be sources of such host shifts is essential to understanding disease threats to both humans and wildlife. However, the factors that determine whether a pathogen can infect a novel host are poorly understood. We have examined the ability of three host-specific RNA-viruses (Drosophila sigma viruses from the family Rhabdoviridae) to persist and replicate in 51 different species of Drosophilidae. Using a novel analytical approach we found that the host phylogeny could explain most of the variation in viral replication and persistence between different host species. This effect is partly driven by viruses reaching a higher titre in those novel hosts most closely related to the original host. However, there is also a strong effect of host phylogeny that is independent of the distance from the original host, with viral titres being similar in groups of related hosts. Most of this effect could be explained by variation in general susceptibility to all three sigma viruses, as there is a strong phylogenetic correlation in the titres of the three viruses. These results suggest that the source of new emerging diseases may often be predictable from the host phylogeny, but that the effect may be more complex than simply causing most host shifts to occur between closely related hosts.

Host Phylogeny Determines Viral Persistence and Replication in Novel Hosts

PubMed Central

Longdon, Ben; Hadfield, Jarrod D.; Webster, Claire L.

2011-01-01

Pathogens switching to new hosts can result in the emergence of new infectious diseases, and determining which species are likely to be sources of such host shifts is essential to understanding disease threats to both humans and wildlife. However, the factors that determine whether a pathogen can infect a novel host are poorly understood. We have examined the ability of three host-specific RNA-viruses (Drosophila sigma viruses from the family Rhabdoviridae) to persist and replicate in 51 different species of Drosophilidae. Using a novel analytical approach we found that the host phylogeny could explain most of the variation in viral replication and persistence between different host species. This effect is partly driven by viruses reaching a higher titre in those novel hosts most closely related to the original host. However, there is also a strong effect of host phylogeny that is independent of the distance from the original host, with viral titres being similar in groups of related hosts. Most of this effect could be explained by variation in general susceptibility to all three sigma viruses, as there is a strong phylogenetic correlation in the titres of the three viruses. These results suggest that the source of new emerging diseases may often be predictable from the host phylogeny, but that the effect may be more complex than simply causing most host shifts to occur between closely related hosts. PMID:21966271

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

Least-Squares Support Vector Machine Approach to Viral Replication Origin Prediction

PubMed Central

Cruz-Cano, Raul; Chew, David S.H.; Kwok-Pui, Choi; Ming-Ying, Leung

2010-01-01

Replication of their DNA genomes is a central step in the reproduction of many viruses. Procedures to find replication origins, which are initiation sites of the DNA replication process, are therefore of great importance for controlling the growth and spread of such viruses. Existing computational methods for viral replication origin prediction have mostly been tested within the family of herpesviruses. This paper proposes a new approach by least-squares support vector machines (LS-SVMs) and tests its performance not only on the herpes family but also on a collection of caudoviruses coming from three viral families under the order of caudovirales. The LS-SVM approach provides sensitivities and positive predictive values superior or comparable to those given by the previous methods. When suitably combined with previous methods, the LS-SVM approach further improves the prediction accuracy for the herpesvirus replication origins. Furthermore, by recursive feature elimination, the LS-SVM has also helped find the most significant features of the data sets. The results suggest that the LS-SVMs will be a highly useful addition to the set of computational tools for viral replication origin prediction and illustrate the value of optimization-based computing techniques in biomedical applications. PMID:20729987

Least-Squares Support Vector Machine Approach to Viral Replication Origin Prediction.

PubMed

Cruz-Cano, Raul; Chew, David S H; Kwok-Pui, Choi; Ming-Ying, Leung

2010-06-01

Replication of their DNA genomes is a central step in the reproduction of many viruses. Procedures to find replication origins, which are initiation sites of the DNA replication process, are therefore of great importance for controlling the growth and spread of such viruses. Existing computational methods for viral replication origin prediction have mostly been tested within the family of herpesviruses. This paper proposes a new approach by least-squares support vector machines (LS-SVMs) and tests its performance not only on the herpes family but also on a collection of caudoviruses coming from three viral families under the order of caudovirales. The LS-SVM approach provides sensitivities and positive predictive values superior or comparable to those given by the previous methods. When suitably combined with previous methods, the LS-SVM approach further improves the prediction accuracy for the herpesvirus replication origins. Furthermore, by recursive feature elimination, the LS-SVM has also helped find the most significant features of the data sets. The results suggest that the LS-SVMs will be a highly useful addition to the set of computational tools for viral replication origin prediction and illustrate the value of optimization-based computing techniques in biomedical applications.

HPV31 Utilizes the ATR-Chk1 Pathway to Maintain Elevated RRM2 Levels and a Replication-Competent Environment in Differentiating Keratinocytes

PubMed Central

Anacker, Daniel C.; Aloor, Heather L.; Shepard, Caitlin N.; Lenzi, Gina M.; Johnson, Bryan A.; Kim, Baek; Moody, Cary A.

2016-01-01

Productive replication of human papillomaviruses (HPV) is restricted to the uppermost layers of the differentiating epithelia. How HPV ensures an adequate supply of cellular substrates for viral DNA synthesis in a differentiating environment is unclear. Here, we demonstrate that HPV31 positive cells exhibit increased dNTP pools and levels of RRM2, a component of the ribonucleotide reductase (RNR) complex, which is required for de novo synthesis of dNTPs. RRM2 depletion blocks productive replication, suggesting RRM2 provides dNTPs for viral DNA synthesis in differentiating cells. We demonstrate that HPV31 regulates RRM2 levels through expression of E7 and activation of the ATR-Chk1-E2F1 DNA damage response, which is essential to combat replication stress upon entry into S-phase, as well as for productive replication. Our findings suggest a novel way in which viral DNA synthesis is regulated through activation of ATR and Chk1 and highlight an intriguing new virus/host interaction utilized for viral replication. PMID:27764728

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.

RNA N6-adenosine methylation (m6A) steers epitranscriptomic control of herpesvirus replication

PubMed Central

Ye, Fengchun

2017-01-01

Latency is a hallmark of all herpesviruses, during which the viral genomes are silenced through DNA methylation and suppressive histone modifications. When latent herpesviruses reactivate to undergo productive lytic replication, the suppressive epigenetic marks are replaced with active ones to allow for transcription of viral genes. Interestingly, by using Kaposi’s sarcoma-associated herpesvirus (KSHV) as a model, we recently demonstrated that the newly transcribed viral RNAs are also subjected to post-transcriptional N6-adenosine methylation (m6A). Blockade of this post-transcriptional event abolishes viral protein expression and halts virion production. We found that m6A modification controls RNA splicing, stability, and protein translation to regulate viral lytic gene expression and replication. Thus, our finding for the first time reveals a critical role of this epitranscriptomic mechanism in the control of herpesviral replication, which shall shed lights on development of novel strategies for the control of herpesviral infection. PMID:29082271

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.

Switch from translation to RNA replication in a positive-stranded RNA virus

PubMed Central

Gamarnik, Andrea V.; Andino, Raul

1998-01-01

In positive-stranded viruses, the genomic RNA serves as a template for both translation and RNA replication. Using poliovirus as a model, we examined the interaction between these two processes. We show that the RNA polymerase is unable to replicate RNA templates undergoing translation. We discovered that an RNA structure at the 5′ end of the viral genome, next to the internal ribosomal entry site, carries signals that control both viral translation and RNA synthesis. The interaction of this RNA structure with the cellular factor PCBP up-regulates viral translation, while the binding of the viral protein 3CD represses translation and promotes negative-strand RNA synthesis. We propose that the interaction of 3CD with this RNA structure controls whether the genomic RNA is used for translation or RNA replication. PMID:9694795

The Molecular Biology of Frog Virus 3 and other Iridoviruses Infecting Cold-Blooded Vertebrates

PubMed Central

Chinchar, V. Gregory; Yu, Kwang H.; Jancovich, James K.

2011-01-01

Frog virus 3 (FV3) is the best characterized member of the family Iridoviridae. FV3 study has provided insights into the replication of other family members, and has served as a model of viral transcription, genome replication, and virus-mediated host-shutoff. Although the broad outlines of FV3 replication have been elucidated, the precise roles of most viral proteins remain unknown. Current studies using knock down (KD) mediated by antisense morpholino oligonucleotides (asMO) and small, interfering RNAs (siRNA), knock out (KO) following replacement of the targeted gene with a selectable marker by homologous recombination, ectopic viral gene expression, and recombinant viral proteins have enabled researchers to systematically ascertain replicative- and virulence-related gene functions. In addition, the application of molecular tools to ecological studies is providing novel ways for field biologists to identify potential pathogens, quantify infections, and trace the evolution of ecologically important viral species. In this review, we summarize current studies using not only FV3, but also other iridoviruses infecting ectotherms. As described below, general principles ascertained using FV3 served as a model for the family, and studies utilizing other ranaviruses and megalocytiviruses have confirmed and extended our understanding of iridovirus replication. Collectively, these and future efforts will elucidate molecular events in viral replication, intrinsic and extrinsic factors that contribute to disease outbreaks, and the role of the host immune system in protection from disease. PMID:22069524

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription

PubMed Central

Cifuentes-Muñoz, Nicolás; Branttie, Jean; Slaughter, Kerri Beth

2017-01-01

ABSTRACT Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications. IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets. PMID:28978704

Human Metapneumovirus Induces Formation of Inclusion Bodies for Efficient Genome Replication and Transcription.

PubMed

Cifuentes-Muñoz, Nicolás; Branttie, Jean; Slaughter, Kerri Beth; Dutch, Rebecca Ellis

2017-12-15

Human metapneumovirus (HMPV) causes significant upper and lower respiratory disease in all age groups worldwide. The virus possesses a negative-sense single-stranded RNA genome of approximately 13.3 kb encapsidated by multiple copies of the nucleoprotein (N), giving rise to helical nucleocapsids. In addition, copies of the phosphoprotein (P) and the large RNA polymerase (L) decorate the viral nucleocapsids. After viral attachment, endocytosis, and fusion mediated by the viral glycoproteins, HMPV nucleocapsids are released into the cell cytoplasm. To visualize the subsequent steps of genome transcription and replication, a fluorescence in situ hybridization (FISH) protocol was established to detect different viral RNA subpopulations in infected cells. The FISH probes were specific for detection of HMPV positive-sense RNA (+RNA) and viral genomic RNA (vRNA). Time course analysis of human bronchial epithelial BEAS-2B cells infected with HMPV revealed the formation of inclusion bodies (IBs) from early times postinfection. HMPV IBs were shown to be cytoplasmic sites of active transcription and replication, with the translation of viral proteins being closely associated. Inclusion body formation was consistent with an actin-dependent coalescence of multiple early replicative sites. Time course quantitative reverse transcription-PCR analysis suggested that the coalescence of inclusion bodies is a strategy to efficiently replicate and transcribe the viral genome. These results provide a better understanding of the steps following HMPV entry and have important clinical implications. IMPORTANCE Human metapneumovirus (HMPV) is a recently discovered pathogen that affects human populations of all ages worldwide. Reinfections are common throughout life, but no vaccines or antiviral treatments are currently available. In this work, a spatiotemporal analysis of HMPV replication and transcription in bronchial epithelial cell-derived immortal cells was performed. HMPV was shown to induce the formation of large cytoplasmic granules, named inclusion bodies, for genome replication and transcription. Unlike other cytoplasmic structures, such as stress granules and processing bodies, inclusion bodies are exclusively present in infected cells and contain HMPV RNA and proteins to more efficiently transcribe and replicate the viral genome. Though inclusion body formation is nuanced, it corresponds to a more generalized strategy used by different viruses, including filoviruses and rhabdoviruses, for genome transcription and replication. Thus, an understanding of inclusion body formation is crucial for the discovery of innovative therapeutic targets. Copyright © 2017 American Society for Microbiology.

Identification of the Essential Role of Viral Bcl-2 for Kaposi's Sarcoma-Associated Herpesvirus Lytic Replication

PubMed Central

Liang, Qiming; Chang, Brian; Lee, Patrick; Brulois, Kevin F.; Ge, Jianning; Shi, Mude; Rodgers, Mary A.; Feng, Pinghui; Oh, Byung-Ha; Liang, Chengyu

2015-01-01

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) evades host defenses through tight suppression of autophagy by targeting each step of its signal transduction: by viral Bcl-2 (vBcl-2) in vesicle nucleation, by viral FLIP (vFLIP) in vesicle elongation, and by K7 in vesicle maturation. By exploring the roles of KSHV autophagy-modulating genes, we found, surprisingly, that vBcl-2 is essential for KSHV lytic replication, whereas vFLIP and K7 are dispensable. Knocking out vBcl-2 from the KSHV genome resulted in decreased lytic gene expression at the mRNA and protein levels, a lower viral DNA copy number, and, consequently, a dramatic reduction in the amount of progeny infectious viruses, as also described in the accompanying article (A. Gelgor, I. Kalt, S. Bergson, K. F. Brulois, J. U. Jung, and R. Sarid, J Virol 89:5298–5307, 2015). More importantly, the antiapoptotic and antiautophagic functions of vBcl-2 were not required for KSHV lytic replication. Using a comprehensive mutagenesis analysis, we identified that glutamic acid 14 (E14) of vBcl-2 is critical for KSHV lytic replication. Mutating E14 to alanine totally blocked KSHV lytic replication but showed little or no effect on the antiapoptotic and antiautophagic functions of vBcl-2. Our study indicates that vBcl-2 harbors at least three important and genetically separable functions to modulate both cellular signaling and the virus life cycle. IMPORTANCE The present study shows for the first time that vBcl-2 is essential for KSHV lytic replication. Removal of the vBcl-2 gene results in a lower level of KSHV lytic gene expression, impaired viral DNA replication, and consequently, a dramatic reduction in the level of progeny production. More importantly, the role of vBcl-2 in KSHV lytic replication is genetically separated from its antiapoptotic and antiautophagic functions, suggesting that the KSHV Bcl-2 carries a novel function in viral lytic replication. PMID:25740994

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.

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.

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.

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

Analysis of HSV viral reactivation in explants of sensory neurons

PubMed Central

Turner, Anne-Marie W.; Kristie, Thomas M.

2014-01-01

As with all Herpesviruses, Herpes simplex virus (HSV) has both a lytic replication phase and a latency-reactivation cycle. During lytic replication, there is an ordered cascade of viral gene expression that leads to the synthesis of infectious viral progeny. In contrast, latency is characterized by the lack of significant lytic gene expression and the absence of infectious virus. Reactivation from latency is characterized by the re-entry of the virus into the lytic replication cycle and the production of recurrent disease. This unit describes the establishment of the mouse sensory neuron model of HSV-1 latency-reactivation as a useful in vivo system for the analysis of mechanisms involved in latency and reactivation. Assays including the determination of viral yields, immunohistochemical/immunofluorescent detection of viral antigens, and mRNA quantitation are used in experiments designed to investigate the network of cellular and viral proteins regulating HSV-1 lytic infection, latency, and reactivation. PMID:25367271

Synaptogyrin-2 Promotes Replication of a Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs*

PubMed Central

Sun, Qiyu; Qi, Xian; Zhang, Yan; Wu, Xiaodong; Liang, Mifang; Li, Chuan; Li, Dexin; Cardona, Carol J.; Xing, Zheng

2016-01-01

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection. PMID:27226560

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

Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment

PubMed Central

Zhong, Ting; Zhang, Li-ying; Wang, Zeng-yan; Wang, Yue; Song, Feng-mei; Zhang, Ya-hong; Yu, Jing-hua

2017-01-01

Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 μmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 μmol/L) nor artemisinin (50 μmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication, thus is a candidate anti-HFMD drug. PMID:27840410

Rheum emodin inhibits enterovirus 71 viral replication and affects the host cell cycle environment.

PubMed

Zhong, Ting; Zhang, Li-Ying; Wang, Zeng-Yan; Wang, Yue; Song, Feng-Mei; Zhang, Ya-Hong; Yu, Jing-Hua

2017-03-01

Human enterovirus 71 (EV71) is the primary causative agent of recent large-scale outbreaks of hand, foot, and mouth disease (HFMD) in Asia. Currently, there are no drugs available for the prevention and treatment of HFMD. In this study, we compared the anti-EV71 activities of three natural compounds, rheum emodin, artemisinin and astragaloside extracted from Chinese herbs Chinese rhubarb, Artemisia carvifolia and Astragalus, respectively, which have been traditionally used for the treatment and prevention of epidemic diseases. Human lung fibroblast cell line MRC5 was mock-infected or infected with EV71, and treated with drugs. The cytotoxicity of the drugs was detected with MTT assay. The cytopathic effects such as cell death and condensed nuclei were morphologically observed. The VP1-coding sequence required for EV71 genome replication was assayed with qRT-PCR. Viral protein expression was analyzed with Western blotting. Viral TCID50 was determined to evaluate EV71 virulence. Flow cytometry analysis of propidium iodide staining was performed to analyze the cell cycle distribution of MRC5 cells. Rheum emodin (29.6 μmol/L) effectively protected MRC5 cells from EV71-induced cytopathic effects, which resulted from the inhibiting viral replication: rheum emodin treatment decreased viral genomic levels by 5.34-fold, viral protein expression by less than 30-fold and EV71 virulence by 0.33107-fold. The fact that inhibition of rheum emodin on viral virulence was much stronger than its effects on genomic levels and viral protein expression suggested that rheum emodin inhibited viral maturation. Furthermore, rheum emodin treatment markedly diminished cell cycle arrest at S phase in MRC5 cells, which was induced by EV71 infection and favored the viral replication. In contrast, neither astragaloside (50 μmol/L) nor artemisinin (50 μmol/L) showed similar anti-EV71 activities. Among the three natural compounds tested, rheum emodin effectively suppressed EV71 viral replication, thus is a candidate anti-HFMD drug.

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.

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.

A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication

PubMed Central

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

2014-01-01

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

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.

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

Primary simian immunodeficiency virus SIVmnd-2 infection in mandrills (Mandrillus sphinx).

PubMed

Onanga, Richard; Souquière, Sandrine; Makuwa, Maria; Mouinga-Ondeme, Augustin; Simon, François; Apetrei, Cristian; Roques, Pierre

2006-04-01

Mandrills are the only nonhuman primate (NHP) naturally infected by two types of simian immunodeficiency virus (SIV): SIVmnd-1 and SIVmnd-2. We have already reported that the high SIVmnd-1 replication during primary infection contrasts with only transient changes in CD4+ and CD8+ cell counts. Since early virus-host interactions predict viral control and disease progression in human immunodeficiency virus-infected patients, we investigated the dynamics of SIVmnd-2 primary infection in mandrills to examine the impact on immune effectors in blood and lymph nodes (LNs). To avoid in vitro strain selection, all mandrills in this study received plasma from SIVmnd-2-infected mandrills. SIVmnd-2 plasma viremia peaked at 10(7) to 10(8) RNA copies/ml between days 7 and 10. This peak was followed in all four monkeys by a decline in virus replication, with a set point level of 10(5) to 10(6) RNA copies/ml at day 42 postinfection (p.i.). Viral DNA load in PBMC and LNs also peaked between days 7 and 10 (10(5) to 10(6) DNA copies/10(6) cells) and stabilized at 10(3) to 10(4) DNA copies/10(6) cells during the chronic phase. Anti-SIVmnd-2 antibodies were detected starting from days 28 to 32. A transitory decline of CD3+ CD4+ cells in the LNs occurred in animals with high peak VLs. CD4+ and CD8+ T-cell activation in blood and LNs was noted between days 5 and 17 p.i., surrounding the peak of viral replication. This was most significant in the LNs. Activation markers then returned to preinfection values despite continuous and active viral replication during the chronic infection. The dynamics of SIVmnd-2 infection in mandrills showed a pattern similar to that of SIVmnd-1 infection. This might be a general feature of nonpathogenic SIV natural African NHP models.

Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of the Viral Genome to Assembly Sites.

PubMed

Boson, Bertrand; Denolly, Solène; Turlure, Fanny; Chamot, Christophe; Dreux, Marlène; Cosset, François-Loïc

2017-03-01

Daclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs, and inhibition of viral particle assembly. Cell-culture-derived HCV particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity and production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy. Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B, and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes. In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins because viral particles and replication complex vesicles cannot form or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout

USGS Publications Warehouse

Wargo, Andrew R.; Scott, Robert J.; Kerr, Benjamin; Kurath, Gael

2017-01-01

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2 days, defining a generally uniform early peak period of shedding from 1 to 4 days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority of fish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur.

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout.

PubMed

Wargo, Andrew R; Scott, Robert J; Kerr, Benjamin; Kurath, Gael

2017-01-02

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2days, defining a generally uniform early peak period of shedding from 1 to 4days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority of fish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur. Copyright © 2016 Elsevier B.V. All rights reserved.

Replication and shedding kinetics of infectious hematopoietic necrosis virus in juvenile rainbow trout

PubMed Central

Wargo, Andrew R.; Scott, Robert J.; Kerr, Benjamin; Kurath, Gael

2016-01-01

Viral replication and shedding are key components of transmission and fitness, the kinetics of which are heavily dependent on virus, host, and environmental factors. To date, no studies have quantified the shedding kinetics of infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss), or how they are associated with replication, making it difficult to ascertain the transmission dynamics of this pathogen of high agricultural and conservation importance. Here, the replication and shedding kinetics of two M genogroup IHNV genotypes were examined in their naturally co-evolved rainbow trout host. Within host virus replication began rapidly, approaching maximum values by day 3 post-infection, after which viral load was maintained or gradually dropped through day 7. Host innate immune response measured as stimulation of Mx-1 gene expression generally followed within host viral loads. Shedding also began very quickly and peaked within 2 days, defining a generally uniform early peak period of shedding from 1 to 4 days after exposure to virus. This was followed by a post-peak period where shedding declined, such that the majority offish were no longer shedding by day 12 post-infection. Despite similar kinetics, the average shedding rate over the course of infection was significantly lower in mixed compared to single genotype infections, suggesting a competition effect, however, this did not significantly impact the total amount of virus shed. The data also indicated that the duration of shedding, rather than peak amount of virus shed, was correlated with fish mortality. Generally, the majority of virus produced during infection appeared to be shed into the environment rather than maintained in the host, although there was more retention of within host virus during the post-peak period. Viral virulence was correlated with shedding, such that the more virulent of the two genotypes shed more total virus. This fundamental understanding of IHNV shedding kinetics and variation at the individual fish level could assist with management decisions about how to respond to disease outbreaks when they occur. PMID:27771253

Replication of poliovirus RNA and subgenomic RNA transcripts in transfected cells.

PubMed Central

Collis, P S; O'Donnell, B J; Barton, D J; Rogers, J A; Flanegan, J B

1992-01-01

Full-length and subgenomic poliovirus RNAs were transcribed in vitro and transfected into HeLa cells to study viral RNA replication in vivo. RNAs with deletion mutations were analyzed for the ability to replicate in either the absence or the presence of helper RNA by using a cotransfection procedure and Northern (RNA) blot analysis. An advantage of this approach was that viral RNA replication and genetic complementation could be characterized without first isolating conditional-lethal mutants. A subgenomic RNA with a large in-frame deletion in the capsid coding region (P1) replicated more efficiently than full-length viral RNA transcripts. In cotransfection experiments, both the full-length and subgenomic RNAs replicated at slightly reduced levels and appeared to interfere with each other's replication. In contrast, a subgenomic RNA with a similarly sized out-of-frame deletion in P1 did not replicate in transfected cells, either alone or in the presence of helper RNA. Similar results were observed with an RNA transcript containing a large in-frame deletion spanning the P1, P2, and P3 coding regions. A mutant RNA with an in-frame deletion in the P1-2A coding sequence was self-replicating but at a significantly reduced level. The replication of this RNA was fully complemented after cotransfection with a helper RNA that provided 2A in trans. A P1-2A-2B in-frame deletion, however, totally blocked RNA replication and was not complemented. Control experiments showed that all of the expected viral proteins were both synthesized and processed when the RNA transcripts were translated in vitro. Thus, our results indicated that 2A was a trans-acting protein and that 2B and perhaps other viral proteins were cis acting during poliovirus RNA replication in vivo. Our data support a model for poliovirus RNA replication which directly links the translation of a molecule of plus-strand RNA with the formation of a replication complex for minus-strand RNA synthesis. Images PMID:1328676

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

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

Viral Interference and Persistence in Mosquito-Borne Flaviviruses.

PubMed

Salas-Benito, Juan Santiago; De Nova-Ocampo, Mónica

2015-01-01

Mosquito-borne flaviviruses are important pathogens for humans, and the detection of two or more flaviviruses cocirculating in the same geographic area has often been reported. However, the epidemiological impact remains to be determined. Mosquito-borne flaviviruses are primarily transmitted through Aedes and Culex mosquitoes; these viruses establish a life-long or persistent infection without apparent pathological effects. This establishment requires a balance between virus replication and the antiviral host response. Viral interference is a phenomenon whereby one virus inhibits the replication of other viruses, and this condition is frequently associated with persistent infections. Viral interference and persistent infection are determined by several factors, such as defective interfering particles, competition for cellular factors required for translation/replication, and the host antiviral response. The interaction between two flaviviruses typically results in viral interference, indicating that these viruses share common features during the replicative cycle in the vector. The potential mechanisms involved in these processes are reviewed here.

Inactivation of Norovirus by Lemongrass Essential Oil Using a Norovirus Surrogate System.

PubMed

Kim, Ye Won; You, Hyun Ju; Lee, Soyoung; Kim, Bomi; Kim, Do Kyung; Choi, Joo-Bong; Kim, Ji-Ah; Lee, Hee Jung; Joo, In Sun; Lee, Jeong Su; Kang, Dong Hyun; Lee, Giljae; Ko, Gwang Pyo; Lee, Sung-Joon

2017-08-01

This study investigated the effect of lemongrass essential oil (LGEO) on the infectivity and viral replication of norovirus. Murine norovirus 1 (MNV-1), a surrogate of human norovirus, was preincubated with LGEO and then used to infect RAW 264.7 cells in a plaque reduction assay. LGEO exhibited a significant reduction in MNV-1 plaque formation in both time- and dose-dependent manners. The quantification of viral genome by quantitative real-time PCR showed similar results in line with those of the plaque reduction assay. It was revealed that citral, a single compound in LGEO, showed dramatic reduction in MNV-1 infectivity (-73.09% when using a treatment of 0.02%, v/v). The inhibitory activity of LGEO on viral replication was further investigated in HG23 cells that harbored a human norovirus replicon. LGEO treatment significantly reduced viral replication in HG23 cells, which suggests that LGEO may have dual inhibitory activities that inactivate viral coat proteins required for viral infection and suppress norovirus genome replication in host cells. In animal experiments, oral administration of murine norovirus preincubated with LGEO significantly suppressed virus infectivity in vivo. Collectively, these results suggest that LGEO, in particular the LGEO component citral, inactivates the norovirus and its subsequent replication in host cells. Thus, LGEO shows promise as a method of inhibiting norovirus within the food industry.

Role of the myeloid differentiation primary response (MYD88) and TIR-domain-containing adapter-inducing interferon-β (TRIF) pathways in dengue.

PubMed

Duran, Anyelo; Valero, Nereida; Mosquera, Jesus; Delgado, Lineth; Alvarez-Mon, Melchor; Torres, Mariana

2016-10-01

Dengue disease courses with high viremia titers and high cytokine production suggesting viral replication and active immune response that could be related to viral evasion. One of the main targets of dengue virus (DENV) is monocyte/macrophage cells; however, little information regarding viral evasive mechanisms and pathway activation in monocytes infected by DENV is available. The aim of this study was to determine the role of myeloid differentiation primary response (MyD88), TIR-domain-containing adapter- inducing interferon-β (TRIF) and NF-kB pathways in viral replication and cytokine production in human monocyte cultures infected by DENV2. In this regard Pepinh- TRIF, Pepinh- MYD and pyrrolidine dithiocarbamate (PDTC) were used to inhibit TRIF, MYD88 and NF-kB pathways. Cytokine production was measured by ELISA. Increased DENV replication and IFNα/β, TNF-α, IL-12 and IL-18 in infected cultures at 24h were found. All of these parameters were significantly decreased after TRIF, MYD88 or NF-kB inhibition. Association analysis between viral replication and cytokine production showed high significant positive correlation in TRIF and MYD88 treated cultures. This study shows that DENV2 induces activation of innate-immune response and transcription factors to drive viral expression and replication in the face of pro-inflammatory antiviral responses in vitro. Copyright © 2016 Elsevier Inc. All rights reserved.

Viruses in close associations with free-living amoebae.

PubMed

Scheid, Patrick

2015-11-01

As both groups of organisms, free-living amoebae (FLA) and viruses, can be found in aquatic environments side by side, it appears obvious that there are multiple interactions with respect to host-endocytobiont relationships. Several relationships between viruses and protozoan hosts are described and it was the discovery of the so called "giant viruses," associated with amoebae, which gave another dimension to these interactions. Mimiviruses, Pandoraviruses and Pithoviruses are examples for interesting viral endocytobionts within FLA. In the Mimivirus viral factories, viral DNA undergoes replication and transcription, and the DNA is prepared to be packed in procapsids. Theses Mimivirus factories can be considered as efficient "production lines" where, at any given moment, all stages of viral generation including membrane biogenesis, capsid assembly and genome encapsidation, are occurring concomitantly. There are some hints that similar replication factories are involved as well during the Pandoravirus development. Some scientists favour the assumption that the giant viruses have received many of their genes from their hosts or from sympatric occurring endocytobionts via lateral gene transfer. This hypothesis would mean that this type of transfer has been an important process in the evolution of genomes in the context of the intracellular parasitic or endocytobiotic lifestyle. In turn, that would migitate against hypothesizing development of a new branch in the tree of life. Based on the described scenarios to explain the presence of genes related to translation, it is also possible that earlier ancestors of today's DNA viruses were involved in the origin of eukaryotes. That possibly could in turn support the idea that cellular organisms could have evolved from viruses with growing autarkic properties. In future we expect the discovery of further (giant) viruses within free-living amoebae and other protozoa through genomic, transcriptomic and proteomic analyses.

Clinical Utility of Urinary Cytology to Detect BK Viral Nephropathy.

PubMed

Nankivell, Brian J; Renthawa, Jasveen; Jeoffreys, Neisha; Kable, Kathy; O'Connell, Philip J; Chapman, Jeremy R; Wong, Germaine; Sharma, Raghwa N

2015-08-01

Reactivation of BK polyoma virus can result in destructive viral allograft nephropathy (BKVAN) with limited treatment options. Screening programs using surrogate markers of viral replication are important preventive strategies, guiding immunosuppression reduction. We prospectively evaluated the diagnostic test performance of urinary decoy cells and urinary SV40T immunochemistry of exfoliated cells, to screen for BKVAN, (defined by reference histology with SV40 immunohistochemistry, n = 704 samples), compared with quantitative viremia, from 211 kidney and 141 kidney-pancreas transplant recipients. The disease prevalence of BKVAN was 2.6%. Decoy cells occurred in 95 of 704 (13.5%) samples, with a sensitivity of 66.7%, specificity of 88.6%, positive predictive value (PPV) of 11.7%, and negative predictive value of 98.5% to predict histologically proven BKVAN. Quantification of decoy cells improved the PPV to 32.1% (10 ≥ cells threshold). Immunohistochemical staining of urinary exfoliated cells for SV40T improved sensitivity to 85.7%, detecting atypical or degenerate infected cells (specificity of 92.3% and PPV of 33.3%), but was hampered by technical failures. Viremia occurred in 90 of 704 (12.8%) with sensitivity of 96.3%, specificity of 90.3%, PPV of 31.5%, and negative predictive value of 99.8%. The receiver-operator curve performance of quantitative viremia surpassed decoy cells (area under the curve of 0.95 and 0.79, respectively, P = 0.0018 for differences). Combining decoy cell and BK viremia in a diagnostic matrix improved prediction of BKVAN and diagnostic risk stratification, especially for high-level positive results. Although quantified decoy cells are acceptable surrogate markers of BK viral replication with unexceptional test performances, quantitative viremia displayed superior test characteristics and is suggested as the screening test of choice.

Inactivation of Dengue and Yellow Fever viruses by heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX.

PubMed

Assunção-Miranda, I; Cruz-Oliveira, C; Neris, R L S; Figueiredo, C M; Pereira, L P S; Rodrigues, D; Araujo, D F F; Da Poian, A T; Bozza, M T

2016-03-01

To investigate the effect of heme, cobalt-protoporphyrin IX and tin-protoporphyrin IX (CoPPIX and SnPPIX), macrocyclic structures composed by a tetrapyrrole ring with a central metallic ion, on Dengue Virus (DENV) and Yellow Fever Virus (YFV) infection. Treatment of HepG2 cells with heme, CoPPIX and SnPPIX after DENV infection reduced infectious particles without affecting viral RNA contents in infected cells. The reduction of viral load occurs only with the direct contact of DENV with porphyrins, suggesting a direct effect on viral particles. Previously incubation of DENV and YFV with heme, CoPPIX and SnPPIX resulted in viral particles inactivation in a dose-dependent manner. Biliverdin, a noncyclical porphyrin, was unable to inactivate the viruses tested. Infection of HepG2 cells with porphyrin-pretreated DENV2 results in a reduced or abolished viral protein synthesis, RNA replication and cell death. Treatment of HepG2 or THP-1 cell lineage with heme or CoPPIX after DENV infection with a very low MOI resulted in a decreased DENV replication and protection from death. Heme, CoPPIX and SnPPIX possess a marked ability to inactivate DENV and YFV, impairing its ability to infect and induce cytopathic effects on target cells. These results open the possibility of therapeutic application of porphyrins or their use as models to design new antiviral drugs against DENV and YFV. © 2016 The Society for Applied Microbiology.

The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication-associated inclusion bodies.

PubMed

Sun, Liying; Andika, Ida Bagus; Shen, Jiangfeng; Yang, Di; Chen, Jianping

2014-06-01

Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa-Pro (nuclear inclusion-a protease) and VPg (viral protein genome-linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa-Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV-infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2-associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV. © 2013 BSPP AND JOHN WILEY & SONS LTD.

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.

Chromosomally Integrated Human Herpesvirus 6: Models of Viral Genome Release from the Telomere and Impacts on Human Health.

PubMed

Wood, Michael L; Royle, Nicola J

2017-07-12

Human herpesvirus 6A and 6B, alongside some other herpesviruses, have the striking capacity to integrate into telomeres, the terminal repeated regions of chromosomes. The chromosomally integrated forms, ciHHV-6A and ciHHV-6B, are proposed to be a state of latency and it has been shown that they can both be inherited if integration occurs in the germ line. The first step in full viral reactivation must be the release of the integrated viral genome from the telomere and here we propose various models of this release involving transcription of the viral genome, replication fork collapse, and t-circle mediated release. In this review, we also discuss the relationship between ciHHV-6 and the telomere carrying the insertion, particularly how the presence and subsequent partial or complete release of the ciHHV-6 genome may affect telomere dynamics and the risk of disease.

Experimentally-induced immune activation in natural hosts of SIV induces significant increases in viral replication and CD4+ T cell depletion

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

Ribeiro, Ruy M

2008-01-01

Chronically SIVagm-infected African green monkeys (AGMs) have a remarkably stable non-pathogenic disease course, with levels of immune activation in chronic SIVagm infection similar to those observed in uninfected monkeys and stable viral loads (VLs) for long periods of time. In vivo administration of lipopolysaccharide (LPS) or an IL-2/diphtheria toxin fusion protein (Ontak) to chronically SIVagm-infected AGMs triggered increases in immune activation and subsequently of viral replication and depletion of intestinal CD4{sup +} T cells. Our study indicates that circulating microbial products can increase viral replication by inducing immune activation and increasing the number of viral target cells, thus demonstrating thatmore » immune activation and T cell prolifeation are key factors in AIDS pathogenesis.« less

High viral load in lymph nodes and latent human immunodeficiency virus (HIV) in peripheral blood cells of HIV-1-infected chimpanzees.

PubMed Central

Saksela, K; Muchmore, E; Girard, M; Fultz, P; Baltimore, D

1993-01-01

We have examined human immunodeficiency virus type 1 (HIV-1) infection in chimpanzees by analyzing HIV-1 DNA and RNA in lymph nodes and peripheral mononuclear cells (PBMCs). Like certain asymptomatic HIV-infected persons, these chimpanzees had no detectable viral replication in their PBMCs. However, viral replication and a high viral load were observed in the lymphatic tissue. Despite the absence of viral replication in PBMCs, 1/1,000 to 1/10,000 of the PBMCs contained HIV-1 proviral DNA, and HIV transcription could be rapidly induced in these cells in vitro. These results provide direct evidence of cellular latency of HIV in vivo and suggest that HIV infection in chimpanzees may be a useful model for clinical latency of HIV infection in humans. Images PMID:8230463

Residual Viremia in Treated HIV+ Individuals

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

Conway, Jessica M.; Perelson, Alan S.

Antiretroviral therapy (ART) effectively controls HIV infection, suppressing HIV viral loads. However, some residual virus remains, below the level of detection, in HIV-infected patients on ART. Furthermore, the source of this viremia is an area of debate: does it derive primarily from activation of infected cells in the latent reservoir, or from ongoing viral replication? Our observations seem to be contradictory: there is evidence of short term evolution, implying that there must be ongoing viral replication, and viral strains should thus evolve. The phylogenetic analyses, and rare emergent drug resistance, suggest no long-term viral evolution, implying that virus derived frommore » activated latent cells must dominate. We use simple deterministic and stochastic models to gain insight into residual viremia dynamics in HIV-infected patients. Our modeling relies on two underlying assumptions for patients on suppressive ART: that latent cell activation drives viral dynamics and that the reproductive ratio of treated infection is less than 1. Nonetheless, the contribution of viral replication to residual viremia in patients on ART may be non-negligible. However, even if the portion of viremia attributable to viral replication is significant, our model predicts (1) that latent reservoir re-seeding remains negligible, and (2) some short-term viral evolution is permitted, but long-term evolution can still be limited: stochastic analysis of our model shows that de novo emergence of drug resistance is rare. Thus, our simple models reconcile the seemingly contradictory observations on residual viremia and, with relatively few parameters, recapitulates HIV viral dynamics observed in patients on suppressive therapy.« less

Residual Viremia in Treated HIV+ Individuals

DOE PAGES

Conway, Jessica M.; Perelson, Alan S.

2016-01-06

Antiretroviral therapy (ART) effectively controls HIV infection, suppressing HIV viral loads. However, some residual virus remains, below the level of detection, in HIV-infected patients on ART. Furthermore, the source of this viremia is an area of debate: does it derive primarily from activation of infected cells in the latent reservoir, or from ongoing viral replication? Our observations seem to be contradictory: there is evidence of short term evolution, implying that there must be ongoing viral replication, and viral strains should thus evolve. The phylogenetic analyses, and rare emergent drug resistance, suggest no long-term viral evolution, implying that virus derived frommore » activated latent cells must dominate. We use simple deterministic and stochastic models to gain insight into residual viremia dynamics in HIV-infected patients. Our modeling relies on two underlying assumptions for patients on suppressive ART: that latent cell activation drives viral dynamics and that the reproductive ratio of treated infection is less than 1. Nonetheless, the contribution of viral replication to residual viremia in patients on ART may be non-negligible. However, even if the portion of viremia attributable to viral replication is significant, our model predicts (1) that latent reservoir re-seeding remains negligible, and (2) some short-term viral evolution is permitted, but long-term evolution can still be limited: stochastic analysis of our model shows that de novo emergence of drug resistance is rare. Thus, our simple models reconcile the seemingly contradictory observations on residual viremia and, with relatively few parameters, recapitulates HIV viral dynamics observed in patients on suppressive therapy.« less

Viruses Roll the Dice: The Stochastic Behavior of Viral Genome Molecules Accelerates Viral Adaptation at the Cell and Tissue Levels

PubMed Central

Miyashita, Shuhei; Ishibashi, Kazuhiro; Kishino, Hirohisa; Ishikawa, Masayuki

2015-01-01

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1–20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence–tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell. PMID:25781391

In vitro inhibition of African swine fever virus-topoisomerase II disrupts viral replication.

PubMed

Freitas, Ferdinando B; Frouco, Gonçalo; Martins, Carlos; Leitão, Alexandre; Ferreira, Fernando

2016-10-01

African swine fever virus (ASFV) is the etiological agent of a highly-contagious and fatal disease of domestic pigs, leading to serious socio-economic impact in affected countries. To date, neither a vaccine nor a selective anti-viral drug are available for prevention or treatment of African swine fever (ASF), emphasizing the need for more detailed studies at the role of ASFV proteins involved in viral DNA replication and transcription. Notably, ASFV encodes for a functional type II topoisomerase (ASFV-Topo II) and we recently showed that several fluoroquinolones (bacterial DNA topoisomerase inhibitors) fully abrogate ASFV replication in vitro. Here, we report that ASFV-Topo II gene is actively transcribed throughout infection, with transcripts being detected as early as 2 hpi and reaching a maximum peak concentration around 16 hpi, when viral DNA synthesis, transcription and translation are more active. siRNA knockdown experiments showed that ASFV-Topo II plays a critical role in viral DNA replication and gene expression, with transfected cells presenting lower viral transcripts (up to 89% decrease) and reduced cytopathic effect (-66%) when compared to the control group. Further, a significant decrease in the number of both infected cells (75.5%) and viral factories per cell and in virus yields (up to 99.7%, 2.5 log) was found only in cells transfected with siRNA targeting ASFV-Topo II. We also demonstrate that a short exposure to enrofloxacin during the late phase of infection (from 15 to 1 hpi) induces fragmentation of viral genomes, whereas no viral genomes were detected when enrofloxacin was added from the early phase of infection (from 2 to 16 hpi), suggesting that fluoroquinolones are ASFV-Topo II poisons. Altogether, our results demonstrate that ASFV-Topo II enzyme has an essential role during viral genome replication and transcription, emphasizing the idea that this enzyme can be a potential target for drug and vaccine development against ASF. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Interplay Among Constitutes of Ebola Virus: Nucleoprotein, Polymerase L, Viral Proteins

NASA Astrophysics Data System (ADS)

Zhang, Minchuan; He, Peiming; Su, Jing; Singh, Dadabhai T.; Su, Hailei; Su, Haibin

Ebola virus is a highly lethal filovirus, claimed thousands of people in its recent outbreak. Seven viral proteins constitute ebola viral structure, and four of them (nucleoprotein (NP), polymerase L, VP35 and VP30) participate majorly in viral replication and transcription. We have elucidated a conformation change of NP cleft by VP35 NP-binding protein domains through superimposing two experimental NP structure images and discussed the function of this conformation change in the replication and transcription with polymerase complex (L, VP35 and VP30). The important roles of VP30 in viral RNA synthesis have also been discussed. A “tapping” model has been proposed in this paper for a better understanding of the interplay among the four viral proteins (NP, polymerase L, VP35 and VP30). Moreover, we have pinpointed some key residue changes on NP (both NP N- and C-terminal) and L between Reston and Zaire by computational studies. Together, this paper provides a description of interactions among ebola viral proteins (NP, L, VP35, VP30 and VP40) in viral replication and transcription, and sheds light on the complex system of viral reproduction.

Cycluridine: A novel antiviral effective against flaviviruses

PubMed Central

Galabov, Angel S; Mukova, Lucia; Abashev, Yuriy P; Wassilewa, Lilia; Tzvetkov, Petko; Minkov, Vassil; Barinskiy, Igor F; Rice, Charles M; Ouzounov, Sergey; Sidzhakova, Dorotea

2017-01-01

This review describes the contemporary state of research for antivirals effective against flaviviruses, especially focusing on inhibitors of the pestivirus causative agent of bovine viral diarrhoea virus. We highlight cycluridine, an originally synthesized Mannich’s base [a tetrahydro-2(1H)-pyrimidinones derivative], as a highly effective antiviral possessing a strong inhibitory effect on bovine viral diarrhoea virus replication. Cycluridine was active against replication of a wide variety of bovine viral diarrhoea virus strains in cell cultures. The drug-sensitive period in the bovine viral diarrhoea virus replication cycle included the latent period and the exponential phase; a 90-min delay in the peak of viral RNA synthesis was observed. Cycluridine administered orally manifested a pronounced protective effect in calves with natural mucosal disease/viral diarrhoea and calves experimentally infected with bovine viral diarrhoea virus. Its magnitude of activity and selectivity places cycluridine in the lead among all known substances with anti- bovine viral diarrhoea virus activity. Additionally, cycluridine applied subcutaneously showed anti-tick-born encephalitis virus activity, manifesting a marked protective effect in mice infected with tick-born encephalitis virus. Cycluridine could be a prospective antiviral in veterinary and medical practice for the treatment of bovine viral diarrhoea virus and other flavivirus infections. PMID:28768435

Single-stranded DNA binding protein Gp5 of Bacillus subtilis phage Φ29 is required for viral DNA replication in growth-temperature dependent fashion.

PubMed

Tone, Takahiro; Takeuchi, Ari; Makino, Osamu

2012-01-01

In the absence of viral single-stranded DNA binding protein gp5, Bacillus subtilis phage φ29 failed to grow and to replicate its genome at 45 °C, while it grew and replicated normally at 30 °C and 42 °C. This indicates that gp5 is dispensable for φ29 DNA replication at 42 °C and lower temperatures.

Sequential structures provide insights into the fidelity of RNA replication.

PubMed

Ferrer-Orta, Cristina; Arias, Armando; Pérez-Luque, Rosa; Escarmís, Cristina; Domingo, Esteban; Verdaguer, Nuria

2007-05-29

RNA virus replication is an error-prone event caused by the low fidelity of viral RNA-dependent RNA polymerases. Replication fidelity can be decreased further by the use of mutagenic ribonucleoside analogs to a point where viral genetic information can no longer be maintained. For foot-and-mouth disease virus, the antiviral analogs ribavirin and 5-fluorouracil have been shown to be mutagenic, contributing to virus extinction through lethal mutagenesis. Here, we report the x-ray structure of four elongation complexes of foot-and-mouth disease virus polymerase 3D obtained in presence of natural substrates, ATP and UTP, or mutagenic nucleotides, ribavirin triphosphate and 5-fluorouridine triphosphate with different RNAs as template-primer molecules. The ability of these complexes to synthesize RNA in crystals allowed us to capture different successive replication events and to define the critical amino acids involved in (i) the recognition and positioning of the incoming nucleotide or analog; (ii) the positioning of the acceptor base of the template strand; and (iii) the positioning of the 3'-OH group of the primer nucleotide during RNA replication. The structures identify key interactions involved in viral RNA replication and provide insights into the molecular basis of the low fidelity of viral RNA polymerases.

Inhibition of adenovirus multiplication by short interfering RNAs directly or indirectly targeting the viral DNA replication machinery.

PubMed

Kneidinger, Doris; Ibrišimović, Mirza; Lion, Thomas; Klein, Reinhard

2012-06-01

Human adenoviruses are a common threat to immunocompromised patients, e.g., HIV-positive individuals or solid-organ and, in particular, allogeneic stem cell transplant recipients. Antiviral drugs have a limited effect on adenoviruses, and existing treatment modalities often fail to prevent fatal outcome. Silencing of viral genes by short interfering RNAs (siRNAs) holds a great promise in the treatment of viral infections. The aim of the present study was to identify adenoviral candidate targets for RNA interference-mediated inhibition of adenoviral replication. We investigated the impact of silencing of a set of early, middle, and late viral genes on the replication of adenovirus 5 in vitro. Adenovirus replication was inhibited by siRNAs directed against the adenoviral E1A, DNA polymerase, preterminal protein (pTP), IVa2, hexon, and protease genes. Silencing of early and middle genes was more effective in inhibiting adenovirus multiplication than was silencing of late genes. A siRNA directed against the viral DNA polymerase mRNA decreased viral genome copy numbers and infectious virus progeny by several orders of magnitude. Since silencing of any of the early genes directly or indirectly affected viral DNA synthesis, our data suggest that reducing viral genome copy numbers is a more promising strategy for the treatment of adenoviral infections than is reducing the numbers of proteins necessary for capsid generation. Thus, adenoviral DNA replication was identified as a key target for RNAi-mediated inhibition of adenovirus multiplication. In addition, the E1A transcripts emerged as a second important target, because its knockdown markedly improved the viability of cells at late stages of infection. Copyright © 2012 Elsevier B.V. All rights reserved.

Synaptogyrin-2 Promotes Replication of a Novel Tick-borne Bunyavirus through Interacting with Viral Nonstructural Protein NSs.

PubMed

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

2016-07-29

Synaptogyrin-2 is a non-neuronal member of the synaptogyrin family involved in synaptic vesicle biogenesis and trafficking. Little is known about the function of synaptogyrin-2. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease characterized by high fever, thrombocytopenia, and leukocytopenia with high mortality, caused by a novel tick-borne phlebovirus in the family Bunyaviridae. Our previous studies have shown that the viral nonstructural protein NSs forms inclusion bodies (IBs) that are involved in viral immune evasion, as well as viral RNA replication. In this study, we sought to elucidate the mechanism by which NSs formed the IBs, a lipid droplet-based structure confirmed by NSs co-localization with perilipin A and adipose differentiation-related protein (ADRP). Through a high throughput screening, we identified synaptogyrin-2 to be highly up-regulated in response to SFTS bunyavirus (SFTSV) infection and to be a promoter of viral replication. We demonstrated that synaptogyrin-2 interacted with NSs and was translocated into the IBs, which were reconstructed from lipid droplets into large structures in infection. Viral RNA replication decreased, and infectious virus titers were lowered significantly when synaptogyrin-2 was silenced in specific shRNA-expressing cells, which correlated with the reduced number of the large IBs restructured from regular lipid droplets. We hypothesize that synaptogyrin-2 is essential to promoting the formation of the IBs to become virus factories for viral RNA replication through its interaction with NSs. These findings unveil the function of synaptogyrin-2 as an enhancer in viral infection. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Stereophysicochemical variability plots highlight conserved antigenic areas in Flaviviruses

PubMed Central

Schein, Catherine H; Zhou, Bin; Braun, Werner

2005-01-01

Background Flaviviruses, which include Dengue (DV) and West Nile (WN), mutate in response to immune system pressure. Identifying escape mutants, variant progeny that replicate in the presence of neutralizing antibodies, is a common way to identify functionally important residues of viral proteins. However, the mutations typically occur at variable positions on the viral surface that are not essential for viral replication. Methods are needed to determine the true targets of the neutralizing antibodies. Results Stereophysicochemical variability plots (SVPs), 3-D images of protein structures colored according to variability, as determined by our PCPMer program, were used to visualize residues conserved in their physical chemical properties (PCPs) near escape mutant positions. The analysis showed 1) that escape mutations in the flavivirus envelope protein are variable residues by our criteria and 2) two escape mutants found at the same position in many flaviviruses sit above clusters of conserved residues from different regions of the linear sequence. Conservation patterns in T-cell epitopes in the NS3- protease suggest a similar mechanism of immune system evasion. Conclusion The SVPs add another dimension to structurally defining the binding sites of neutralizing antibodies. They provide a useful aid for determining antigenically important regions and designing vaccines. PMID:15845145

Prereplicative events involving simian virus 40 DNA in permissive cells

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

Rinaldy, A.; Feunteun, J.; Rosenberg, B.H.

1982-01-01

Simian virus 40 DNA molecules were found to be unable to replicate for 9 h after infection, even in cells that were already replicating the DNA of preinfecting simian virus 40; after 9 h, the ability of the DNA to replicate began to rise sharply. The kinetics of activation indicated that each DNA molecule undergoes a series of slow consecutive reactions, not involving T-antigen, before it can replicate. These pre-replicative molecular transformations probably involve configurational changes; their nature and their relation to the initiation of viral DNA synthesis is discussed. Observation of the replicative behavior of one viral DNA inmore » the presence of another was made possible by the use of two different mutants with distinguishable DNAs: a viable deletion mutant containing DNA insensitive to TaqI restriction enzyme was used to provide viral functions required for replication, and is a tsA mutant with TaqI-sensitive DNA was introduced at various times as a probe to determine the ability of the DNA to replicate under different conditions.« less

The M33 G Protein-Coupled Receptor Encoded by Murine Cytomegalovirus Is Dispensable for Hematogenous Dissemination but Is Required for Growth within the Salivary Gland

PubMed Central

Bittencourt, Fabiola M.; Wu, Shu-En; Bridges, James P.

2014-01-01

ABSTRACT Human cytomegalovirus (HCMV) is a pathogen found worldwide and is a serious threat to immunocompromised individuals and developing fetuses. Due to the species specificity of cytomegaloviruses, murine cytomegalovirus (MCMV) has been used as a model for in vivo studies of HCMV pathogenesis. The MCMV genome, like the genomes of other beta- and gammaherpesviruses, encodes G protein-coupled receptors (GPCRs) that modulate host signaling pathways presumably to facilitate viral replication and dissemination. Among these viral receptors, the M33 GPCR carried by MCMV is an activator of CREB, NF-κB, and phospholipase C-β signaling pathways and has been implicated in aspects of pathogenesis in vivo, including persistence in the salivary glands of BALB/c mice. In this study, we used immunocompetent nonobese diabetic (NOD) and immunocompromised NOD-scid-gamma (NSG) mice to further investigate the salivary gland defect exhibited by M33 deficiency. Interestingly, we demonstrate that virus with an M33 deletion (ΔM33) can replicate in the salivary gland of immunocompromised animals, albeit with a 400-fold growth defect compared with the growth of wild-type virus. Moreover, we determined that M33 does not have a role in cell-associated hematogenous dissemination but is required for viral amplification once the virus reaches the salivary gland. We conclude that the reduced replicative capacity of the ΔM33 virus is due to a specific defect occurring within the localized environment of the salivary gland. Importantly, since the salivary gland represents a site essential for persistence and horizontal transmission, an understanding of the mechanisms of viral replication within this site could lead to the generation of novel therapeutics useful for the prevention of HCMV spread. IMPORTANCE Human cytomegalovirus infects the majority of the American people and can reside silently in infected individuals for the duration of their lives. Under a number of circumstances, the virus can reactivate, leading to a variety of diseases in both adults and developing babies, and therefore, identifying the function of viral proteins is essential to understand how the virus spreads and causes disease. We aim to utilize animal models to study the function of an important class of viral proteins termed G protein-coupled receptors with the ultimate goal of developing inhibitors to these proteins that could one day be used to prevent viral spread. PMID:25100846

Results of interferon treatment in children with chronic hepatitis B.

PubMed

Grigorescu-Sido, Paula; Călin, Lazăr; Manasia, Rodica; Mireştean, Stefan; Creţ, Victoria; Skorka, Cristina; Grigorescu-Sido, Anca

2002-12-01

Many observations report a variable therapeutical response to interferon in children with chronic hepatitis B. In order to evaluate the efficiency of alpha-interferon treatment in the downregulation of viral replication and in the eradication of infection in these patients, we assessed HBeAg/HBeAb and HBsAg/HBsAb seroconversion (as well as with clinical outcome and the changes in the plasma level of aminotransferases) in 61 treated patients. The diagnosis was established by means of the usual clinical, biochemical and histopathological criteria. There was no possibility to viral DNA test and no control group was included. Patients were selected for interferon treatment who displayed at least a two fold rise in the plasma level of aminotransferases as compared to normal values, as well as necroinflammatory activity (score > or = 6) and positive HBeAg as a marker of viral replication. Treatment was carried out with alpha-2a interferon or alpha-2b interferon in a dose of 3 million U/m2/dose in 3 weekly doses for a period of 4-6 months. The monitoring interval was 6.6+/-3 years. HBeAg/HBeAb seroconversion was registered in 77.2% of the patients and mainly occurred during the first year of follow-up (50.9 %). HBsAg/HBsAb seroconversion was revealed in 1.75% of the cases. The therapeutical response was complete, incomplete, transient and absent in 1.75%, 64.9%, 10.5% and 22.8% of the patients, respectively. The results show that the eradication of HBV infection is insignificant, but the downregulation of viral replication and, subsequently the halt of further progression of hepatic lesions is obtained in a high percentage of cases, highlighting the efficiency of this treatment in children with chronic hepatitis B

Inhibitory Effect of a Nucleotide Analog on Infectious Salmon Anemia Virus Infection ▿

PubMed Central

Rivas-Aravena, Andrea; Vallejos-Vidal, Eva; Cortez-San Martin, Marcelo; Reyes-Lopez, Felipe; Tello, Mario; Mora, Patricia; Sandino, Ana María; Spencer, Eugenio

2011-01-01

The infectious salmon anemia virus (ISAV), which belongs to the Orthomyxoviridae family, has been responsible for major losses in the salmon industry, with mortalities close to 100% in areas where Atlantic salmon (Salmo salar) is grown. This work studied the effect of ribavirin (1-β-d-ribofuranosyl-1,2,3-triazole-3-carbaxaide), a broad-spectrum antiviral compound with proven ability to inhibit the replicative cycle of the DNA and RNA viruses. The results show that ribavirin was able to inhibit the infectivity of ISAV in in vitro assays. In these assays, a significant inhibition of the replicative viral cycle was observed with a 50% inhibitory concentration (IC50) of 0.02 μg/ml and an IC90 of 0.4 μg/ml of ribavirin. After ribavirin treatment, viral proteins were not detectable and a reduction of viral mRNA association with ribosomes was observed. Ribavirin does not affect the levels of EF1a, nor its association with polysomes, suggesting that the inhibition of RNA synthesis occurs specifically for the virus mRNAs and not for cellular mRNAs. Moreover, ribavirin caused a significant reduction in genomic and viral RNA messenger levels. The study of the inhibitory mechanism showed that it was not reversed by the addition of guanosine. Furthermore, in vivo assays showed a reduction in the mortality of Salmo salar by more than 90% in fish infected with ISAV and treated with ribavirin without adverse effects. In fact, these results show that ribavirin is an antiviral that could be used to prevent ISAV replication either in vitro or in vivo. PMID:21653663

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

Modulation of human beta-defensin-1 (hBD-1) in plasmacytoid dendritic cells (PDC), monocytes, and epithelial cells by influenza virus, Herpes simplex virus, and Sendai virus and its possible role in innate immunity.

PubMed

Ryan, Lisa K; Dai, Jihong; Yin, Zhiwei; Megjugorac, Nicholas; Uhlhorn, Victoria; Yim, Sunghan; Schwartz, Kyell D; Abrahams, Joshua M; Diamond, Gill; Fitzgerald-Bocarsly, Patricia

2011-08-01

hBD comprise a family of antimicrobial peptides that plays a role in bridging the innate and adaptive immune responses to infection. The expression of hBD-2 increases upon stimulation of numerous cell types with LPS and proinflammatory cytokines. In contrast, hBD-1 remains constitutively expressed in most cells in spite of cytokine or LPS stimulation; however, its presence in human PDC suggests it plays a role in viral host defense. To examine this, we characterized the expression of hBD-1 in innate immune cells in response to viral challenge. PDC and monocytes increased production of hBD-1 peptide and mRNA as early as 2 h following infection of purified cells and PBMCs with PR8, HSV-1, and Sendai virus. However, treatment of primary NHBE cells with influenza resulted in a 50% decrease in hBD-1 mRNA levels, as measured by qRT-PCR at 3 h following infection. A similar inhibition occurred with HSV-1 challenge of human gingival epithelial cells. Studies with HSV-1 showed that replication occurred in epithelial cells but not in PDC. Together, these results suggest that hBD-1 may play a role in preventing viral replication in immune cells. To test this, we infected C57BL/6 WT mice and mBD-1((-/-)) mice with mouse-adapted HK18 (300 PFU/mouse). mBD-1((-/-)) mice lost weight earlier and died sooner than WT mice (P=0.0276), suggesting that BD-1 plays a role in early innate immune responses against influenza in vivo. However, lung virus titers were equal between the two mouse strains. Histopathology showed a greater inflammatory influx in the lungs of mBD-1((-/-)) mice at Day 3 postinfection compared with WT C57BL/6 mice. The results suggest that BD-1 protects mice from influenza pathogenesis with a mechanism other than inhibition of viral replication.

Viral carcinogenesis: revelation of molecular mechanisms and etiology of human disease

NASA Technical Reports Server (NTRS)

Butel, J. S.

2000-01-01

The RNA and DNA tumor viruses have made fundamental contributions to two major areas of cancer research. Viruses were vital, first, to the discovery and analysis of cellular growth control pathways and the synthesis of current concepts of cancer biology and, second, to the recognition of the etiology of some human cancers. Transforming retroviruses carry oncogenes derived from cellular genes that are involved in mitogenic signalling and growth control. DNA tumor viruses encode oncogenes of viral origin that are essential for viral replication and cell transformation; viral oncoproteins complex with cellular proteins to stimulate cell cycle progression and led to the discovery of tumor suppressors. Viral systems support the concept that cancer development occurs by the accumulation of multiple cooperating events. Viruses are now accepted as bona fide etiologic factors of human cancer; these include hepatitis B virus, Epstein-Barr virus, human papillomaviruses, human T-cell leukemia virus type I and hepatitis C virus, plus several candidate human cancer viruses. It is estimated that 15% of all human tumors worldwide are caused by viruses. The infectious nature of viruses distinguishes them from all other cancer-causing factors; tumor viruses establish long-term persistent infections in humans, with cancer an accidental side effect of viral replication strategies. Viruses are usually not complete carcinogens, and the known human cancer viruses display different roles in transformation. Many years may pass between initial infection and tumor appearance and most infected individuals do not develop cancer, although immunocompromised individuals are at elevated risk of viral-associated cancers. Variable factors that influence viral carcinogenesis are reviewed, including possible synergy between viruses and environmental cofactors. The difficulties in establishing an etiologic role for a virus in human cancer are discussed, as well as the different approaches that proved viral links to cancer. Future directions for tumor virus studies are considered.

Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method.

PubMed

Dou, Dan; Hernández-Neuta, Iván; Wang, Hao; Östbye, Henrik; Qian, Xiaoyan; Thiele, Swantje; Resa-Infante, Patricia; Kouassi, Nancy Mounogou; Sender, Vicky; Hentrich, Karina; Mellroth, Peter; Henriques-Normark, Birgitta; Gabriel, Gülsah; Nilsson, Mats; Daniels, Robert

2017-07-05

Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics in situ, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Automatic detection and measurement of viral replication compartments by ellipse adjustment

PubMed Central

Garcés, Yasel; Guerrero, Adán; Hidalgo, Paloma; López, Raul Eduardo; Wood, Christopher D.; Gonzalez, Ramón A.; Rendón-Mancha, Juan Manuel

2016-01-01

Viruses employ a variety of strategies to hijack cellular activities through the orchestrated recruitment of macromolecules to specific virus-induced cellular micro-environments. Adenoviruses (Ad) and other DNA viruses induce extensive reorganization of the cell nucleus and formation of nuclear Replication Compartments (RCs), where the viral genome is replicated and expressed. In this work an automatic algorithm designed for detection and segmentation of RCs using ellipses is presented. Unlike algorithms available in the literature, this approach is deterministic, automatic, and can adjust multiple RCs using ellipses. The proposed algorithm is non iterative, computationally efficient and is invariant to affine transformations. The method was validated over both synthetic images and more than 400 real images of Ad-infected cells at various timepoints of the viral replication cycle obtaining relevant information about the biogenesis of adenoviral RCs. As proof of concept the algorithm was then used to quantitatively compare RCs in cells infected with the adenovirus wild type or an adenovirus mutant that is null for expression of a viral protein that is known to affect activities associated with RCs that result in deficient viral progeny production. PMID:27819325

Automatic detection and measurement of viral replication compartments by ellipse adjustment

NASA Astrophysics Data System (ADS)

Garcés, Yasel; Guerrero, Adán; Hidalgo, Paloma; López, Raul Eduardo; Wood, Christopher D.; Gonzalez, Ramón A.; Rendón-Mancha, Juan Manuel

2016-11-01

Viruses employ a variety of strategies to hijack cellular activities through the orchestrated recruitment of macromolecules to specific virus-induced cellular micro-environments. Adenoviruses (Ad) and other DNA viruses induce extensive reorganization of the cell nucleus and formation of nuclear Replication Compartments (RCs), where the viral genome is replicated and expressed. In this work an automatic algorithm designed for detection and segmentation of RCs using ellipses is presented. Unlike algorithms available in the literature, this approach is deterministic, automatic, and can adjust multiple RCs using ellipses. The proposed algorithm is non iterative, computationally efficient and is invariant to affine transformations. The method was validated over both synthetic images and more than 400 real images of Ad-infected cells at various timepoints of the viral replication cycle obtaining relevant information about the biogenesis of adenoviral RCs. As proof of concept the algorithm was then used to quantitatively compare RCs in cells infected with the adenovirus wild type or an adenovirus mutant that is null for expression of a viral protein that is known to affect activities associated with RCs that result in deficient viral progeny production.

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

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.

Divergent Requirement for a DNA Repair Enzyme during Enterovirus Infections

PubMed Central

Maciejewski, Sonia; Nguyen, Joseph H. C.; Gómez-Herreros, Fernando; Cortés-Ledesma, Felipe; Caldecott, Keith W.

2015-01-01

ABSTRACT Viruses of the Enterovirus genus of picornaviruses, including poliovirus, coxsackievirus B3 (CVB3), and human rhinovirus, commandeer the functions of host cell proteins to aid in the replication of their small viral genomic RNAs during infection. One of these host proteins is a cellular DNA repair enzyme known as 5′ tyrosyl-DNA phosphodiesterase 2 (TDP2). TDP2 was previously demonstrated to mediate the cleavage of a unique covalent linkage between a viral protein (VPg) and the 5′ end of picornavirus RNAs. Although VPg is absent from actively translating poliovirus mRNAs, the removal of VPg is not required for the in vitro translation and replication of the RNA. However, TDP2 appears to be excluded from replication and encapsidation sites during peak times of poliovirus infection of HeLa cells, suggesting a role for TDP2 during the viral replication cycle. Using a mouse embryonic fibroblast cell line lacking TDP2, we found that TDP2 is differentially required among enteroviruses. Our single-cycle viral growth analysis shows that CVB3 replication has a greater dependency on TDP2 than does poliovirus or human rhinovirus replication. During infection, CVB3 protein accumulation is undetectable (by Western blot analysis) in the absence of TDP2, whereas poliovirus protein accumulation is reduced but still detectable. Using an infectious CVB3 RNA with a reporter, CVB3 RNA could still be replicated in the absence of TDP2 following transfection, albeit at reduced levels. Overall, these results indicate that TDP2 potentiates viral replication during enterovirus infections of cultured cells, making TDP2 a potential target for antiviral development for picornavirus infections. PMID:26715620

Visualization of Arenavirus RNA Species in Individual Cells by Single-Molecule Fluorescence In Situ Hybridization Suggests a Model of Cyclical Infection and Clearance during Persistence.

PubMed

King, Benjamin R; Samacoits, Aubin; Eisenhauer, Philip L; Ziegler, Christopher M; Bruce, Emily A; Zenklusen, Daniel; Zimmer, Christophe; Mueller, Florian; Botten, Jason

2018-06-15

Lymphocytic choriomeningitis mammarenavirus (LCMV) is an enveloped, negative-strand RNA virus that causes serious disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. Different models have been proposed to describe how arenaviruses regulate the replication and transcription of their bisegmented, single-stranded RNA genomes, particularly during persistent infection. However, these models were based largely on viral RNA profiling data derived from entire populations of cells. To better understand LCMV replication and transcription at the single-cell level, we established a high-throughput, single-molecule fluorescence in situ hybridization (smFISH) image acquisition and analysis pipeline and examined viral RNA species at discrete time points from virus entry through the late stages of persistent infection in vitro We observed the transcription of viral nucleoprotein and polymerase mRNAs from the incoming S and L segment genomic RNAs, respectively, within 1 h of infection, whereas the transcription of glycoprotein mRNA from the S segment antigenome required ∼4 to 6 h. This confirms the temporal separation of viral gene expression expected due to the ambisense coding strategy of arenaviruses and also suggests that antigenomic RNA contained in virions is not transcriptionally active upon entry. Viral replication and transcription peaked at 36 h postinfection, followed by a progressive loss of viral RNAs over the next several days. During persistence, the majority of cells showed repeating cyclical waves of viral transcription and replication followed by the clearance of viral RNA. Thus, our data support a model of LCMV persistence whereby infected cells can spontaneously clear infection and become reinfected by viral reservoir cells that remain in the population. IMPORTANCE Arenaviruses are human pathogens that can establish asymptomatic, lifelong infections in their rodent reservoirs. Several models have been proposed to explain how arenavirus spread is restricted within host rodents, including the periodic accumulation and loss of replication-competent, but transcriptionally incompetent, viral genomes. A limitation of previous studies was the inability to enumerate viral RNA species at the single-cell level. We developed a high-throughput, smFISH assay and used it to quantitate lymphocytic choriomeningitis mammarenavirus (LCMV) replicative and transcriptional RNA species in individual cells at distinct time points following infection. Our findings support a model whereby productively infected cells can clear infection, including viral RNAs and antigen, and later be reinfected. This information improves our understanding of the timing and possible regulation of LCMV genome replication and transcription during infection. Importantly, the smFISH assay and data analysis pipeline developed here is easily adaptable to other RNA viruses. Copyright © 2018 American Society for Microbiology.

Interferon lambda inhibits dengue virus replication in epithelial cells.

PubMed

Palma-Ocampo, Helen K; Flores-Alonso, Juan C; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio; Flores-Mendoza, Lilian; Herrera-Camacho, Irma; Rosas-Murrieta, Nora H; Santos-López, Gerardo

2015-09-28

In viral disease, infection is controlled at the cellular level by type I interferon (IFN-I), but dengue virus (DENV) has the ability to inhibit this response. Type III interferon, also known as lambda IFN (IFN-III or IFN-λ), is a complementary pathway to the antiviral response by IFN-I. This work analyzed the IFN-λ (IFN-III) mediated antiviral response against DENV serotype 2 (DENV-2) infection. Dengue fever patients were sampled to determine their IFN-λ levels by ELISA. To study the IFN-λ response during DENV infection we selected the epithelial cell line C33-A, and we demonstrated that it is permissive to DENV-2 infection. The effect of IFN-λ on virus replication was determined in these cells, in parallel to the expression of IFN-stimulated genes (ISGs), and Suppressor of Cytokine Signaling (SOCS), genes measured by RT-qPCR. We found increased (~1.8 times) serological IFN-λ in dengue fever patients compared to healthy blood donors. IFN-λ inhibited DENV-2 replication in a dose-dependent manner in vitro. The reduction of viral titer corresponded with increased ISG mRNA levels (MX1 and OAS1), with the highest inhibition occurring at ISG's peak expression. Presence of IFN-negative regulators, SOCS1 and SOCS3, during DENV-2 infection was associated with reduced IFN-λ1 expression. Evidence described here suggests that IFN-λ is a good candidate inhibitor of viral replication in dengue infection. Mechanisms for the cellular and organismal interplay between DENV and IFN- λ need to be further studied as they could provide insights into strategies to treat this disease. Furthermore, we report a novel epithelial model to study dengue infection in vitro.

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters.

PubMed

Baseler, Laura; Scott, Dana P; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz; de Wit, Emmie

2016-11-01

Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology.

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.

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.

The Role of Electron Microscopy in Studying the Continuum of Changes in Membranous Structures during Poliovirus Infection

PubMed Central

Rossignol, Evan D.; Yang, Jie E.; Bullitt, Esther

2015-01-01

Replication of the poliovirus genome is localized to cytoplasmic replication factories that are fashioned out of a mixture of viral proteins, scavenged cellular components, and new components that are synthesized within the cell due to viral manipulation/up-regulation of protein and phospholipid synthesis. These membranous replication factories are quite complex, and include markers from multiple cytoplasmic cellular organelles. This review focuses on the role of electron microscopy in advancing our understanding of poliovirus RNA replication factories. Structural data from the literature provide the basis for interpreting a wide range of biochemical studies that have been published on virus-induced lipid biosynthesis. In combination, structural and biochemical experiments elucidate the dramatic membrane remodeling that is a hallmark of poliovirus infection. Temporal and spatial membrane modifications throughout the infection cycle are discussed. Early electron microscopy studies of morphological changes following viral infection are re-considered in light of more recent data on viral manipulation of lipid and protein biosynthesis. These data suggest the existence of distinct subcellular vesicle populations, each of which serves specialized roles in poliovirus replication processes. PMID:26473912

Rapid and highly fieldable viral diagnostic

DOEpatents

McKnight, Timothy E.

2016-12-20

The present invention relates to a rapid, highly fieldable, nearly reagentless diagnostic to identify active RNA viral replication in a live, infected cells, and more particularly in leukocytes and tissue samples (including biopsies and nasal swabs) using an array of a plurality of vertically-aligned nanostructures that impale the cells and introduce a DNA reporter construct that is expressed and amplified in the presence of active viral replication.

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.

Kinetics of liver macrophages (Kupffer cells) in SIV-infected macaques

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

Ahsan, Muhammad H.; Gill, Amy F.; Alvarez, Xavier

Since the liver drains antigens from the intestinal tract, and since the intestinal tract is a major site of viral replication, we examined the dynamics of liver macrophages (Kupffer cells) throughout SIV infection. Absolute numbers of Kupffer cells increased in the livers in acute infection, and in animals with AIDS. Significantly higher percentages of proliferating (BrdU+) Kupffer cells were detected in acute infection and in AIDS with similar trends in blood monocytes. Significantly higher percentages of apoptotic (AC3+) Kupffer cells were also found in acute and AIDS stages. However, productively infected cells were not detected in liver of 41/42 animalsmore » examined, despite abundant infected cells in gut and lymph nodes of all animals. Increased rates of Kupffer cell proliferation resulting in an increase in Kupffer cells without productive infection indicate SIV infection affects Kupffer cells, but the liver does not appear to be a major site of productive viral replication. - Highlights: • Kupffer cells increase in the liver of SIV-infected macaques. • Increased proliferation and apoptosis of Kupffer cells occurs in SIV infection. • Productively infected cells are rarely detected in the liver. • The liver is not a major site for SIV replication.« less

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

PubMed

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

2018-01-01

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

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.

Safety mechanism assisted by the repressor of tetracycline (SMART) vaccinia virus vectors for vaccines and therapeutics.

PubMed

Grigg, Patricia; Titong, Allison; Jones, Leslie A; Yilma, Tilahun D; Verardi, Paulo H

2013-09-17

Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.

The logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes

PubMed Central

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-01-01

Abstract Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. PMID:27112572

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

HSV-1-induced activation of NF-κB protects U937 monocytic cells against both virus replication and apoptosis

PubMed Central

Marino-Merlo, Francesca; Papaianni, Emanuela; Medici, Maria Antonietta; Macchi, Beatrice; Grelli, Sandro; Mosca, Claudia; Borner, Christoph; Mastino, Antonio

2016-01-01

The transcription factor nuclear factor-kappa B (NF-κB) is a crucial player of the antiviral innate response. Intriguingly, however, NF-κB activation is assumed to favour herpes simplex virus (HSV) infection rather than restrict it. Apoptosis, a form of innate response to viruses, is completely inhibited by HSV in fully permissive cells, but not in cells incapable to fully sustain HSV replication, such as immunocompetent cells. To resolve the intricate interplay among NF-κB signalling, apoptosis and permissiveness to HSV-1 in monocytic cells, we utilized U937 monocytic cells in which NF-κB activation was inhibited by expressing a dominant-negative IκBα. Surprisingly, viral production was increased in monocytic cells in which NF-κB was inhibited. Moreover, inhibition of NF-κB led to increased apoptosis following HSV-1 infection, associated with lysosomal membrane permeabilization. High expression of late viral proteins and induction of apoptosis occurred in distinct cells. Transcriptional analysis of known innate response genes by real-time quantitative reverse transcription-PCR excluded a contribution of the assayed genes to the observed phenomena. Thus, in monocytic cells NF-κB activation simultaneously serves as an innate process to restrict viral replication as well as a mechanism to limit the damage of an excessive apoptotic response to HSV-1 infection. This finding may clarify mechanisms controlling HSV-1 infection in monocytic cells. PMID:27584793

Bioactive activities of natural products against herpesvirus infection.

PubMed

Son, Myoungki; Lee, Minjung; Sung, Gi-Ho; Lee, Taeho; Shin, Yu Su; Cho, Hyosun; Lieberman, Paul M; Kang, Hyojeung

2013-10-01

More than 90% of adults have been infected with at least one human herpesvirus, which establish long-term latent infection for the life of the host. While anti-viral drugs exist that limit herpesvirus replication, many of these are ineffective against latent infection. Moreover, drug-resistant strains of herpesvirus emerge following chemotherapeutic treatment. For example, resistance to acyclovir and related nucleoside analogues can occur when mutations arise in either HSV thymidine kinase or DNA polymerases. Thus, there exists an unmet medical need to develop new anti-herpesvirus agents with different mechanisms of action. In this Review, we discuss the promise of anti-herpetic substances derived from natural products including extracts and pure compounds from potential herbal medicines. One example is Glycyrrhizic acid isolated from licorice that shows promising antiviral activity towards human gammaherpesviruses. Secondly, we discuss anti-herpetic mechanisms utilized by several natural products in molecular level. While nucleoside analogues inhibit replicating herpesviruses in lytic replication, some natural products can disrupt the herpesvirus latent infection in the host cell. In addition, natural products can stimulate immune responses against herpesviral infection. These findings suggest that natural products could be one of the best choices for development of new treatments for latent herpesvirus infection, and may provide synergistic anti-viral activity when supplemented with nucleoside analogues. Therefore, it is important to identify which natural products are more efficacious anti-herpetic agents, and to understand the molecular mechanism in detail for further advance in the anti-viral therapies.

Viral drug sensitivity testing using quantitative PCR: effect of tyrosine kinase inhibitors on polyomavirus BK replication.

PubMed

Randhawa, Parmjeet S; Farasati, Noush A; Huang, Yuchen; Mapara, Markus Y; Shapiro, Ron

2010-12-01

Our objective was to determine whether quantitative polymerase chain reaction (PCR) can be used to measure the effect of tyrosine kinase (TK) inhibition on polyomavirus BK (BKV) replication. The BKV was grown in a cell culture system. The rate of viral replication in the presence or absence of the drug being tested was assessed by amplifying the viral genome using primers directed against the viral capsid 1 protein. Dasatinib, erlotinib, gefitinib, imatinib, sunitinib, and sorafenib all showed antiviral activity at micromolar concentrations. The 50% effective concentration for erlotinib and sorafenib was within blood concentrations readily achieved in human subjects. Quantitative PCR is a convenient method for viral drug sensitivity testing for slow-growing viruses that do not readily produce cytopathic effect. TK inhibitors deserve further consideration as a potential therapeutic option for BKV-associated nephropathy and hemorrhagic cystitis.

Protease-Mediated Maturation of HIV: Inhibitors of Protease and the Maturation Process.

PubMed

Adamson, Catherine S

2012-01-01

Protease-mediated maturation of HIV-1 virus particles is essential for virus infectivity. Maturation occurs concomitant with immature virus particle release and is mediated by the viral protease (PR), which sequentially cleaves the Gag and Gag-Pol polyproteins into mature protein domains. Maturation triggers a second assembly event that generates a condensed conical capsid core. The capsid core organizes the viral RNA genome and viral proteins to facilitate viral replication in the next round of infection. The fundamental role of proteolytic maturation in the generation of mature infectious particles has made it an attractive target for therapeutic intervention. Development of small molecules that target the PR active site has been highly successful and nine protease inhibitors (PIs) have been approved for clinical use. This paper provides an overview of their development and clinical use together with a discussion of problems associated with drug resistance. The second-half of the paper discusses a novel class of antiretroviral drug termed maturation inhibitors, which target cleavage sites in Gag not PR itself. The paper focuses on bevirimat (BVM) the first-in-class maturation inhibitor: its mechanism of action and the implications of naturally occurring polymorphisms that confer reduced susceptibility to BVM in phase II clinical trials.

Chronic and persistent viral hemorrhagic septicemia virus infections in Pacific herring

USGS Publications Warehouse

Hershberger, P.K.; Gregg, J.L.; Grady, C.A.; Taylor, L.; Winton, J.R.

2010-01-01

Chronic viral hemorrhagic septicemia virus (VHSV) infections were established in a laboratory stock of Pacific herring Clupea pallasii held in a large-volume tank supplied with pathogenfree seawater at temperatures ranging from 6.8 to 11.6??C. The infections were characterized by viral persistence for extended periods and near-background levels of host mortality. Infectious virus was recovered from mortalities occurring up to 167 d post-exposure and was detected in normal-appearing herring for as long as 224 d following initial challenge. Geometric mean viral titers were generally as high as or higher in brain tissues than in pools of kidney and spleen tissues, with overall prevalence of infection being higher in the brain. Upon re-exposure to VHSV in a standard laboratory challenge, negligible mortality occurred among groups of herring that were either chronically infected or fully recovered, indicating that survival from chronic manifestations conferred protection against future disease. However, some survivors of chronic VHS infections were capable of replicating virus upon re-exposure. Demonstration of a chronic manifestation of VHSV infection among Pacific herring maintained at ambient seawater temperatures provides insights into the mechanisms by which the virus is maintained among populations of endemic hosts. ?? 2010 Inter-Research.

Chronic and persistent viral hemorrhagic septicemia virus infections in Pacific herring

USGS Publications Warehouse

Hershberger, Paul K.; Gregg, Jacob L.; Winton, James R.; Grady, Cortney A.; Taylor, L.

2010-01-01

Chronic viral hemorrhagic septicemia virus (VHSV) infections were established in a laboratory stock of Pacific herring Clupea pallasii held in a large-volume tank supplied with pathogen-free seawater at temperatures ranging from 6.8 to 11.6°C. The infections were characterized by viral persistence for extended periods and near-background levels of host mortality. Infectious virus was recovered from mortalities occurring up to 167 d post-exposure and was detected in normal-appearing herring for as long as 224 d following initial challenge. Geometric mean viral titers were generally as high as or higher in brain tissues than in pools of kidney and spleen tissues, with overall prevalence of infection being higher in the brain. Upon re-exposure to VHSV in a standard laboratory challenge, negligible mortality occurred among groups of herring that were either chronically infected or fully recovered, indicating that survival from chronic manifestations conferred protection against future disease. However, some survivors of chronic VHS infections were capable of replicating virus upon re-exposure. Demonstration of a chronic manifestation of VHSV infection among Pacific herring maintained at ambient seawater temperatures provides insights into the mechanisms by which the virus is maintained among populations of endemic hosts.

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.

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

PubMed Central

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

2015-01-01

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

SIRT1 inhibits EV71 genome replication and RNA translation by interfering with the viral polymerase and 5′UTR RNA

PubMed Central

Han, Yang; Wang, Lvyin; Cui, Jin; Song, Yu; Luo, Zhen; Chen, Junbo; Xiong, Ying; Zhang, Qi; Liu, Fang; Ho, Wenzhe; Liu, Yingle; Wu, Jianguo

2016-01-01

ABSTRACT Enterovirus 71 (EV71) possesses a single-stranded positive RNA genome that contains a single open reading frame (ORF) flanked by a 5′ untranslated region (5′UTR) and a polyadenylated 3′UTR. Here, we demonstrated that EV71 activates the production of silent mating type information regulation 2 homolog 1 (SIRT1), a histone deacetylase (HDAC). EV71 further stimulates SIRT1 sumoylation and deacetylase activity, and enhances SIRT1 translocation from the nucleus to the cytoplasm. More interestingly, activated SIRT1 subsequently binds with the EV71 3Dpol protein (a viral RNA-dependent RNA polymerase, RdRp) to repress the acetylation and RdRp activity of 3Dpol, resulting in the attenuation of viral genome replication. Moreover, SIRT1 interacts with the cloverleaf structure of the EV71 RNA 5′UTR to inhibit viral RNA transcription, and binds to the internal ribosome entry site (IRES) of the EV71 5′UTR to attenuate viral RNA translation. Thus, EV71 stimulates SIRT1 production and activity, which in turn represses EV71 genome replication by inhibiting viral polymerase, and attenuates EV71 RNA transcription and translation by interfering with viral RNA. These results uncover a new function of SIRT1 and reveal a new mechanism underlying the regulation of EV71 replication. PMID:27875274

Molecular Detection of Tobacco rattle virus in Bleeding Heart [Dicentra spectabilis (L.) Lem.] Growing in Alaska

USDA-ARS?s Scientific Manuscript database

Tobacco rattle virus (TRV) is a single-stranded positive RNA plant virus that in nature may occur as M-type isolates with RNA1 and RNA2, or as NM-type isolates with only RNA1. RNA1 is responsible for viral replication and transmission, while the formation of the coat protein (CP) is dependent on RNA...

Transcriptome analysis of the Spodoptera frugiperda ascovirus in vivo provides insights into how its apoptosis inhibitors and caspase promote increased synthesis of viral vesicles and virion progeny.

PubMed

Zaghloul, Heba; Hice, Robert; Arensburger, Peter; Federici, Brian A

2017-09-27

Ascoviruses are ds DNA viruses that attack caterpillars and differ from all other viruses by inducing nuclear lysis followed by cleavage of host cells into numerous anucleate vesicles in which virus replication continues as these grow in the blood. Ascoviruses are also unusual in that most encode apoptosis inhibitors and caspase or caspase-like proteins. A robust cell line to study the novel molecular biology of ascovirus replication in vitro is lacking. Therefore, we used strand-specific RNA-Seq to study transcription in vivo in third instars of Spodoptera frugiperda infected with the Spodoptera frugiperda ascovirus, a member of the type species, Spodoptera frugiperda ascovirus (SfAV-1a), sampling transcripts at different time points after infection. We targeted transcription of two types of SfAV-1a genes; first, 44 core genes that occur in several ascovirus species, and second, 26 genes predicted in silico to have metabolic functions likely involved in synthesizing viral vesicle membranes. Gene cluster analysis showed differences in temporal expression of SfAV-1a genes, enabling their assignment to three temporal classes; early, late and very late. Inhibitors of apoptosis (IAP-like proteins; ORF016, ORF025 and ORF074) were expressed early, whereas its caspase (ORF073) was expressed very late, which correlated with apoptotic events leading to viral vesicle formation. Expression analysis revealed that a Diedel gene homolog (ORF121), the only known "virokine," was highly expressed, implying this ascovirus protein helps evade innate host immunity. Lastly, single-nucleotide resolution of RNA-Seq data revealed 15 bicistronic and tricistronic messages along the genome, an unusual occurrence for large ds DNA viruses. IMPORTANCE Unlike all other DNA viruses, ascoviruses code for an executioner caspase, apparently involved in a novel cytopathology in which viral replication induces nuclear lysis followed by cell cleavage yielding numerous large anucleate viral vesicles that continue to produce virions. Our transcriptome analysis of genome expression in vivo by the Spodoptera frugiperda ascovirus shows that inhibitors of apoptosis are expressed first enabling viral replication to proceed, after which the SfAV-1a caspase is synthesized, leading to viral vesicle synthesis and subsequent extensive production of progeny virions. Moreover, we detected numerous bicistronic and tricistronic mRNA messages in the ascovirus transcriptome, implying ascoviruses use other non-canonical translational mechanisms such as Internal Ribosome Entry Site (IRES). These results provide the first insights into the molecular biology of a unique coordinated gene expression pattern in which cell architecture is markedly modified, more than in any other known eukaryotic virus, to promote viral reproduction and transmission. Copyright © 2017 American Society for Microbiology.

In vitro anti-reovirus activity of kuraridin isolated from Sophora flavescens against viral replication and hemagglutination.

PubMed

Kwon, Hyung-Jun; Jeong, Jae-Ho; Lee, Seung Woong; Ryu, Young Bae; Jeong, Hyung Jae; Jung, Kyungsook; Lim, Jae Sung; Cho, Kyoung-Oh; Lee, Woo Song; Rho, Mun-Chual; Park, Su-Jin

2015-08-01

In this study, we evaluated the anti-reovirus activity of kuraridin isolated from the roots of Sophora flavescens. In particular, we focused on whether this property is attributable to direct inhibition of reovirus attachment and/or inhibition of viral replication with the aid of time-of-addition (pre-treatment, simultaneous treatment, and post-treatment) experiments. No significant antiviral activity of kuraridin was detected in the pre-treatment assay. In the simultaneous assay, the 50% effective inhibitory concentrations (EC50) of kuraridin were 15.3-176.9 μM against human type 1-3 reoviruses (HRV1-3) and Korean porcine reovirus (PRV). Kuraridin completely blocked binding of viral sigma 1 protein to sialic acids at concentrations lower than 82.5 μM in the hemagglutination inhibition assay. Moreover, kuraridin inhibited HRV1-3 and PRV viral replication with EC50 values of 14.0-62.0 μM. Quantitative real-time PCR analysis disclosed strong suppression of reovirus RNA synthesis at the late stage (18 h) of virus replication by kuraridin. The viral yields of kuraridin-treated cells were significantly reduced at 24 h post-infection, compared with DMSO-treated cells. Our results collectively suggest that kuraridin inhibits virus adsorption and replication by inhibiting hemagglutination, viral RNA and protein synthesis and virus shedding, supporting its utility as a viable candidate antiviral drug against reoviruses. Copyright © 2015 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

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.

Adenovirus Core Protein VII Protects the Viral Genome from a DNA Damage Response at Early Times after Infection▿

PubMed Central

Karen, Kasey A.; Hearing, Patrick

2011-01-01

Adenovirus has a linear, double-stranded DNA genome that is perceived by the cellular Mre11-Rad50-Nbs1 (MRN) DNA repair complex as a double-strand break. If unabated, MRN elicits a double-strand break repair response that blocks viral DNA replication and ligates the viral genomes into concatemers. There are two sets of early viral proteins that inhibit the MRN complex. The E1B-55K/E4-ORF6 complex recruits an E3 ubiquitin ligase and targets MRN proteins for proteasome-dependent degradation. The E4-ORF3 protein inhibits MRN through sequestration. The mechanism that prevents MRN recognition of the viral genome prior to the expression of these early proteins was previously unknown. Here we show a temporal correlation between the loss of viral core protein VII from the adenovirus genome and a gain of checkpoint signaling due to the double-strand break repair response. While checkpoint signaling corresponds to the recognition of the viral genome, core protein VII binding to and checkpoint signaling at viral genomes are largely mutually exclusive. Transcription is known to release protein VII from the genome, and the inhibition of transcription shows a decrease in checkpoint signaling. Finally, we show that the nuclease activity of Mre11 is dispensable for the inhibition of viral DNA replication during a DNA damage response. These results support a model involving the protection of the incoming viral genome from checkpoint signaling by core protein VII and suggest that the induction of an MRN-dependent DNA damage response may inhibit adenovirus replication by physically masking the origins of DNA replication rather than altering their integrity. PMID:21345950

Viral Replication Complexes Are Targeted by LC3-Guided Interferon-Inducible GTPases.

PubMed

Biering, Scott B; Choi, Jayoung; Halstrom, Rachel A; Brown, Hailey M; Beatty, Wandy L; Lee, Sanghyun; McCune, Broc T; Dominici, Erin; Williams, Lelia E; Orchard, Robert C; Wilen, Craig B; Yamamoto, Masahiro; Coers, Jörn; Taylor, Gregory A; Hwang, Seungmin

2017-07-12

All viruses with positive-sense RNA genomes replicate on membranous structures in the cytoplasm called replication complexes (RCs). RCs provide an advantageous microenvironment for viral replication, but it is unknown how the host immune system counteracts these structures. Here we show that interferon-gamma (IFNG) disrupts the RC of murine norovirus (MNV) via evolutionarily conserved autophagy proteins and the induction of IFN-inducible GTPases, which are known to destroy the membrane of vacuoles containing bacteria, protists, or fungi. The MNV RC was marked by the microtubule-associated-protein-1-light-chain-3 (LC3) conjugation system of autophagy and then targeted by immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs) upon their induction by IFNG. Further, the LC3 conjugation system and the IFN-inducible GTPases were necessary to inhibit MNV replication in mice and human cells. These data suggest that viral RCs can be marked and antagonized by a universal immune defense mechanism targeting diverse pathogens replicating in cytosolic membrane structures. Copyright © 2017 Elsevier Inc. All rights reserved.

THE E1 PROTEINS

PubMed Central

Bergvall, Monika; Melendy, Thomas; Archambault, Jacques

2013-01-01

E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner. PMID:24029589

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.

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

HIV life cycle, innate immunity and autophagy in the central nervous system.

PubMed

Meulendyke, Kelly A; Croteau, Joshua D; Zink, M Christine

2014-11-01

In this era of modern combination antiretroviral therapy (cART) HIV-associated neurocognitive disorders (HAND) continue to affect a large portion of the infected population. In this review, we highlight recent discoveries that help to define the interplay between HIV life cycle, the innate immune system and cellular autophagy in the context of the central nervous system (CNS). Investigators have recently elucidated themes in HAND, which place it in a unique framework. Cells of macrophage lineage and probably astrocytes play a role in disseminating virus through the CNS. Each of these cell types responds to a diverse population of constantly evolving virus existing in an inflammatory environment. This occurs though the failure of both host antiviral mechanisms, such as autophagy, and innate immunological signalling pathways to control viral replication. The newest findings detailed in this review help define why HIV CNS disease is a difficult target for therapeutics and create hope that these new mechanisms may be exploited to attenuate viral replication and eliminate disease.

The second chance story of HIV-1 DNA: Unintegrated? Not a problem!

PubMed

Wu, Yuntao

2008-07-09

Accumulation of high levels of unintegrated viral DNA is a common feature of retroviral infection. It was recently discovered that coinfection of cells with integrated and unintegrated HIV-1 can result in complementation, allowing viral replication in the absence of integration. This new mode of HIV-1 replication has numerous implications for the function of unintegrated viral DNA and its application as a therapeutic vector.

HIV-1 Nef protein in the nucleus influences adipogenesis as well as viral transcription through the peroxisome proliferator-activated receptors.

PubMed

Otake, Kaori; Omoto, Shinya; Yamamoto, Takuya; Okuyama, Harumi; Okada, Hidechika; Okada, Noriko; Kawai, Masahiro; Saksena, Nitin K; Fujii, Yoichi R

2004-01-23

Although the HIV-1 Nef protein (27 kDa) localizes primarily in cytoplasm, there is considerable evidence suggesting its occasional localization in the nucleus. Nef is known to play an important role in transcriptional events and viral replication, but the actual target of Nef in the nucleus remains to be identified. To examine the functional roles of Nef in the nucleus and its possible interactions with other unknown factors in the nucleus. High-density microarray analysis was used to screen directly the unique functions of Nef on host gene transcription. The nuclear localization of Nef and its effects on the expression of peroxisome proliferator-activated receptors (PPAR) was examined using PPAR promoter/reporter assay and immunoblotting. A long terminal repeat/reporter assay was used to investigated the effects of Nef and PPAR on viral transcription. Nef in the nucleus suppressed PPAR gamma expression and reduced fatty acid levels in human T and macrophage cell lines. Expression of Nef or PPAR suppressed viral replication; the effect of PPAR gamma or retinoid X receptor-alpha on viral replication were reduced by coexpression of Nef in MT(-)4 T cells. Nef may be involved in both viral replication and the wasting syndrome associated with AIDS.

Efficacy of hepatitis B virus ribonuclease H inhibitors, a new class of replication antagonists, in FRG human liver chimeric mice.

PubMed

Long, Kelly R; Lomonosova, Elena; Li, Qilan; Ponzar, Nathan L; Villa, Juan A; Touchette, Erin; Rapp, Stephen; Liley, R Matt; Murelli, Ryan P; Grigoryan, Alexandre; Buller, R Mark; Wilson, Lisa; Bial, John; Sagartz, John E; Tavis, John E

2018-01-01

Chronic hepatitis B virus infection cannot be cured by current therapies, so new treatments are urgently needed. We recently identified novel inhibitors of the hepatitis B virus ribonuclease H that suppress viral replication in cell culture. Here, we employed immunodeficient FRG KO mice whose livers had been engrafted with primary human hepatocytes to ask whether ribonuclease H inhibitors can suppress hepatitis B virus replication in vivo. Humanized FRG KO mice infected with hepatitis B virus were treated for two weeks with the ribonuclease H inhibitors #110, an α-hydroxytropolone, and #208, an N-hydroxypyridinedione. Hepatitis B virus viral titers and S and e antigen plasma levels were measured. Treatment with #110 and #208 caused significant reductions in plasma viremia without affecting hepatitis B virus S or e antigen levels, and viral titers rebounded following treatment cessation. This is the expected pattern for inhibitors of viral DNA synthesis. Compound #208 suppressed viral titers of both hepatitis B virus genotype A and C isolates. These data indicate that Hepatitis B virus replication can be suppressed during infection in an animal by inhibiting the viral ribonuclease H, validating the ribonuclease H as a novel target for antiviral drug development. Copyright © 2017 Elsevier B.V. All rights reserved.

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

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.

In vitro infection of salmonid epidermal tissues by infectious hematopoietic necrosis virus and viral hemorrhagic septicemia virus

USGS Publications Warehouse

Yamamoto, T.; Batts, W.N.; Winton, J.R.

1992-01-01

The ability of two rhabdoviruses, infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV), to infect fish skin was investigated by in vitro infection of excised tissues. Virus replication was determined by plaque assay of homogenized tissue extracts, and the virus antigen was detected by immunohistology of tissue sections. Gill, fin, and ventral abdominal skin tissues of rainbow trout Oncorhynchus mykiss that had been infected in vitro with a virulent strain of IHNV (193–110) produced substantial increases in virus titer within 24 h. Titers continued to increase up until day 3 of incubation; by this time, virus had increased 1,000-fold or more. This increase in IHNV titer occurred in epidermal tissues of fingerlings and of older fish. In another experiment, IHNV replicated in excised rainbow trout tissues whether the fish had been subject to prior infection with a virulent strain of IHNV (Western Regional Aquaculture Consortium isolate) or whether the fish had been infected previously with an attenuated strain of the virus (Nan Scott Lake, with 100 passes in culture). A virulent strain of VHSV (23/75) replicated effectively in excised gill tissues and epidermal tissues of rainbow trout and chinook salmon O. tshawytscha; however, the avirulent North American strain of VHSV (Makah) replicated poorly or not at all.

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

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

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.

Construction of a subgenomic CV-B3 replicon expressing emerald green fluorescent protein to assess viral replication of a cardiotropic enterovirus strain in cultured human cells.

PubMed

Wehbe, Michel; Huguenin, Antoine; Leveque, Nicolas; Semler, Bert L; Hamze, Monzer; Andreoletti, Laurent; Bouin, Alexis

2016-04-01

Coxsackieviruses B (CV-B) (Picornaviridae) are a common infectious cause of acute myocarditis in children and young adults, a disease, which is a precursor to 10-20% of chronic myocarditis and dilated cardiomyopathy (DCM) cases. The mechanisms involved in the disease progression from acute to chronic myocarditis phase and toward the DCM clinical stage are not fully understood but are influenced by both viral and host factors. Subgenomic replicons of CV-B can be used to assess viral replication mechanisms in human cardiac cells and evaluate the effects of potential antiviral drugs on viral replication activities. Our objectives were to generate a reporter replicon from a cardiotropic prototype CV-B3/28 strain and to characterize its replication properties into human cardiac primary cells. To obtain this replicon, a cDNA plasmid containing the full CV-B3/28 genome flanked by a hammerhead ribozyme sequence and an MluI restriction site was generated and used as a platform for the insertion of sequences encoding emerald green fluorescent protein (EmGFP) in place of those encoding VP3. In vitro transcribed RNA from this plasmid was transfected into HeLa cells and human primary cardiac cells and was able to produce EmGFP and VP1-containing polypeptides. Moreover, non-structural protein biological activity was assessed by the specific cleavage of eIF4G1 by viral 2A(pro). Viral RNA replication was indirectly demonstrated by inhibition assays, fluoxetine was added to cell culture and prevented the EmGFP synthesis. Our results indicated that the EmGFP CV-B3 replicon was able to replicate and translate as well as the CV-B3/28 prototype strain. Our EmGFP CV-B3 replicon will be a valuable tool to readily investigate CV-B3 replication activities in human target cell models. Copyright © 2016 Elsevier B.V. All rights reserved.

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.

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

Poliovirus Proteins Induce Membrane Association of GTPase ADP-Ribosylation Factor

PubMed Central

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

2005-01-01

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

Opposite Roles of RNase and Kinase Activities of Inositol-Requiring Enzyme 1 (IRE1) on HSV-1 Replication

PubMed Central

Su, Airong; Wang, Huanru; Li, Yanlei; Wang, Xiaohui; Chen, Deyan; Wu, Zhiwei

2017-01-01

In response to the endoplasmic reticulum (ER) stress induced by herpes simplex virus type 1 (HSV-1) infection, host cells activate the unfolded protein response (UPR) to reduce the protein-folding burden in the ER. The regulation of UPR upon HSV-1 infection is complex, and the downstream effectors can be detrimental to viral replication. Therefore, HSV-1 copes with the UPR to create a beneficial environment for its replication. UPR has three branches, including protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activated transcription factor 6 (ATF6). IRE1α is the most conserved branch of UPR which has both RNase and kinase activities. Previous studies have shown that IRE1α RNase activity was inactivated during HSV-1 infection. However, the effect of the two activities of IRE1α on HSV-1 replication remains unknown. Results in this study showed that IRE1α expression was up-regulated during HSV-1 infection. We found that in HEC-1-A cells, increasing RNase activity, or inhibiting kinase activity of IRE1α led to viral suppression, indicating that the kinase activity of IRE1α was beneficial, while the RNase activity was detrimental to viral replication. Further evidence showed that the kinase activity of IRE1α leads to the activation of the JNK (c-Jun N-terminal kinases) pathway, which enhances viral replication. Taken together, our evidence suggests that IRE1α is involved in HSV-1 replication, and its RNase and kinase activities play differential roles during viral infection. PMID:28832521

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.

Association between HIV replication and serum leptin levels: an observational study of a cohort of HIV-1-infected South African women

PubMed Central

2010-01-01

Background Advanced HIV infection can result in lipoatrophy and wasting, even in the absence of ongoing opportunistic infections, suggesting that HIV may directly affect adipose tissue amount and distribution. Methods We assessed the relationship of fat (measured using anthropometry, DEXA, MRI scans) or markers related to glucose and lipid metabolism with viral load in a cross-sectional sample of 83 antiretroviral-naïve HIV-1-infected South African women. A multivariable linear model was fitted to log10VL to assess the combined effect of these variables. Results In addition to higher T cell activation, women with viral load greater than the population median had lower waist circumference, body mass index and subcutaneous abdominal fat, as well as lower serum leptin. We demonstrate that leptin serum levels are inversely associated with viral replication, independent of the amount of adipose tissue. This association is maintained after adjusting for multiple variables associated with disease progression (i.e., cellular activation and innate immunity effector levels). Conclusions Our results demonstrate that serum leptin levels are inversely associated with viral replication, independent of disease progression: we postulate that leptin may affect viral replication. PMID:20822522

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 logic of DNA replication in double-stranded DNA viruses: insights from global analysis of viral genomes.

PubMed

Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

2016-06-02

Genomic DNA replication is a complex process that involves multiple proteins. Cellular DNA replication systems are broadly classified into only two types, bacterial and archaeo-eukaryotic. In contrast, double-stranded (ds) DNA viruses feature a much broader diversity of DNA replication machineries. Viruses differ greatly in both completeness and composition of their sets of DNA replication proteins. In this study, we explored whether there are common patterns underlying this extreme diversity. We identified and analyzed all major functional groups of DNA replication proteins in all available proteomes of dsDNA viruses. Our results show that some proteins are common to viruses infecting all domains of life and likely represent components of the ancestral core set. These include B-family polymerases, SF3 helicases, archaeo-eukaryotic primases, clamps and clamp loaders of the archaeo-eukaryotic type, RNase H and ATP-dependent DNA ligases. We also discovered a clear correlation between genome size and self-sufficiency of viral DNA replication, the unanticipated dominance of replicative helicases and pervasive functional associations among certain groups of DNA replication proteins. Altogether, our results provide a comprehensive view on the diversity and evolution of replication systems in the DNA virome and uncover fundamental principles underlying the orchestration of viral DNA replication. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Hepatitis B Virus Core Protein Dephosphorylation Occurs during Pregenomic RNA Encapsidation.

PubMed

Zhao, Qiong; Hu, Zhanying; Cheng, Junjun; Wu, Shuo; Luo, Yue; Chang, Jinhong; Hu, Jianming; Guo, Ju-Tao

2018-07-01

Hepatitis B virus (HBV) core protein consists of an N-terminal assembly domain and a C-terminal domain (CTD) with seven conserved serines or threonines that are dynamically phosphorylated/dephosphorylated during the viral replication cycle. Sulfamoylbenzamide derivatives are small molecular core protein allosteric modulators (CpAMs) that bind to the heteroaryldihydropyrimidine (HAP) pocket between the core protein dimer-dimer interfaces. CpAM binding alters the kinetics and pathway of capsid assembly and can result in the formation of morphologically "normal" capsids devoid of viral pregenomic RNA (pgRNA) and DNA polymerase. In order to investigate the mechanism underlying CpAM inhibition of pgRNA encapsidation, we developed an immunoblotting assay that can resolve core protein based on its phosphorylation status and demonstrated, for the first time, that core protein is hyperphosphorylated in free dimers and empty capsids from both mock-treated and CpAM-treated cells but is hypophosphorylated in pgRNA- and DNA-containing nucleocapsids. Interestingly, inhibition of pgRNA encapsidation by a heat shock protein 90 (HSP90) inhibitor prevented core protein dephosphorylation. Moreover, core proteins with point mutations at the wall of the HAP pocket, V124A and V124W, assembled empty capsids and nucleocapsids with altered phosphorylation status. The results thus suggest that core protein dephosphorylation occurs in the assembly of pgRNA and that interference with the interaction between core protein subunits at dimer-dimer interfaces during nucleocapsid assembly alters not only capsid structure, but also core protein dephosphorylation. Hence, inhibition of pgRNA encapsidation by CpAMs might be due to disruption of core protein dephosphorylation during nucleocapsid assembly. IMPORTANCE Dynamic phosphorylation of HBV core protein regulates multiple steps of viral replication. However, the regulatory function was mainly investigated by phosphomimetic mutagenesis, which disrupts the natural dynamics of core protein phosphorylation/dephosphorylation. Development of an immunoblotting assay capable of resolving hyper- and hypophosphorylated core proteins allowed us to track the phosphorylation status of core proteins existing as free dimers and the variety of intracellular capsids and to investigate the role of core protein phosphorylation/dephosphorylation in viral replication. Here, we found that disruption of core protein interaction at dimer-dimer interfaces during nucleocapsid assembly (by CpAMs or mutagenesis) inhibited core protein dephosphorylation and pgRNA packaging. Our work has thus revealed a novel function of core protein dephosphorylation in HBV replication and the mechanism by which CpAMs, a class of compounds that are currently in clinical trials for treatment of chronic hepatitis B, induce the assembly of empty capsids. Copyright © 2018 American Society for Microbiology.

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.

Inhibition of coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand.

PubMed

Yuan, Ji; Cheung, Paul K M; Zhang, Huifang M; Chau, David; Yang, Decheng

2005-02-01

Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic potential. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4 was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence match in the central region of the target, but mismatches were more tolerated near the 3' end than the 5' end of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new possibility for antiviral intervention.

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.

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.

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

PubMed

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

2018-01-01

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

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

PubMed Central

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

2018-01-01

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

Inhibition of rotavirus replication by downregulation of fatty acid synthesis.

PubMed

Gaunt, Eleanor R; Cheung, Winsome; Richards, James E; Lever, Andrew; Desselberger, Ulrich

2013-06-01

Recently the recruitment of lipid droplets (LDs) to sites of rotavirus (RV) replication was reported. LDs are polymorphic organelles that store triacylglycerols, cholesterol and cholesterol esters. The neutral fats are derived from palmitoyl-CoA, synthesized via the fatty acid biosynthetic pathway. RV-infected cells were treated with chemical inhibitors of the fatty acid biosynthetic pathway, and the effects on viral replication kinetics were assessed. Treatment with compound C75, an inhibitor of the fatty acid synthase enzyme complex (FASN), reduced RV infectivity 3.2-fold (P = 0.07) and modestly reduced viral RNA synthesis (1.2-fold). Acting earlier in the fatty acid synthesis pathway, TOFA [5-(Tetradecyloxy)-2-furoic acid] inhibits the enzyme acetyl-CoA carboxylase 1 (ACC1). TOFA reduced the infectivity of progeny RV 31-fold and viral RNA production 6-fold. The effect of TOFA on RV infectivity and RNA replication was dose-dependent, and infectivity was reduced by administering TOFA up to 4 h post-infection. Co-treatment of RV-infected cells with C75 and TOFA synergistically reduced viral infectivity. Knockdown by siRNA of FASN and ACC1 produced findings similar to those observed by inhibiting these proteins with the chemical compounds. Inhibition of fatty acid synthesis using a range of approaches uniformly had a more marked impact on viral infectivity than on viral RNA yield, inferring a role for LDs in virus assembly and/or egress. Specific inhibitors of fatty acid metabolism may help pinpoint the critical structural and biochemical features of LDs that are essential for RV replication, and facilitate the development of antiviral therapies.

Suppression of AcMNPV replication by adf and thymosin protein up-regulation in a new testis cell line, Ha-shl-t

USDA-ARS?s Scientific Manuscript database

Host cytoskeletons facilitate the entry, replication and egress of viruses; because cytoskeletons are essential for viral survival, one mechanism of resisting viral infections involves regulating cytoskeletal polymerization/depolymerization. However, the molecular mechanisms of regulating these chan...

Assessing Zika virus replication and the development of Zika-specific antibodies after a mid-gestation viral challenge in guinea pigs

PubMed Central

Fernández-Alarcón, Claudia; Hernandez-Alvarado, Nelmary; Zabeli, Jason C.; Janus, Bradley C.; Putri, Dira S.; Schleiss, Mark R.

2017-01-01

Primary Zika virus (ZIKV) infections that occur during pregnancy can cause spontaneous abortion and profoundly disrupt fetal development. While the full range of developmental abnormalities associated with congenital Zika syndrome is not yet known, severe cases of the syndrome can present with microcephaly, extensive neurologic and ocular damage, and pronounced joint malformations. Animal models that accurately recapitulate congenital Zika syndrome are urgently needed for vaccine development and for the study of ZIKV pathogenesis. As guinea pigs have successfully been used to model transplacental infections by cytomegalovirus, syphilis, and Listeria monocytogenes, we sought to test whether ZIKV could productively infect guinea pigs and whether viral transmission with attendant fetal pathology would occur after a mid-gestation viral challenge. We found that guinea pig cells supported ZIKV replication in vitro. Experimental infection of non-pregnant animals did not result in overt disease but low-level, detectable viremia was observed. When pregnant guinea pigs were challenged with ZIKV at between 18 and 21 days gestational age, ZIKV was not detected in maternal or pup blood, plasma, or tissues and no significant differences in maternal weight gain or pup size were observed following challenge. Nonetheless, a robust antibody response against ZIKV was detected in both the pups and dams. These results suggest that, while guinea pigs can model aspects of the immune response to ZIKV infection during pregnancy, naturally circulating ZIKV strains are not pathogenic during the pregnancy of immunocompetent guinea pigs and do not interfere with normal pup development. PMID:29099873

Inhibition of West Nile Virus replication by retrovirus-delivered small interfering RNA in human neuroblastoma cells.

PubMed

Yang, Yongbo; Wu, Chengxiang; Wu, Jianguo; Nerurkar, Vivek R; Yanagihara, Richard; Lu, Yuanan

2008-05-01

West Nile virus (WNV) has been responsible for the largest outbreaks of arboviral encephalitis in U.S. history. No specific drug is currently available for the effective treatment of WNV infection. To exploit RNA interference as a potential therapeutic approach, a Moloney murine leukemia virus-based retrovirus vector was used to effectively deliver WNV-specific small interfering RNA (siRNA) into human neuroblastoma HTB-11 cells. Viral plaque assays demonstrated that transduced cells were significantly refractory to WNV replication, as compared to untransduced control cells (P < 0.05), which correlated with the reduced expression of target viral genes and respective viral proteins. Therefore, retrovirus-mediated delivery of siRNA for gene silencing can be used to study the specific functions of viral genes associated with replication and may have potential therapeutic applications.

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

PubMed

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

2016-04-01

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

Oncolytic Herpes Simplex Viral Therapy: A Stride toward Selective Targeting of Cancer Cells.

PubMed

Sanchala, Dhaval S; Bhatt, Lokesh K; Prabhavalkar, Kedar S

2017-01-01

Oncolytic viral therapy, which makes use of replication-competent lytic viruses, has emerged as a promising modality to treat malignancies. It has shown meaningful outcomes in both solid tumor and hematologic malignancies. Advancements during the last decade, mainly genetic engineering of oncolytic viruses have resulted in improved specificity and efficacy of oncolytic viruses in cancer therapeutics. Oncolytic viral therapy for treating cancer with herpes simplex virus-1 has been of particular interest owing to its range of benefits like: (a) large genome and power to infiltrate in the tumor, (b) easy access to manipulation with the flexibility to insert multiple transgenes, (c) infecting majority of the malignant cell types with quick replication in the infected cells and (d) as Anti-HSV agent to terminate HSV replication. This review provides an exhaustive list of oncolytic herpes simplex virus-1 along with their genetic alterations. It also encompasses the major developments in oncolytic herpes simplex-1 viral therapy and outlines the limitations and drawbacks of oncolytic herpes simplex viral therapy.

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.

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.

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.

The S230R Integrase Substitution Associated with Viral Rebound during DTG Monotherapy Confers Low Levels INSTI Drug Resistance.

PubMed

Pham, Hanh T; Labrie, Lydia; Wijting, Ingeborg E A; Hassounah, Said; Lok, Ka Yee; Portna, Inna; Goring, Mark; Han, Yingshan; Lungu, Cynthia; van der Ende, Marchina E; Brenner, Bluma G; Boucher, Charles A; Rijnders, Bart J A; van Kampen, Jeroen J A; Mesplède, Thibault; Wainberg, Mark A

2018-03-29

Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of HIV-infected individuals. Due to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virological failure while using DTG monotherapy. We evaluated the effect of S230R substitution in regard to IN enzyme activity, viral infectivity, replicative capacity and susceptibility to different INSTIs by biochemical and cell-based assays. S230R substitution conferred 63% reduction in enzyme efficiency. The S230R virus was 1.29-fold less infectious than wildtype (WT), but could replicate in PM1 cells without significant delay. Resistance levels against DTG, CAB, RAL and EVG in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively. Our data indicate that the S230R substitution is comparable to the previously reported R263K in some respects. Virological failure under DTG monotherapy can occur through the development of such S230R or R263K mutations without the need for high levels DTG resistance.

Inside the lifestyle of the virophage.

PubMed

Desnues, C; Raoult, D

2010-01-01

We sought to better characterize Sputnik, the first isolated virophage, and to analyze its parasitic lifestyle during co-infection with Marseillevirus (a new giant virus) in Acanthamoeba castellanii. A combination of electron microscopy, immunofluorescence microscopy, and real-time PCR was used to characterize the kinetics of the viral replication cycle. RT-PCR was performed to detect RNAs inside the Sputnik virions. Sputnik is a new viral entity carrying an almost complete ready-to-use set of viral RNAs (20 out of 21). Sputnik does not replicate with Marseillevirus but delays its replication cycle. While Marseillevirus is successfully internalized by A. castellanii following co-infections with Mamavirus and Sputnik, it does not initiate a replication cycle. In contrast, both Marseillevirus and Mamavirus can replicate in the amoeba in case of co-infection, but the development of one is exclusive from the other inside a single amoeba cell. This work provides new insight into the Sputnik replication cycle with another giant virus and confirms that Sputnik is a virophage. It shows new dimensions of the interactions existing among giant viruses. Copyright 2010 S. Karger AG, Basel.

Use of a highly sensitive strand-specific quantitative PCR to identify abortive replication in the mouse model of respiratory syncytial virus disease

PubMed Central

2010-01-01

Background The BALB/c mouse is commonly used to study RSV infection and disease. However, despite the many advantages of this well-characterised model, the inoculum is large, viral replication is restricted and only a very small amount of virus can be recovered from infected animals. A key question in this model is the fate of the administered virus. Is replication really being measured or is the model measuring the survival of the virus over time? To answer these questions we developed a highly sensitive strand-specific quantitative PCR (QPCR) able to accurately quantify the amount of RSV replication in the BALB/c mouse lung, allowing characterisation of RSV negative and positive strand RNA dynamics. Results In the mouse lung, no increase in RSV genome was seen above the background of the original inoculum whilst only a limited transient increase (< 1 log) in positive strand, replicative intermediate (RI) RNA occurred. This RNA did however persist at detectable levels for 59 days post infection. As expected, ribavirin therapy reduced levels of infectious virus and RI RNA in the mouse lung. However, whilst Palivizumab therapy was also able to reduce levels of infectious virus, it failed to prevent production of intracellular RI RNA. A comparison of RSV RNA kinetics in human (A549) and mouse (KLN205) cell lines demonstrated that RSV replication was also severely delayed and impaired in vitro in the mouse cells. Conclusions This is the first time that such a sensitive strand-specific QPCR technique has been to the RSV mouse system. We have accurately quantified the restricted and abortive nature of RSV replication in the mouse. Further in vitro studies in human and mouse cells suggest this restricted replication is due at least in part to species-specific host cell-viral interactions. PMID:20860795

Attenuation of virus production at high multiplicities of infection in Aureococcus anophagefferens

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

Brown, Christopher M.; Bidle, Kay D., E-mail: bidle@marine.rutgers.edu

2014-10-15

Infection dynamics (saturation kinetics, infection efficiency, adsorption and burst size) for the Aureococcus anophagefferens-Brown Tide virus (AaV) system were investigated using susceptible and resistant strains. Adsorption assays revealed that virus affinity to the cell surface is a key determinant of infectivity. Saturation of infection occurred at a multiplicity of infection (MOI) of 8 viruses per host and resulted in ∼90–95% of infected cells, with burst sizes ranging from 164 to 191. Insight from the AaV genome implicates recycling of host nucleotides rather than de novo synthesis as a constraint on viral replication. Viral yields and mean burst sizes were significantlymore » diminished with increasing MOI. This phenomenon, which was reminiscent of phage-induced ‘lysis from without’, appeared to be caused by viral contact and was unrelated to bacteria, signaling/toxic compounds, or defective interfering viruses. We posit that high-MOI effects attenuate viral proliferation in natural systems providing a negative feedback on virus-induced bloom collapse.« less

Next Generation Respiratory Viral Vaccine System: Advanced and Emerging Bioengineered Human Lung Epithelia Model (HLEM) Organoid Technology

NASA Technical Reports Server (NTRS)

Goodwin, Thomas J.; Schneider, Sandra L.; MacIntosh, Victor; Gibbons, Thomas F.

2010-01-01

Acute respiratory infections, including pneumonia and influenza, are the S t" leading cause of United States and worldwide deaths. Newly emerging pathogens signaled the need for an advanced generation of vaccine technology.. Human bronchial-tracheal epithelial tissue was bioengineered to detect, identify, host and study the pathogenesis of acute respiratory viral disease. The 3-dimensional (3D) human lung epithelio-mesechymal tissue-like assemblies (HLEM TLAs) share characteristics with human respiratory epithelium: tight junctions, desmosomes, microvilli, functional markers villin, keratins and production of tissue mucin. Respiratory Syntial Virus (RSV) studies demonstrate viral growth kinetics and membrane bound glycoproteins up to day 20 post infection in the human lung-orgainoid infected cell system. Peak replication of RSV occurred on day 10 at 7 log10 particles forming units per ml/day. HLEM is an advanced virus vaccine model and biosentinel system for emergent viral infectious diseases to support DoD global surveillance and military readiness.

Dog response to inactivated canine parvovirus and feline panleukopenia virus vaccines.

PubMed

Pollock, R V; Carmichael, L E

1982-01-01

Inactivated canine parvovirus (CPV) and inactivated feline panleukopenia virus (FPV) vaccines were evaluated in dogs. Maximal serologic response occurred within 1-2 weeks after vaccination. Antibody titers then declined rapidly to low levels that persisted at least 20 weeks. Immunity to CPV, defined as complete resistance to infection, was correlated with serum antibody titer and did not persist longer than 6 weeks after vaccination with inactivated virus. However, protection against generalized infection was demonstrated 20 weeks after vaccination. In unvaccinated dogs, viremia and generalized infection occurred after oronasal challenge with virulent CPV. In contrast, viral replication was restricted to the intestinal tract and gut-associated lymphoid tissue of vaccinated dogs. Canine parvovirus was inactivated by formalin, beta-propiolactone (BPL), and binary ethylenimine (BEI) in serum-free media; inactivation kinetics were determined. Formalin resulted in a greater loss of viral HA than either BEI of BPL, and antigenicity was correspondingly reduced.

Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

PubMed Central

Hirata, Yuichi; Ikeda, Kazutaka; Sudoh, Masayuki; Tokunaga, Yuko; Suzuki, Akemi; Weng, Leiyun; Ohta, Masatoshi; Tobita, Yoshimi; Okano, Ken; Ozeki, Kazuhisa; Kawasaki, Kenichi; Tsukuda, Takuo; Katsume, Asao; Aoki, Yuko; Umehara, Takuya; Sekiguchi, Satoshi; Toyoda, Tetsuya; Shimotohno, Kunitada; Soga, Tomoyoshi; Nishijima, Masahiro; Taguchi, Ryo; Kohara, Michinori

2012-01-01

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle. PMID:22916015

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

PubMed

Hirata, Yuichi; Ikeda, Kazutaka; Sudoh, Masayuki; Tokunaga, Yuko; Suzuki, Akemi; Weng, Leiyun; Ohta, Masatoshi; Tobita, Yoshimi; Okano, Ken; Ozeki, Kazuhisa; Kawasaki, Kenichi; Tsukuda, Takuo; Katsume, Asao; Aoki, Yuko; Umehara, Takuya; Sekiguchi, Satoshi; Toyoda, Tetsuya; Shimotohno, Kunitada; Soga, Tomoyoshi; Nishijima, Masahiro; Taguchi, Ryo; Kohara, Michinori

2012-01-01

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Amiloride inhibits the initiation of Coxsackievirus and poliovirus RNA replication by inhibiting VPg uridylylation.

PubMed

Ogram, Sushma A; Boone, Christopher D; McKenna, Robert; Flanegan, James B

2014-09-01

The mechanism of amiloride inhibition of Coxsackievirus B3 (CVB3) and poliovirus type 1 (PV1) RNA replication was investigated using membrane-associated RNA replication complexes. Amiloride was shown to inhibit viral RNA replication and VPgpUpU synthesis. However, the drug had no effect on polymerase elongation activity during either (-) strand or (+) strand synthesis. These findings indicated that amiloride inhibited the initiation of RNA synthesis by inhibiting VPg uridylylation. In addition, in silico binding studies showed that amiloride docks in the VPg binding site on the back of the viral RNA polymerase, 3D(pol). Since VPg binding at this site on PV1 3D(pol) was previously shown to be required for VPg uridylylation, our results suggest that amiloride inhibits VPg binding to 3D(pol). In summary, our findings are consistent with a model in which amiloride inhibits VPgpUpU synthesis and viral RNA replication by competing with VPg for binding to 3D(pol). Copyright © 2014 Elsevier Inc. All rights reserved.

Polyamines and Hypusination Are Required for Ebolavirus Gene Expression and Replication

PubMed Central

Olsen, Michelle E.; Filone, Claire Marie; Rozelle, Dan; Mire, Chad E.; Agans, Krystle N.; Hensley, Lisa

2016-01-01

ABSTRACT Ebolavirus (EBOV) is an RNA virus that is known to cause severe hemorrhagic fever in humans and other primates. EBOV successfully enters and replicates in many cell types. This replication is dependent on the virus successfully coopting a number of cellular factors. Many of these factors are currently unidentified but represent potential targets for antiviral therapeutics. Here we show that cellular polyamines are critical for EBOV replication. We found that small-molecule inhibitors of polyamine synthesis block gene expression driven by the viral RNA-dependent RNA polymerase. Short hairpin RNA (shRNA) knockdown of the polyamine pathway enzyme spermidine synthase also resulted in reduced EBOV replication. These findings led us to further investigate spermidine, a polyamine that is essential for the hypusination of eukaryotic initiation factor 5A (eIF5A). Blocking the hypusination of eIF5A (and thereby inhibiting its function) inhibited both EBOV gene expression and viral replication. The mechanism appears to be due to the importance of hypusinated eIF5A for the accumulation of VP30, an essential component of the viral polymerase. The same reduction in hypusinated eIF5A did not alter the accumulation of other viral polymerase components. This action makes eIF5A function an important gate for proper EBOV polymerase assembly and function through the control of a single virus protein. PMID:27460797

Myeloid Cell Interaction with HIV: A Complex Relationship

PubMed Central

Rodrigues, Vasco; Ruffin, Nicolas; San-Roman, Mabel; Benaroch, Philippe

2017-01-01

Cells of the myeloid lineage, particularly macrophages, serve as primary hosts for HIV in vivo, along with CD4 T lymphocytes. Macrophages are present in virtually every tissue of the organism, including locations with negligible T cell colonization, such as the brain, where HIV-mediated inflammation may lead to pathological sequelae. Moreover, infected macrophages are present in multiple other tissues. Recent evidence obtained in humanized mice and macaque models highlighted the capacity of macrophages to sustain HIV replication in vivo in the absence of T cells. Combined with the known resistance of the macrophage to the cytopathic effects of HIV infection, such data bring a renewed interest in this cell type both as a vehicle for viral spread as well as a viral reservoir. While our understanding of key processes of HIV infection of macrophages is far from complete, recent years have nevertheless brought important insight into the uniqueness of the macrophage infection. Productive infection of macrophages by HIV can occur by different routes including from phagocytosis of infected T cells. In macrophages, HIV assembles and buds into a peculiar plasma membrane-connected compartment that preexists to the infection. While the function of such compartment remains elusive, it supposedly allows for the persistence of infectious viral particles over extended periods of time and may play a role on viral transmission. As cells of the innate immune system, macrophages have the capacity to detect and respond to viral components. Recent data suggest that such sensing may occur at multiple steps of the viral cycle and impact subsequent viral spread. We aim to provide an overview of the HIV–macrophage interaction along the multiple stages of the viral life cycle, extending when pertinent such observations to additional myeloid cell types such as dendritic cells or blood monocytes. PMID:29250073

Early viral replication and induced or constitutive immunity in rainbow trout families with differential resistance to Infectious hematopoietic necrosis virus (IHNV)

USGS Publications Warehouse

Purcell, M.K.; LaPatra, S.E.; Woodson, J.C.; Kurath, G.; Winton, J.R.

2010-01-01

The main objective of this study was to assess correlates of innate resistance in rainbow trout full-sibling families that differ in susceptibility to Infectious hematopoietic necrosis virus (IHNV). As part of a commercial breeding program, full-sibling families were challenged with IHNV by waterborne exposure at the 1 g size to determine susceptibility to IHNV. Progeny from select families (N = 7 families) that varied in susceptibility (ranging from 32 to 90% cumulative percent mortality (CPM)) were challenged again at the 10 g size by intra-peritoneal injection and overall mortality, early viral replication and immune responses were evaluated. Mortality challenges included 20–40 fish per family while viral replication and immune response studies included 6 fish per family at each time point (24, 48 and 72 h post-infection (hpi)). CPM at the 1 g size was significantly correlated with CPM at the 10 g size, indicating that inherent resistance was a stable trait irrespective of size. In the larger fish, viral load was measured by quantitative reverse-transcriptase PCR in the anterior kidney and was a significant predictor of family disease outcome at 48 hpi. Type I interferon (IFN) transcript levels were significantly correlated with an individual's viral load at 48 and 72 hpi, while type II IFN gene expression was significantly correlated with an individual's viral load at 24 and 48 hpi. Mean family type I but not type II IFN gene expression was weakly associated with susceptibility at 72 hpi. There was no association between mean family susceptibility and the constitutive expression of a range of innate immune genes (e.g. type I and II IFN pathway genes, cytokine and viral recognition receptor genes). The majority of survivors from the challenge had detectable serum neutralizing antibody titers but no trend was observed among families. This result suggests that even the most resistant families experienced sufficient levels of viral replication to trigger specific immunity. In summary, disease outcome for each family was determined very early in the infection process and resistance was associated with lower early viral replication.

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.

G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV

PubMed Central

Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P.; Robertson, Erle S.; Schildkraut, Carl L.; Verma, Subhash C.

2016-01-01

Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. PMID:26837574

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

Plant RNA Regulatory Network and RNA Granules in Virus Infection.

PubMed

Mäkinen, Kristiina; Lõhmus, Andres; Pollari, Maija

2017-01-01

Regulation of post-transcriptional gene expression on mRNA level in eukaryotic cells includes translocation, translation, translational repression, storage, mRNA decay, RNA silencing, and nonsense-mediated decay. These processes are associated with various RNA-binding proteins and cytoplasmic ribonucleoprotein complexes many of which are conserved across eukaryotes. Microscopically visible aggregations formed by ribonucleoprotein complexes are termed RNA granules. Stress granules where the translationally inactive mRNAs are stored and processing bodies where mRNA decay may occur present the most studied RNA granule types. Diverse RNP-granules are increasingly being assigned important roles in viral infections. Although the majority of the molecular level studies on the role of RNA granules in viral translation and replication have been conducted in mammalian systems, some studies link also plant virus infection to RNA granules. An increasing body of evidence indicates that plant viruses require components of stress granules and processing bodies for their replication and translation, but how extensively the cellular mRNA regulatory network is utilized by plant viruses has remained largely enigmatic. Antiviral RNA silencing, which is an important regulator of viral RNA stability and expression in plants, is commonly counteracted by viral suppressors of RNA silencing. Some of the RNA silencing suppressors localize to cellular RNA granules and have been proposed to carry out their suppression functions there. Moreover, plant nucleotide-binding leucine-rich repeat protein-mediated virus resistance has been linked to enhanced processing body formation and translational repression of viral RNA. Many interesting questions relate to how the pathways of antiviral RNA silencing leading to viral RNA degradation and/or repression of translation, suppression of RNA silencing and viral RNA translation converge in plants and how different RNA granules and their individual components contribute to these processes. In this review we discuss the roles of cellular RNA regulatory mechanisms and RNA granules in plant virus infection in the light of current knowledge and compare the findings to those made in animal virus studies.

Evaluation of an antimicrobial surgical glove to inactivate live human immunodeficiency virus following simulated glove puncture.

PubMed

Edmiston, Charles E; Zhou, S Steve; Hoerner, Pierre; Krikorian, Raffi; Krepel, Candace J; Lewis, Brian D; Brown, Kellie R; Rossi, Peter J; Graham, Mary Beth; Seabrook, Gary R

2013-02-01

Percutaneous injuries associated with cutting instruments, needles, and other sharps (eg, metallic meshes, bone fragments, etc) occur commonly during surgical procedures, exposing members of surgical teams to the risk for contamination by blood-borne pathogens. This study evaluated the efficacy of an innovative integrated antimicrobial glove to reduce transmission of the human immunodeficiency virus (HIV) following a simulated surgical-glove puncture injury. A pneumatically activated puncturing apparatus was used in a surgical-glove perforation model to evaluate the passage of live HIV-1 virus transferred via a contaminated blood-laden needle, using a reference (standard double-layer glove) and an antimicrobial benzalkonium chloride (BKC) surgical glove. The study used 2 experimental designs. In method A, 10 replicates were used in 2 cycles to compare the mean viral load following passage through standard and antimicrobial gloves. In method B, 10 replicates were pooled into 3 aliquots and were used to assess viral passage though standard and antimicrobial test gloves. In both methods, viral viability was assessed by observing the cytopathic effects in human lymphocytic C8166 T-cell tissue culture. Concurrent viral and cell culture viability controls were run in parallel with the experiment's studies. All controls involving tissue culture and viral viability were performed according to study design. Mean HIV viral loads (log(10)TCID(50)) were significantly reduced (P < .01) following passage through the BKC surgical glove compared to passage through the nonantimicrobial glove. The reduction (log reduction and percent viral reduction) of the HIV virus ranged from 1.96 to 2.4 and from 98.9% to 99.6%, respectively, following simulated surgical-glove perforation. Sharps injuries in the operating room pose a significant occupational risk for surgical practitioners. The findings of this study suggest that an innovative antimicrobial glove was effective at significantly (P < .01) reducing the risk for blood-borne virus transfer in a model of simulated glove perforation. Copyright © 2013 Mosby, Inc. All rights reserved.

Manipulating 3D-Printed and Paper Models Enhances Student Understanding of Viral Replication

ERIC Educational Resources Information Center

Couper, Lisa; Johannes, Kristen; Powers, Jackie; Silberglitt, Matt; Davenport, Jodi

2016-01-01

Understanding key concepts in molecular biology requires reasoning about molecular processes that are not directly observable and, as such, presents a challenge to students and teachers. We ask whether novel interactive physical models and activities can help students understand key processes in viral replication. Our 3D tangible models are…

Replication of Minute Virus of Mice in Murine Cells Is Facilitated by Virally Induced Depletion of p21

PubMed Central

Adeyemi, Richard O.

2012-01-01

The DNA damage response to infection with minute virus of mice (MVM) leads to activated p53; however, p21 levels are reduced via a proteasome-mediated mechanism. This loss was sustained, as virus replicated in infected cells held at the G2/M border. Addition of the cyclin-dependent kinase (CDK) inhibitor roscovitine after S-phase entry reduced MVM replication, suggesting that CDK activity was critical for continued viral replication and virus-induced reduction of p21 may thus be necessary to prevent inhibition of CDK. PMID:22623787

Mutational analysis of three predicted 5'-proximal stem-loop structures in the genome of tick-borne encephalitis virus indicates different roles in RNA replication and translation

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

Rouha, Harald; Hoenninger, Verena M.; Thurner, Caroline

2011-08-15

Flavivirus gene expression is modulated by RNA secondary structure elements at the terminal ends of the viral RNA molecule. For tick-borne encephalitis virus (TBEV), four stem-loop (SL) elements have been predicted in the first 180 nucleotides of the viral genome: 5'-SL1, 5'-SL2, 5'-SL3 and 5'-SL4. The last three of these appear to be unique to tick-borne flaviviruses. Here, we report their characterization by mutagenesis in a TBEV luciferase reporter system. By manipulating their thermodynamic properties, we found that an optimal stability of the 5'-SL2 is required for efficient RNA replication. 5'-SL3 formation is also important for viral RNA replication, butmore » although it contains the viral start codon, its formation is dispensable for RNA translation. 5'-SL4 appears to facilitate both RNA translation and replication. Our data suggest that maintenance of the balanced thermodynamic stability of these SL elements is important for temporal regulation of its different functions.« less

Nanotherapeutics Using an HIV-1 Poly A and Transactivator of the HIV-1 LTR-(TAR-) Specific siRNA

PubMed Central

Mahajan, Supriya D.; Aalinkeel, Ravikumar; Reynolds, Jessica L.; Nair, Bindukumar; Sykes, Donald E.; Law, Wing-Cheung; Ding, Hong; Bergey, Earl J.; Prasad, Paras N.; Schwartz, Stanley A.

2011-01-01

HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. We used a well-validated siRNA (si510) which targets the poly A/TAR (transactivator of the HIV-1 LTR) site and suppresses viral replication. Nanotechnology holds much potential for impact in the field of HIV-1 therapeutics, and nanoparticles such as quantum rods (QRs) can be easily functionalized to incorporate siRNA forming stable nanoplexes that can be used for gene silencing. We evaluated the efficacy of the QR-si510 HIV-1 siRNA nanoplex in suppressing viral replication in the HIV-1-infected monocytic cell line THP-1 by measuring p24 antigen levels and gene expression levels of HIV-1 LTR. Our results suggest that the QR-si510 HIV-1 siRNA nanoplex is not only effective in delivering siRNA, but also in suppressing HIV-1 viral replication for a longer time period. HIV-1 nanotherapeutics can thus enhance systemic bioavailability and offer multifunctionality. PMID:21660279

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

Antiretroviral treatment start-time during primary SIV(mac) infection in macaques exerts a different impact on early viral replication and dissemination.

PubMed

Sellier, Pierre; Mannioui, Abdelkrim; Bourry, Olivier; Dereuddre-Bosquet, Nathalie; Delache, Benoit; Brochard, Patricia; Calvo, Julien; Prévot, Sophie; Roques, Pierre

2010-05-11

The time of infection is rarely known in human cases; thus, the effects of delaying the initiation of antiretroviral therapy (ART) on the peripheral viral load and the establishment of viral reservoirs are poorly understood. Six groups of macaques, infected intravenously with SIV(mac251), were given placebo or antiretroviral therapy to explore reservoir establishment; macaques were treated for 2 weeks, with treatment starting 4 hours, 7 or 14 days after infection. Viral replication and dissemination were measured in the gut (rectum), in the lung and in blood and lymphoid tissues (peripheral lymph nodes), by quantifying viral RNA, DNA and 2LTR circles. We used immunohistochemistry (CD4 and CD68) to assess the impact of these treatments on the relative amount of virus target cells in tissue. Treatment that was started 4 hours post-infection (pi) decreased viral replication and dissemination in blood and tissue samples, which were assessed on day 14 (RNA/DNA/2LTR circles). The virus remained detectable and lymphoid tissues were activated in LN and the gut in both placebo- and ART-treated animals. Viral RNA in plasma continued to be lower in macaques treated seven days after infection; however, this was not the case for viral DNA in peripheral blood mononuclear cells. There was a small but significant difference in RNA and DNA levels in tissues between placebo- and ART-treated animals on day 21. When started 14 days after infection, treatment resulted in a limited decrease in the plasma viral load. Treatment that was started 4 hours after infection significantly reduced viral replication and dissemination. When started 7 days after infection, it was of slight virological benefit in peripheral blood and in tissues, and treatment was even less effective if started 14 days pi. These data favor starting ART no longer than one week after intravenous SIV(mac251) exposure.

Identification of a novel multiple kinase inhibitor with potent antiviral activity against influenza virus by reducing viral polymerase activity

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

Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn

Highlights: • Screening of 50,000 compounds and subsequent lead optimization identified WV970. • WV970 has antiviral effects against influenza A, B and highly pathogenic viral strains. • WV970 inhibits viral genome replication and transcription. • A target database search suggests that WV970 may bind to a number of kinases. • KINOMEscan screening revealed that WV970 has inhibitory effects on 15 kinases. - Abstract: Neuraminidase inhibitors are the only currently available influenza treatment, although resistant viruses to these drugs have already been reported. Thus, new antiviral drugs with novel mechanisms of action are urgently required. In this study, we identified amore » novel antiviral compound, WV970, through cell-based screening of a 50,000 compound library and subsequent lead optimization. This compound exhibited potent antiviral activity with nanomolar IC{sub 50} values against both influenza A and B viruses but not non-influenza RNA viruses. Time-of-addition and indirect immunofluorescence assays indicated that WV970 acted at an early stage of the influenza life cycle, but likely after nuclear entry of viral ribonucleoprotein (vRNP). Further analyses of viral RNA expression and viral polymerase activity indicated that WV970 inhibited vRNP-mediated viral genome replication and transcription. Finally, structure-based virtual screening and comprehensive human kinome screening were used to demonstrate that WV970 acts as a multiple kinase inhibitor, many of which are associated with influenza virus replication. Collectively, these results strongly suggest that WV970 is a promising anti-influenza drug candidate and that several kinases associated with viral replication are promising drug targets.« less

Enhancement of Herpes Simplex Virus (HSV) Infection by Seminal Plasma and Semen Amyloids Implicates a New Target for the Prevention of HSV Infection

PubMed Central

Torres, Lilith; Ortiz, Tatiana; Tang, Qiyi

2015-01-01

Human herpesviruses cause different infectious diseases, resulting in world-wide health problems. Sexual transmission is a major route for the spread of both herpes simplex virus-1 (HSV-1) and -2. Semen plays an important role in carrying the viral particle that invades the vaginal or rectal mucosa and, thereby, initiates viral replication. Previously, we demonstrated that the amyloid fibrils semenogelin (SEM) and semen-derived enhancer of viral infection (SEVI), and seminal plasma (SP) augment cytomegalovirus infection (Tang et al., J. Virol 2013). Whether SEM or SEVI amyloids or SP could also enhance other herpesvirus infections has not been examined. In this study, we found that the two amyloids as well as SP strongly enhance both HSV-1 and -2 infections in cell culture. Along with SP, SEM and SEVI amyloids enhanced viral entry and increased infection rates by more than 10-fold, as assessed by flow cytometry assay and fluorescence microscopy. Viral replication was increased by about 50- to 100-fold. Moreover, viral growth curve assays showed that SEM and SEVI amyloids, as well as SP, sped up the kinetics of HSV replication such that the virus reached its replicative peak more quickly. The interactions of SEM, SEVI, and SP with HSVs are direct. Furthermore, we discovered that the enhancing effects of SP, SEM, and SEVI can be significantly reduced by heparin, a sulfated polysaccharide with an anionic charge. It is probable that heparin abrogates said enhancing effects by interfering with the interaction of the viral particle and the amyloids, which interaction results in the binding of the viral particles and both SEM and SEVI. PMID:25903833

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

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

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

Severity of Disease in Humanized Mice Infected With Ebola Virus or Reston Virus Is Associated With Magnitude of Early Viral Replication in Liver.

PubMed

Spengler, Jessica R; Saturday, Greg; Lavender, Kerry J; Martellaro, Cynthia; Keck, James G; Nichol, Stuart T; Spiropoulou, Christina F; Feldmann, Heinz; Prescott, Joseph

2017-12-27

Both Ebola virus (EBOV) and Reston virus (RESTV) cause disease in nonhuman primates, yet only EBOV causes disease in humans. To investigate differences in viral pathogenicity, humanized mice (hu-NSG-SGM3) were inoculated with EBOV or RESTV. Consistent with differences in disease in human infection, pronounced weight loss and markers of hepatic damage and disease were observed exclusively in EBOV-infected mice. These abnormalities were associated with significantly higher EBOV replication in the liver but not in the spleen, suggesting that in this model, efficiency of viral replication in select tissues early in infection may contribute to differences in viral pathogenicity. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

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

Active Ebola Virus Replication and Heterogeneous Evolutionary Rates in EVD Survivors.

PubMed

Whitmer, Shannon L M; Ladner, Jason T; Wiley, Michael R; Patel, Ketan; Dudas, Gytis; Rambaut, Andrew; Sahr, Foday; Prieto, Karla; Shepard, Samuel S; Carmody, Ellie; Knust, Barbara; Naidoo, Dhamari; Deen, Gibrilla; Formenty, Pierre; Nichol, Stuart T; Palacios, Gustavo; Ströher, Ute

2018-01-30

Following cessation of continuous Ebola virus (EBOV) transmission within Western Africa, sporadic EBOV disease (EVD) cases continued to re-emerge beyond the viral incubation period. Epidemiological and genomic evidence strongly suggests that this represented transmission from EVD survivors. To investigate whether persistent infections are characterized by ongoing viral replication, we sequenced EBOV from the semen of nine EVD survivors and a subset of corresponding acute specimens. EBOV evolutionary rates during persistence were either similar to or reduced relative to acute infection rates. Active EBOV replication/transcription continued during convalescence, but decreased over time, consistent with viral persistence rather than viral latency. Patterns of genetic divergence suggest a moderate relaxation of selective constraints within the sGP carboxy-terminal tail during persistent infections, but do not support widespread diversifying selection. Altogether, our data illustrate that EBOV persistence in semen, urine, and aqueous humor is not a quiescent or latent infection. Published by Elsevier Inc.

HCV replication in gastrointestinal mucosa: Potential extra-hepatic viral reservoir and possible role in HCV infection recurrence after liver transplantation

PubMed Central

Pizzillo, Paola; Iannolo, Gioacchin; Barbera, Floriana; Liotta, Rosa; Traina, Mario; Vizzini, Giovanni; Gridelli, Bruno

2017-01-01

Purpose Hepatitis C virus (HCV) predominantly infects hepatocytes, although it is known that receptors for viral entry are distributed on a wide array of target cells. Chronic HCV infection is indeed characterized by multiple non-liver manifestations, suggesting a more complex HCV tropism extended to extrahepatic tissues and remains to be fully elucidated. In this study, we investigated the gastrointestinal mucosa (GIM) as a potential extrahepatic viral replication site and its contribution to HCV recurrence. Methods We analyzed GIM biopsies from a cohort of 76 patients, 11 of which were HCV-negative and 65 HCV-positive. Of these, 54 biopsies were from liver-transplanted patients. In 29 cases, we were able to investigate gastrointestinal biopsies from the same patient before and after transplant. To evaluate the presence of HCV, we looked for viral antigens and genome RNA, whilst to assess viral replicative activity, we searched for the replicative intermediate minus-strand RNA. We studied the genetic diversity and the phylogenetic relationship of HCV quasispecies from plasma, liver and gastrointestinal mucosa of HCV-liver-transplanted patients in order to assess HCV compartmentalization and possible contribution of gastrointestinal variants to liver re-infection after transplantation. Results Here we show that HCV infects and replicates in the cells of the GIM and that the favorite hosts were mostly enteroendocrine cells. Interestingly, we observed compartmentalization of the HCV quasispecies present in the gastrointestinal mucosa compared to other tissues of the same patient. Moreover, the phylogenetic analysis revealed a high similarity between HCV variants detected in gastrointestinal mucosa and those present in the re-infected graft. Conclusions Our results demonstrated that the gastrointestinal mucosa might be considered as an extrahepatic reservoir of HCV and that could contribute to viral recurrence. Moreover, the finding that HCV infects and replicates in neuroendocrine cells opens new perspectives on the role of these cells in the natural history of HCV infection. PMID:28750044

Requirement of Sur2 for Efficient Replication of Mouse Adenovirus Type 1

PubMed Central

Fang, Lei; Stevens, Jennitte L.; Berk, Arnold J.; Spindler, Katherine R.

2004-01-01

Mouse adenovirus type 1 (MAV-1) early region 1A (E1A) encodes a virulence gene in viral infection of mice. To broaden our understanding of the functions of E1A in MAV-1 pathogenesis, an unbiased experimental approach, glutathione S-transferase (GST) pulldown, was used to screen for cellular proteins that interact with E1A protein. We identified mouse Sur2, a subunit of Mediator complex, as a protein that binds to MAV-1 E1A. The interaction between Sur2 and MAV-1 E1A was confirmed in virus-infected cells. Conserved region 3 (CR3) of MAV-1 E1A was mapped as the region required for Sur2-E1A interaction, as is the case for human adenovirus E1A. Although it has been proposed that human adenovirus E1A recruits the Mediator complex to transactivate transcription of viral early genes, Sur2 function in adenovirus replication has not been directly tested previously. Studies on the functions of Sur2 with mouse embryonic fibroblasts (MEFs) showed that there was a multiplicity-dependent growth defect of MAV-1 in Sur2−/− MEFs compared to Sur2+/+ MEFs. Comparison of the viral DNA and viral mRNA levels in Sur2+/+ and Sur2−/− MEFs confirmed that Sur2 was important for efficient viral replication. The viral replication defects in Sur2−/− MEFs appeared to be due at least in part to a defect in viral early gene transcription. PMID:15542641

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

The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1

PubMed Central

Houzet, Laurent; Klase, Zachary; Yeung, Man Lung; Wu, Annie; Le, Shu-Yun; Quiñones, Mariam; Jeang, Kuan-Teh

2012-01-01

MicroRNAs (miRNAs) are 22-nt non-coding RNAs involved in the regulation of cellular gene expression and potential cellular defense against viral infection. Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome. Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication. Using one of these five miRNAs, human miR-326 as an example, we demonstrated that the degree of complementarity between the predicted viral sequence and cellular miR-326 correlates, in a Dicer-dependent manner, with the potency of miRNA-mediated restriction of viral replication. Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells. PMID:23042677

Host Range Factor 1 from Lymantria dispar Nucleopolyhedrovirus (NPV) Is an Essential Viral Factor Required for Productive Infection of NPVs in IPLB-Ld652Y Cells Derived from L. dispar

PubMed Central

Ishikawa, Hiroki; Ikeda, Motoko; Felipe Alves, Cristiano A.; Thiem, Suzanne M.; Kobayashi, Michihiro

2004-01-01

Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells. PMID:15507661

Host range factor 1 from Lymantria dispar Nucleopolyhedrovirus (NPV) is an essential viral factor required for productive infection of NPVs in IPLB-Ld652Y cells derived from L. dispar.

PubMed

Ishikawa, Hiroki; Ikeda, Motoko; Alves, Cristiano A Felipe; Thiem, Suzanne M; Kobayashi, Michihiro

2004-11-01

Host range factor 1 (HRF-1) of Lymantria dispar multinucleocapsid nucleopolyhedrovirus promotes Autographa californica MNPV replication in nonpermissive Ld652Y cells derived from L. dispar. Here we demonstrate that restricted Hyphantria cunea NPV replication in Ld652Y cells was not due to apoptosis but was likely due to global protein synthesis arrest that could be restored by HRF-1. Our data also showed that HRF-1 promoted the production of progeny virions for two other baculoviruses, Bombyx mori NPV and Spodoptera exigua MNPV, whose replication in Ld652Y cells is limited to replication of viral DNA without successful production of infectious progeny virions. Thus, HRF-1 is an essential viral factor required for productive infection of NPVs in Ld652Y cells.

Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection.

PubMed

Subramanian, T; Zhao, Ling-Jun; Chinnadurai, G

2013-09-01

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP-E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP-E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection

PubMed Central

Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G.

2013-01-01

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP-E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP-E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. PMID:23747199

cis-acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5' nontranslated region chimeras.

PubMed Central

Frolov, I; McBride, M S; Rice, C M

1998-01-01

Pestiviruses, such as bovine viral diarrhea virus (BVDV), share many similarities with hepatitis C virus (HCV) yet are more amenable to virologic and genetic analysis. For both BVDV and HCV, translation is initiated via an internal ribosome entry site (IRES). Besides IRES function, the viral 5' nontranslated regions (NTRs) may also contain cis-acting RNA elements important for viral replication. A series of chimeric RNAs were used to examine the function of the BVDV 5' NTR. Our results show that: (1) the HCV and the encephalomyocarditis virus (EMCV) IRES element can functionally replace that of BVDV; (2) two 5' terminal hairpins in BVDV genomic RNA are important for efficient replication; (3) replacement of the entire BVDV 5' NTR with those of HCV or EMCV leads to severely impaired replication; (4) such replacement chimeras are unstable and efficiently replicating pseudorevertants arise; (5) pseudorevertant mutations involve deletion of 5' sequences and/or acquisition of novel 5' sequences such that the 5' terminal 3-4 bases of BVDV genome RNA are restored. Besides providing new insight into functional elements in the BVDV 5' NTR, these chimeras may prove useful as pestivirus vaccines and for screening and evaluation of anti-HCV IRES antivirals. PMID:9814762

Porcine circovirus type 2 replication is impaired by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway

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

Wei Li; Liu Jue

Postweaning multisystemic wasting syndrome, which is primarily caused by porcine circovirus type 2 (PCV2), is an emerging and important swine disease. We have recently shown that PCV2 induces nuclear factor kappa B activation and its activation is required for active replication, but the other cellular factors involved in PCV2 replication are not well defined. The extracellular signal-regulated kinase (ERK) which served as an important component of cellular signal transduction pathways has been shown to regulate many viral infections. In this report, we show that PCV2 activates ERK1/2 in PCV2-infected PK15 cells dependent on viral replication. The PCV2-induced ERK1/2 leads tomore » phosphorylation of the ternary complex factor Elk-1, which kinetically paralleled ERK1/2 activation. Inhibition of ERK activation with U0126, a specific MEK1/2 inhibitor, significantly reduced viral progeny release. Investigations into the mechanism of ERK1/2 regulation revealed that inhibition of ERK activation leads to decreased viral transcription and lower virus protein expression. These data indicate that the ERK signaling pathway is involved in PCV2 infection and beneficial to PCV2 replication in the cultured cells.« less

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

Electron Microscopy of Ebola Virus-Infected Cells.

PubMed

Noda, Takeshi

2017-01-01

Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

Pathogenic Events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis

PubMed Central

Chen, Crystal Y.; Huang, Dan; Wang, Richard; Zhang, Meihong; Qian, Lixia; Zhu, Yanfen; Zhang, Alvin Zhuoran; Yang, Enzhuo; Qaqish, Arwa; Kouiavskaia, Diana; Nathanson, Neal; Macadam, Andrew J.; Andino, Raul; Kew, Olen; Xu, Junfa

2017-01-01

ABSTRACT Despite a great deal of prior research, the early pathogenic events in natural oral poliovirus infection remain poorly defined. To establish a model for study, we infected 39 macaques by feeding them single high doses of the virulent Mahoney strain of wild type 1 poliovirus. Doses ranging from 107 to 109 50% tissue culture infective doses (TCID50) consistently infected all the animals, and many monkeys receiving 108 or 109 TCID50 developed paralysis. There was no apparent difference in the susceptibilities of the three macaque species (rhesus, cynomolgus, and bonnet) used. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia, and virus was isolated from tonsils, gut mucosa, and draining lymph nodes. Viral replication proteins were detected in both epithelial and lymphoid cell populations expressing CD155 in the tonsil and intestine, as well as in spinal cord neurons. Necrosis was observed in these three cell types, and viral replication in the tonsil/gut was associated with histopathologic destruction and inflammation. The sustained response of neutralizing antibody correlated temporally with resolution of viremia and termination of virus shedding in oropharynges and feces. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), extending previous studies of poliovirus pathogenesis in humans. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis and to assess the efficacy of candidate antiviral drugs and new vaccines. IMPORTANCE Early pathogenic events of poliovirus infection remain largely undefined, and there is a lack of animal models mimicking natural oral human infection leading to paralytic poliomyelitis. All 39 macaques fed with single high doses ranging from 107 to 109 TCID50 Mahoney type 1 virus were infected, and many of the monkeys developed paralysis. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia; tonsil, mesentery lymph nodes, and intestinal mucosa served as major target sites of viral replication. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), thereby supplementing historical reconstructions of poliovirus pathogenesis. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis, candidate antiviral drugs, and the efficacy of new vaccines. PMID:28356537

Secretion of Hepatitis C Virus Replication Intermediates Reduces Activation of Toll-Like Receptor 3 in Hepatocytes.

PubMed

Grünvogel, Oliver; Colasanti, Ombretta; Lee, Ji-Young; Klöss, Volker; Belouzard, Sandrine; Reustle, Anna; Esser-Nobis, Katharina; Hesebeck-Brinckmann, Jasper; Mutz, Pascal; Hoffmann, Katrin; Mehrabi, Arianeb; Koschny, Ronald; Vondran, Florian W R; Gotthardt, Daniel; Schnitzler, Paul; Neumann-Haefelin, Christoph; Thimme, Robert; Binder, Marco; Bartenschlager, Ralf; Dubuisson, Jean; Dalpke, Alexander H; Lohmann, Volker

2018-06-01

Hepatitis C virus (HCV) infections most often result in chronic outcomes, although the virus constantly produces replication intermediates, in particular double-stranded RNA (dsRNA), representing potent inducers of innate immunity. We aimed to characterize the fate of HCV dsRNA in hepatocyte cultures to identify mechanisms contributing to viral persistence in presence of an active innate immune response. We analyzed hepatocyte-based culture models for HCV for induction of innate immunity, secretion of virus positive- or negative-strand RNA, and viral replication using different quantification methods and microscopy techniques. Expression of pattern recognition receptors was reconstituted in hepatoma cells by lentiviral transduction. HCV-infected cells secrete substantial amounts of virus positive- and negative-strand RNAs in extracellular vesicles (EVs), toward the apical and basolateral domain of hepatocytes. Secretion of negative-strand RNA was independent from virus production, and viral RNA secreted in EVs contained higher relative amounts of negative-strands, indicating that mostly virus dsRNA is released. A substantial part of viral replication complexes and dsRNA was found in the endosomal compartment and multivesicular bodies, indicating that secretion of HCV replication intermediates is mediated by the exosomal pathway. Block of vesicle release in HCV-positive cells increased intracellular dsRNA levels and increased activation of toll-like receptor 3, inhibiting HCV replication. Using hepatocyte-based culture models for HCV, we found a portion of HCV dsRNA intermediates to be released from infected cells in EVs, which reduces activation of toll-like receptor 3. This represents a novel mechanism how HCV evades host immune responses, potentially contributing to viral persistence. Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.

Placental macrophage contact potentiates the complete replicative cycle of human cytomegalovirus in syncytiotrophoblast cells: role of interleukin-8 and transforming growth factor-beta1.

PubMed

Bácsi, A; Aranyosi, J; Beck, Z; Ebbesen, P; Andirkó, I; Szabó, J; Lampé, L; Kiss, J; Gergely, L; Tóth, F D

1999-10-01

Although syncytiotrophoblast (ST) cells can be infected by human cytomegalovirus (HCMV), in vitro studies have indicated that ST cells do not support the complete viral reproductive cycle, or HCMV replication may occur in less than 3% of ST cells. The present study tested the possibility that placental macrophages might enhance activation of HCMV carried in ST cells and, further, that infected ST cells would be capable of transmitting virus to neighboring macrophages. For this purpose, we studied HCMV replication in ST cells grown alone or cocultured with uninfected placental macrophages. Our results demonstrated that HCMV gene expression in ST cells was markedly upregulated by coculture with macrophages, resulting in release of substantial amounts of infectious virus from HCMV-infected ST cells. After having become permissive for viral replication, ST cells delivered HCMV to the cocultured macrophages, as evidenced by detection of virus-specific antigens in these cells. The stimulatory effect of coculture on HCMV gene expression in ST cells was mediated by marked interleukin-8 (IL-8) and transforming growth factor-beta1 (TGF-beta1) release from macrophages, an effect caused by contact between the different placental cells. Our findings indicate an interactive role for the ST layer and placental macrophages in the dissemination of HCMV among placental tissue. Eventually, these interactions may contribute to the transmission of HCMV from mother to the fetus.

Targeting RNA–Protein Interactions within the Human Immunodeficiency Virus Type 1 Lifecycle

PubMed Central

2013-01-01

RNA–protein interactions are vital throughout the HIV-1 life cycle for the successful production of infectious virus particles. One such essential RNA–protein interaction occurs between the full-length genomic viral RNA and the major structural protein of the virus. The initial interaction is between the Gag polyprotein and the viral RNA packaging signal (psi or Ψ), a highly conserved RNA structural element within the 5′-UTR of the HIV-1 genome, which has gained attention as a potential therapeutic target. Here, we report the application of a target-based assay to identify small molecules, which modulate the interaction between Gag and Ψ. We then demonstrate that one such molecule exhibits potent inhibitory activity in a viral replication assay. The mode of binding of the lead molecules to the RNA target was characterized by 1H NMR spectroscopy. PMID:24358934

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

HCMV Reprogramming of Infected Monocyte Survival and Differentiation: A Goldilocks Phenomenon

PubMed Central

Stevenson, Emily V.; Collins-McMillen, Donna; Kim, Jung Heon; Cieply, Stephen J.; Bentz, Gretchen L.; Yurochko, Andrew D.

2014-01-01

The wide range of disease pathologies seen in multiple organ sites associated with human cytomegalovirus (HCMV) infection results from the systemic hematogenous dissemination of the virus, which is mediated predominately by infected monocytes. In addition to their role in viral spread, infected monocytes are also known to play a key role in viral latency and life-long persistence. However, in order to utilize infected monocytes for viral spread and persistence, HCMV must overcome a number of monocyte biological hurdles, including their naturally short lifespan and their inability to support viral gene expression and replication. Our laboratory has shown that HCMV is able to manipulate the biology of infected monocytes in order to overcome these biological hurdles by inducing the survival and differentiation of infected monocytes into long-lived macrophages capable of supporting viral gene expression and replication. In this current review, we describe the unique aspects of how HCMV promotes monocyte survival and differentiation by inducing a “finely-tuned” macrophage cell type following infection. Specifically, we describe the induction of a uniquely polarized macrophage subset from infected monocytes, which we argue is the ideal cellular environment for the initiation of viral gene expression and replication and, ultimately, viral spread and persistence within the infected host. PMID:24531335

Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance

PubMed Central

Fenton-May, Angharad E.; Dilernia, Dario A.; Kilembe, William; Allen, Susan A.; Borrow, Persephone; Hunter, Eric

2015-01-01

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential. PMID:26378795

Sp100 isoform-specific regulation of human adenovirus 5 gene expression.

PubMed

Berscheminski, Julia; Wimmer, Peter; Brun, Juliane; Ip, Wing Hang; Groitl, Peter; Horlacher, Tim; Jaffray, Ellis; Hay, Ron T; Dobner, Thomas; Schreiner, Sabrina

2014-06-01

Promyelocytic leukemia nuclear bodies (PML-NBs) are nuclear structures that accumulate intrinsic host factors to restrict viral infections. To ensure viral replication, these must be limited by expression of viral early regulatory proteins that functionally inhibit PML-NB-associated antiviral effects. To benefit from the activating capabilities of Sp100A and simultaneously limit repression by Sp100B, -C, and -HMG, adenoviruses (Ads) employ several features to selectively and individually target these isoforms. Ads induce relocalization of Sp100B, -C, and -HMG from PML-NBs prior to association with viral replication centers. In contrast, Sp100A is kept at the PML tracks that surround the newly formed viral replication centers as designated sites of active transcription. We concluded that the host restriction factors Sp100B, -C, and -HMG are potentially inactivated by active displacement from these sites, whereas Sp100A is retained to amplify Ad gene expression. Ad-dependent loss of Sp100 SUMOylation is another crucial part of the virus repertoire to counteract intrinsic immunity by circumventing Sp100 association with HP1, therefore limiting chromatin condensation. We provide evidence that Ad selectively counteracts antiviral responses and, at the same time, benefits from PML-NB-associated components which support viral gene expression by actively recruiting them to PML track-like structures. Our findings provide insights into novel strategies for manipulating transcriptional regulation to either inactivate or amplify viral gene expression. We describe an adenoviral evasion strategy that involves isoform-specific and active manipulation of the PML-associated restriction factor Sp100. Recently, we reported that the adenoviral transactivator E1A targets PML-II to efficiently activate viral transcription. In contrast, the PML-associated proteins Daxx and ATRX are inhibited by early viral factors. We show that this concept is more intricate and significant than originally believed, since adenoviruses apparently take advantage of specific PML-NB-associated proteins and simultaneously inhibit antiviral measures to maintain the viral infectious program. Specifically, we observed Ad-induced relocalization of the Sp100 isoforms B, C, and HMG from PML-NBs juxtaposed with viral replication centers. In contrast, Sp100A is retained at Ad-induced PML tracks that surround the newly formed viral replication centers, acting as designated sites of active transcription. The host restriction factors Sp100B, -C, and -HMG are potentially inactivated by active displacement from these sites, whereas Sp100A is retained to amplify Ad gene expression.

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.

CNOT4-Mediated Ubiquitination of Influenza A Virus Nucleoprotein Promotes Viral RNA Replication

PubMed Central

Lin, Yu-Chen; Jeng, King-Song

2017-01-01

ABSTRACT Influenza A virus (IAV) RNA segments are individually packaged with viral nucleoprotein (NP) and RNA polymerases to form a viral ribonucleoprotein (vRNP) complex. We previously reported that NP is a monoubiquitinated protein which can be deubiquitinated by a cellular ubiquitin protease, USP11. In this study, we identified an E3 ubiquitin ligase, CNOT4 (Ccr4-Not transcription complex subunit 4), which can ubiquitinate NP. We found that the levels of viral RNA, protein, viral particles, and RNA polymerase activity in CNOT4 knockdown cells were lower than those in the control cells upon IAV infection. Conversely, overexpression of CNOT4 rescued viral RNP activity. In addition, CNOT4 interacted with the NP in the cell. An in vitro ubiquitination assay also showed that NP could be ubiquitinated by in vitro-translated CNOT4, but ubiquitination did not affect the protein stability of NP. Significantly, CNOT4 increased NP ubiquitination, whereas USP11 decreased it. Mass spectrometry analysis of ubiquitinated NP revealed multiple ubiquitination sites on the various lysine residues of NP. Three of these, K184, K227, and K273, are located on the RNA-binding groove of NP. Mutations of these sites to arginine reduced viral RNA replication. These results indicate that CNOT4 is a ubiquitin ligase of NP, and ubiquitination of NP plays a positive role in viral RNA replication. PMID:28536288

Viral Epitranscriptomics

PubMed Central

Kennedy, Edward M.; Courtney, David G.; Tsai, Kevin

2017-01-01

ABSTRACT Although it has been known for over 40 years that eukaryotic mRNAs bear internal base modifications, it is only in the last 5 years that the importance of these modifications has begun to come into focus. The most common mRNA modification, the addition of a methyl group to the N6 position of adenosine (m6A), has been shown to affect splicing, translation, and stability, and m6A is also essential for embryonic development in organisms ranging from plants to mice. While all viral transcripts examined so far have been found to be extensively m6A modified, the role, if any, of m6A in regulating viral gene expression and replication was previously unknown. However, recent data generated using HIV-1 as a model system strongly suggest that sites of m6A addition not only are evolutionarily conserved but also enhance virus replication. It is therefore likely that the field of viral epitranscriptomics, which can be defined as the study of functionally relevant posttranscriptional modifications of viral RNA transcripts that do not change the nucleotide sequence of that RNA, is poised for a major expansion in scientific interest and may well fundamentally change our understanding of how viral replication is regulated. PMID:28250115

The influence of temperature on viral replication and antiviral-related genes response in hirame rhabdovirus-infected flounder (Paralichthys olivaceus).

PubMed

Zhang, Jialin; Tang, Xiaoqian; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

2017-09-01

Hirame rhabdovirus (HIRRV) is a rhabdovirus that causes severe disease in fish. The mortality due to HIRRV infection occurs at temperatures below 15 °C, but no mortality is observed over 20 °C. In this study, Japanese flounder (Paralichthys olivaceus) was artificially infected with the HIRRV CNPo2015 strain at 10 °C or 20 °C. Absolute quantitative real-time PCR was employed to examine the viral replication in spleens after HIRRV infection. Expression profiles of four interferon-related genes (type I IFN, Mx, ISG15, MDA5) and two proinflammatory genes (TNF-α and IL-1β) were also investigated by quantitative real-time PCR. Results showed that viral copies in spleens increased gradually over time and peaked at 72 h post infection (hpi) in the 10 °C group, while viral copies in the 20 °C group increased within 24 hpi, but afterwards decreased to very low levels. Moreover, the expressions of IFNs in the 10 °C group reached the highest levels at 72 hpi, whereas their peak levels appeared much earlier in the 20 °C group, at 12 hpi. The expression levels of TNF-α and IL-1β in the 10 °C group peaked at 12 hpi and then quickly declined. However, the two genes were highly expressed during 6-24 hpi in the 20 °C group. Based on these findings, we concluded that HIRRV infection induced an efficient antiviral immune response at 20 °C, which might inhibit the viral transcription at early stages and finally prevent HIRRV infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

Competing Death Programs in Poliovirus-Infected Cells: Commitment Switch in the Middle of the Infectious Cycle

PubMed Central

Agol, Vadim I.; Belov, George A.; Bienz, Kurt; Egger, Denise; Kolesnikova, Marina S.; Romanova, Lyudmila I.; Sladkova, Larissa V.; Tolskaya, Elena A.

2000-01-01

Productive poliovirus infection of HeLa cells leads to the canonical cytopathic effect (CPE), whereas certain types of abortive infection result in apoptosis. To define the time course of commitment to the different types of poliovirus-induced death, inhibitors of viral replication (guanidine HCl) or translation (cycloheximide) were added at different times postinfection (p.i.). Early in the infection (during the first ∼2 h p.i.), predominantly proapoptotic viral function was expressed, rendering the cells committed to apoptosis, which developed several hours after viral expression was arrested. In the middle of infection, concomitantly with the onset of fast generation of viral progeny, the implementation of the viral apoptotic program was abruptly interrupted. In particular, activation of an Asp-Glu-Val-Asp (DEVD)-specific caspase(s) occurring in the apoptosis-committed cells was prevented by the ongoing productive infection. Simultaneously, the cells retaining normal or nearly normal morphology became committed to CPE, which eventually developed regardless of whether or not further viral expression was allowed to proceed. The implementation of the poliovirus-induced apoptotic program was suppressed in HeLa cells overexpressing the Bcl-2 protein, indicating that the fate of poliovirus-infected cells depends on the balance of host and viral pro- and antiapoptotic factors. PMID:10823859

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

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...

Viral Characteristics Associated with the Clinical Nonprogressor Phenotype Are Inherited by Viruses from a Cluster of HIV-1 Elite Controllers

PubMed Central

2018-01-01

ABSTRACT A small group of HIV-1-infected individuals, called long-term nonprogressors (LTNPs), and in particular a subgroup of LTNPs, elite controllers (LTNP-ECs), display permanent control of viral replication and lack of clinical progression. This control is the result of a complex interaction of host, immune, and viral factors. We identified, by phylogenetic analysis, a cluster of LTNP-ECs infected with very similar low-replication HIV-1 viruses, suggesting the contribution of common viral features to the clinical LTNP-EC phenotype. HIV-1 envelope (Env) glycoprotein mediates signaling and promotes HIV-1 fusion, entry, and infection, being a key factor of viral fitness in vitro, cytopathicity, and infection progression in vivo. Therefore, we isolated full-length env genes from viruses of these patients and from chronically infected control individuals. Functional characterization of the initial events of the viral infection showed that Envs from the LTNP-ECs were ineffective in the binding to CD4 and in the key triggering of actin/tubulin-cytoskeleton modifications compared to Envs from chronic patients. The viral properties of the cluster viruses result in a defective viral fusion, entry, and infection, and these properties were inherited by every virus of the cluster. Therefore, inefficient HIV-1 Env functions and signaling defects may contribute to the low viral replication capacity and transmissibility of the cluster viruses, suggesting a direct role in the LTNP-EC phenotype of these individuals. These results highlight the important role of viral characteristics in the LTNP-EC clinical phenotype. These Env viral properties were common to all the cluster viruses and thus support the heritability of the viral characteristics. PMID:29636433

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

CMV immune evasion and manipulation of the immune system with aging.

PubMed

Jackson, Sarah E; Redeker, Anke; Arens, Ramon; van Baarle, Debbie; van den Berg, Sara P H; Benedict, Chris A; Čičin-Šain, Luka; Hill, Ann B; Wills, Mark R

2017-06-01

Human cytomegalovirus (HCMV) encodes numerous proteins and microRNAs that function to evade the immune response and allow the virus to replicate and disseminate in the face of a competent innate and acquired immune system. The establishment of a latent infection by CMV, which if completely quiescent at the level of viral gene expression would represent an ultimate in immune evasion strategies, is not sufficient for lifelong persistence and dissemination of the virus. CMV needs to reactivate and replicate in a lytic cycle of infection in order to disseminate further, which occurs in the face of a fully primed secondary immune response. Without reactivation, latency itself would be redundant for the virus. It is also becoming clear that latency is not a totally quiescent state, but is characterized by limited viral gene expression. Therefore, the virus also needs immune evasion strategies during latency. An effective immune response to CMV is required or viral replication will cause morbidity and ultimately mortality in the host. There is clearly a complex balance between virus immune evasion and host immune recognition over a lifetime. This poses the important question of whether long-term evasion or manipulation of the immune response driven by CMV is detrimental to health. In this meeting report, three groups used the murine model of CMV (MCMV) to examine if the contribution of the virus to immune senescence is set by the (i) initial viral inoculum, (ii) inflation of T cell responses, (iii) or the balance between functionally distinct effector CD4+ T cells. The work of other groups studying the CMV response in humans is discussed. Their work asks whether the ability to make immune responses to new antigens is compromised by (i) age and HCMV carriage, (ii) long-term exposure to HCMV giving rise to an overall immunosuppressive environment and increased levels of latent virus, or (iii) adapted virus mutants (used as potential vaccines) that have the capacity to elicit conventional and unconventional T cell responses.

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

Lentiviral diseases of sheep and goats: chronic pneumonia leukoencephalomyelitis and arthritis.

PubMed

Narayan, O; Cork, L C

1985-01-01

This review describes the pathogenesis of a slowly progressive disease complex caused by naturally occurring nononcogenic retroviruses in sheep and goats. In nature, infections are usually clinically silent, but disease may manifest itself after prolonged incubation periods. Clinically, this is seen as dyspnea, progressive paralysis, and/or progressive arthritis. In all organs the basic lesion is inflammatory with infiltration and proliferation of lymphocytes, plasma cells, and macrophages. Other organ-specific pathologic changes such as primary demyelination in the central nervous system and degeneration of cartilaginous structures in joints accompany inflammation. The viruses infect tissue-specific macrophage populations in vivo. Viral replication in these cells is restricted to minimal levels but continues indefinitely in the animal as a result of either failure to induce specific neutralizing antibodies or antigenic drift when neutralizing antibodies develop. Consistent low-grade viral replication sets the pace for disease by providing continuous antigenic stimulation for the inflammatory cellular immune response or antibodies that localize in the target tissues. These cells and immune complexes may have adverse effects on indigenous cell populations.

Central and peripheral reservoirs of feline immunodeficiency virus in cats: a review.

PubMed

Eckstrand, Chrissy D; Sparger, Ellen E; Murphy, Brian G

2017-08-01

Infection with feline immunodeficiency virus (FIV), a lentivirus similar to human immunodeficiency virus (HIV), results in lifelong viral persistence and progressive immunopathology in the cat. FIV has the ability to infect and produce infectious virus in a number of different cell types. FIV provirus can also be maintained in a replication-competent but transcriptionally quiescent state, facilitating viral persistence over time. Immediately after the initial infection, FIV infection quickly disseminates to many anatomical compartments within the host including lymphoid organs, gastrointestinal tract and brain. Collectively, the anatomic and cellular compartments that harbour FIV provirus constitute the viral reservoir and contain foci of both ongoing viral replication and transcriptionally restricted virus that may persist over time. The relative importance of the different phenotypes observed for infected cells, anatomic compartment, replication status and size of the reservoir represent crucial areas of investigation for developing effective viral suppression and eradication therapies. In this review, we discuss what is currently known about FIV reservoirs, and emphasize the utility of the FIV-infected cat as a model for the HIV-infected human.

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

PubMed Central

Courtney, David G.

2018-01-01

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

Enhanced inhibition of parvovirus B19 replication by cidofovir in extendedly exposed erythroid progenitor cells.

PubMed

Bonvicini, Francesca; Bua, Gloria; Manaresi, Elisabetta; Gallinella, Giorgio

2016-07-15

Human parvovirus B19 (B19V) commonly induces self-limiting infections but can also cause severe clinical manifestations in patients with underlying haematological disorders or with immune system deficits. Currently, therapeutic options for B19V entirely rely on symptomatic and supportive treatments since a specific antiviral therapy is not yet available. Recently a first step in the research for active compounds inhibiting B19V replication has allowed identifying the acyclic nucleoside phosphonate cidofovir (CDV). Herein, the effect of CDV against B19V replication was characterized in human erythroid progenitor cells (EPCs) cultured and infected following different experimental approaches to replicate in vitro the infection of an expanding erythroid cell population in the bone marrow. B19V replication was selectively inhibited both in infected EPCs extendedly exposed to CDV 500μM (viral inhibition 82%) and in serially infected EPCs cultures with passage of the virus progeny, constantly under drug exposure (viral inhibition 99%). In addition, a potent inhibitory effect against B19V (viral inhibition 92%) was assessed in a short-term infection of EPCs treated with CDV 500μM 1day before viral infection. In the evaluated experimental conditions, the enhanced effect of CDV against B19V might be ascribed both to the increased intracellular drug concentration achieved by extended exposure, and to a progressive reduction in efficiency of the replicative process within treated EPCs population. Copyright © 2016 Elsevier B.V. All rights reserved.

Identifying Early Target Cells of Nipah Virus Infection in Syrian Hamsters

PubMed Central

Baseler, Laura; Scott, Dana P.; Saturday, Greg; Horne, Eva; Rosenke, Rebecca; Thomas, Tina; Meade-White, Kimberly; Haddock, Elaine; Feldmann, Heinz

2016-01-01

Background Nipah virus causes respiratory and neurologic disease with case fatality rates up to 100% in individual outbreaks. End stage lesions have been described in the respiratory and nervous systems, vasculature and often lymphoid organs in fatal human cases; however, the initial target organs of Nipah virus infection have not been identified. Here, we detected the initial target tissues and cells of Nipah virus and tracked virus dissemination during the early phase of infection in Syrian hamsters inoculated with a Nipah virus isolate from Malaysia (NiV-M) or Bangladesh (NiV-B). Methodology/Principal Findings Syrian hamsters were euthanized between 4 and 48 hours post intranasal inoculation and tissues were collected and analyzed for the presence of viral RNA, viral antigen and infectious virus. Virus replication was first detected at 8 hours post inoculation (hpi). Nipah virus initially targeted type I pneumocytes, bronchiolar respiratory epithelium and alveolar macrophages in the lung and respiratory and olfactory epithelium lining the nasal turbinates. By 16 hpi, virus disseminated to epithelial cells lining the larynx and trachea. Although the pattern of viral dissemination was similar for both virus isolates, the rate of spread was slower for NiV-B. Infectious virus was not detected in the nervous system or blood and widespread vascular infection and lesions within lymphoid organs were not observed, even at 48 hpi. Conclusions/Significance Nipah virus initially targets the respiratory system. Virus replication in the brain and infection of blood vessels in non-respiratory tissues does not occur during the early phase of infection. However, virus replicates early in olfactory epithelium and may serve as the first step towards nervous system dissemination, suggesting that development of vaccines that block virus dissemination or treatments that can access the brain and spinal cord and directly inhibit virus replication may be necessary for preventing central nervous system pathology. PMID:27812087

Host Pah1p phosphatidate phosphatase limits viral replication by regulating phospholipid synthesis

PubMed Central

Zhang, Zhenlu; He, Guijuan; Catanzaro, Nicholas; Wu, Zujian; Xie, Lianhui

2018-01-01

Replication of positive-strand RNA viruses [(+)RNA viruses] takes place in membrane-bound viral replication complexes (VRCs). Formation of VRCs requires virus-mediated manipulation of cellular lipid synthesis. Here, we report significantly enhanced brome mosaic virus (BMV) replication and much improved cell growth in yeast cells lacking PAH1 (pah1Δ), the sole yeast ortholog of human LIPIN genes. PAH1 encodes Pah1p (phosphatidic acid phosphohydrolase), which converts phosphatidate (PA) to diacylglycerol that is subsequently used for the synthesis of the storage lipid triacylglycerol. Inactivation of Pah1p leads to altered lipid composition, including high levels of PA, total phospholipids, ergosterol ester, and free fatty acids, as well as expansion of the nuclear membrane. In pah1Δ cells, BMV replication protein 1a and double-stranded RNA localized to the extended nuclear membrane, there was a significant increase in the number of VRCs formed, and BMV genomic replication increased by 2-fold compared to wild-type cells. In another yeast mutant that lacks both PAH1 and DGK1 (encodes diacylglycerol kinase converting diacylglycerol to PA), which has a normal nuclear membrane but maintains similar lipid compositional changes as in pah1Δ cells, BMV replicated as efficiently as in pah1Δ cells, suggesting that the altered lipid composition was responsible for the enhanced BMV replication. We further showed that increased levels of total phospholipids play an important role because the enhanced BMV replication required active synthesis of phosphatidylcholine, the major membrane phospholipid. Moreover, overexpression of a phosphatidylcholine synthesis gene (CHO2) promoted BMV replication. Conversely, overexpression of PAH1 or plant PAH1 orthologs inhibited BMV replication in yeast or Nicotiana benthamiana plants. Competing with its host for limited resources, BMV inhibited host growth, which was markedly alleviated in pah1Δ cells. Our work suggests that Pah1p promotes storage lipid synthesis and thus represses phospholipid synthesis, which in turn restricts both viral replication and cell growth during viral infection. PMID:29649282

Inhibition of herpesvirus and influenza virus replication by blocking polymerase subunit interactions.

PubMed

Palù, Giorgio; Loregian, Arianna

2013-09-01

Protein-protein interactions (PPIs) play a key role in many biological processes, including virus replication in the host cell. Since most of the PPIs are functionally essential, a possible strategy to inhibit virus replication is based on the disruption of viral protein complexes by peptides or small molecules that interfere with subunit interactions. In particular, an attractive target for antiviral drugs is the binding between the subunits of essential viral enzymes. This review describes the development of new antiviral compounds that inhibit herpesvirus and influenza virus replication by blocking interactions between subunit proteins of their polymerase complexes. Copyright © 2013 Elsevier B.V. All rights reserved.

The E1 Protein of Human Papillomavirus Type 16 Is Dispensable for Maintenance Replication of the Viral Genome

PubMed Central

Egawa, Nagayasu; Nakahara, Tomomi; Ohno, Shin-ichi; Narisawa-Saito, Mako; Yugawa, Takashi; Fujita, Masatoshi; Yamato, Kenji; Natori, Yukikazu

2012-01-01

Papillomavirus genomes are thought to be amplified to about 100 copies per cell soon after infection, maintained constant at this level in basal cells, and amplified for viral production upon keratinocyte differentiation. To determine the requirement for E1 in viral DNA replication at different stages, an E1-defective mutant of the human papillomavirus 16 (HPV16) genome featuring a translation termination mutation in the E1 gene was used. The ability of the mutant HPV16 genome to replicate as nuclear episomes was monitored with or without exogenous expression of E1. Unlike the wild-type genome, the E1-defective HPV16 genome became established in human keratinocytes only as episomes in the presence of exogenous E1 expression. Once established, it could replicate with the same efficiency as the wild-type genome, even after the exogenous E1 was removed. However, upon calcium-induced keratinocyte differentiation, once again amplification was dependent on exogenous E1. These results demonstrate that the E1 protein is dispensable for maintenance replication but not for initial and productive replication of HPV16. PMID:22238312

The Function of Herpes Simplex Virus Genes: A Primer for Genetic Engineering of Novel Vectors

NASA Astrophysics Data System (ADS)

Roizman, Bernard

1996-10-01

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

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.

DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription.

PubMed

Frouco, Gonçalo; Freitas, Ferdinando B; Coelho, João; Leitão, Alexandre; Martins, Carlos; Ferreira, Fernando

2017-06-15

African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (-78.3%), and reduced transcription of a late viral gene (-47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV. IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV. Copyright © 2017 American Society for Microbiology.

Cyclooxygenase-2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents.

PubMed

Lin, Chun-Kuang; Tseng, Chin-Kai; Wu, Yu-Hsuan; Liaw, Chih-Chuang; Lin, Chun-Yu; Huang, Chung-Hao; Chen, Yen-Hsu; Lee, Jin-Ching

2017-03-20

Cyclooxygenase-2 (COX-2) is one of the important mediators of inflammation in response to viral infection, and it contributes to viral replication, for example, cytomegalovirus or hepatitis C virus replication. The role of COX-2 in dengue virus (DENV) replication remains unclear. In the present study, we observed an increased level of COX-2 in patients with dengue fever compared with healthy donors. Consistent with the clinical data, an elevated level of COX-2 expression was also observed in DENV-infected ICR suckling mice. Using cell-based experiments, we revealed that DENV-2 infection significantly induced COX-2 expression and prostaglandin E 2 (PGE 2 ) production in human hepatoma Huh-7 cells. The exogenous expression of COX-2 or PGE 2 treatment dose-dependently enhanced DENV-2 replication. In contrast, COX-2 gene silencing and catalytic inhibition sufficiently suppressed DENV-2 replication. In an ICR suckling mouse model, we identified that the COX-2 inhibitor NS398 protected mice from succumbing to life-threatening DENV-2 infection. By using COX-2 promoter-based analysis and specific inhibitors against signaling molecules, we identified that NF-κB and MAPK/JNK are critical factors for DENV-2-induced COX-2 expression and viral replication. Altogether, our results reveal that COX-2 is an important factor for DENV replication and can serve as a potential target for developing therapeutic agents against DENV infection.

G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV.

PubMed

Madireddy, Advaitha; Purushothaman, Pravinkumar; Loosbroock, Christopher P; Robertson, Erle S; Schildkraut, Carl L; Verma, Subhash C

2016-05-05

Kaposi's sarcoma associated herpesvirus (KSHV) establishes life-long latent infection by persisting as an extra-chromosomal episome in the infected cells and by maintaining its genome in dividing cells. KSHV achieves this by tethering its epigenome to the host chromosome by latency associated nuclear antigen (LANA), which binds in the terminal repeat (TR) region of the viral genome. Sequence analysis of the TR, a GC-rich DNA element, identified several potential Quadruplex G-Rich Sequences (QGRS). Since quadruplexes have the tendency to obstruct DNA replication, we used G-quadruplex stabilizing compounds to examine their effect on latent DNA replication and the persistence of viral episomes. Our results showed that these G-quadruplex stabilizing compounds led to the activation of dormant origins of DNA replication, with preferential bi-directional pausing of replications forks moving out of the TR region, implicating the role of the G-rich TR in the perturbation of episomal DNA replication. Over time, treatment with PhenDC3 showed a loss of viral episomes in the infected cells. Overall, these data show that G-quadruplex stabilizing compounds retard the progression of replication forks leading to a reduction in DNA replication and episomal maintenance. These results suggest a potential role for G-quadruplex stabilizers in the treatment of KSHV-associated diseases. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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.

Vertical transmission of sublethal granulovirus infection in the Indian meal moth, Plodia interpunctella.

PubMed

Burden, J P; Griffiths, C M; Cory, J S; Smith, P; Sait, S M

2002-03-01

Knowledge of the mechanisms of pathogen persistence in relation to fluctuations in host density is crucial to our understanding of disease dynamics. In the case of insect baculoviruses, which are typically transmitted horizontally via a lifestage that can persist outside the host, a key issue that remains to be elucidated is whether the virus can also be transmitted vertically as a sublethal infection. We show that RNA transcripts for the Plodia interpunctella GV granulin gene are present in a high proportion of P. interpunctella insects that survive virus challenge. Granulin is a late-expressed gene that is only transcribed after viral genome replication, its presence thus strongly indicates that viral genome replication has occurred. Almost all insects surviving the virus challenge tested positive for viral RNA in the larval and pupal stage. However, this proportion declined in the emerging adults. Granulin mRNA was also detected in both the ovaries and testes, which may represent a putative mechanism by which reduced fecundity in sublethally affected hosts might be manifested. RNA transcripts were also detected in 60-80% of second-generation larvae that were derived from mating surviving adults, but there was no difference between the sexes, with both males and females capable of transmitting a sublethal infection to their offspring. The data indicate that low-level persistent infection, with at least limited gene expression, can occur in P. interpunctella following survival of a granulovirus challenge. We believe that this is the first demonstration of a persistent, sublethal infection by a baculovirus to be initiated by a sublethal virus dose. We hypothesize that the 'latent' baculovirus infections frequently referred to in the literature may also be low level persistent, sublethal infections resulting from survival from initial baculovirus exposure.

Adaptive Mutations That Occurred during Circulation in Humans of H1N1 Influenza Virus in the 2009 Pandemic Enhance Virulence in Mice.

PubMed

Otte, A; Sauter, M; Daxer, M A; McHardy, A C; Klingel, K; Gabriel, G

2015-07-01

During the 2009 H1N1 influenza pandemic, infection attack rates were particularly high among young individuals who suffered from pneumonia with occasional death. Moreover, previously reported determinants of mammalian adaptation and pathogenicity were not present in 2009 pandemic H1N1 influenza A viruses. Thus, it was proposed that unknown viral factors might have contributed to disease severity in humans. In this study, we performed a comparative analysis of two clinical 2009 pandemic H1N1 strains that belong to the very early and later phases of the pandemic. We identified mutations in the viral hemagglutinin (HA) and the nucleoprotein (NP) that occurred during pandemic progression and mediate increased virulence in mice. Lethal disease outcome correlated with elevated viral replication in the alveolar epithelium, increased proinflammatory cytokine and chemokine responses, pneumonia, and lymphopenia in mice. These findings show that viral mutations that have occurred during pandemic circulation among humans are associated with severe disease in mice. In this study, novel determinants of 2009 pandemic H1N1 influenza pathogenicity were identified in the viral hemagglutinin (HA) and the nucleoprotein (NP) genes. In contrast to highly pathogenic avian influenza viruses, increased virulence in mice did not correlate with enhanced polymerase activity but with reduced activity. Lethal 2009 pandemic H1N1 infection in mice correlated with lymphopenia and severe pneumonia. These studies suggest that molecular mechanisms that mediate 2009 pandemic H1N1 influenza pathogenicity are distinct from those that mediate avian influenza virus pathogenicity in mice. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Dengue virus in sub-tropical northern and central Viet Nam: population immunity and climate shape patterns of viral invasion and maintenance.

PubMed

Rabaa, Maia A; Simmons, Cameron P; Fox, Annette; Le, Mai Quynh; Nguyen, Thuy Thi Thu; Le, Hai Yen; Gibbons, Robert V; Nguyen, Xuyen Thanh; Holmes, Edward C; Aaskov, John G

2013-01-01

Dengue virus transmission occurs in both epidemic and endemic cycles across tropical and sub-tropical regions of the world. Incidence is particularly high in much of Southeast Asia, where hyperendemic transmission plagues both urban and rural populations. However, endemicity has not been established in some areas with climates that may not support year-round viral transmission. An understanding of how dengue viruses (DENV) enter these environments and whether the viruses persist in inapparent local transmission cycles is central to understanding how dengue emerges in areas at the margins of endemic transmission. Dengue is highly endemic in tropical southern Vietnam, while increasingly large seasonal epidemics have occurred in northern Viet Nam over the last decade. We have investigated the spread of DENV-1 throughout Vietnam to determine the routes by which the virus enters northern and central regions of the country. Phylogeographic analysis of 1,765 envelope (E) gene sequences from Southeast Asia revealed frequent movement of DENV between neighboring human populations and strong local clustering of viral lineages. Long-distance migration of DENV between human population centers also occurred regularly and on short time-scales, indicating human-mediated viral invasion into northern Vietnam. Human populations in southern Vietnam were found to be the primary source of DENV circulating throughout the country, while central and northern Vietnam acted as sink populations, likely due to reduced connectedness to other populations in the case of the central regions and to the influence of temperature variability on DENV replication and vector survival and competence in the north. Finally, phylogeographic analyses suggested that viral movement follows a gravity model and indicates that population immunity and physical and economic connections between populations may play important roles in shaping patterns of DENV transmission.

Dengue Virus in Sub-tropical Northern and Central Viet Nam: Population Immunity and Climate Shape Patterns of Viral Invasion and Maintenance

PubMed Central

Rabaa, Maia A.; Simmons, Cameron P.; Fox, Annette; Le, Mai Quynh; Nguyen, Thuy Thi Thu; Le, Hai Yen; Gibbons, Robert V.; Nguyen, Xuyen Thanh; Holmes, Edward C.; Aaskov, John G.

2013-01-01

Dengue virus transmission occurs in both epidemic and endemic cycles across tropical and sub-tropical regions of the world. Incidence is particularly high in much of Southeast Asia, where hyperendemic transmission plagues both urban and rural populations. However, endemicity has not been established in some areas with climates that may not support year-round viral transmission. An understanding of how dengue viruses (DENV) enter these environments and whether the viruses persist in inapparent local transmission cycles is central to understanding how dengue emerges in areas at the margins of endemic transmission. Dengue is highly endemic in tropical southern Vietnam, while increasingly large seasonal epidemics have occurred in northern Viet Nam over the last decade. We have investigated the spread of DENV-1 throughout Vietnam to determine the routes by which the virus enters northern and central regions of the country. Phylogeographic analysis of 1,765 envelope (E) gene sequences from Southeast Asia revealed frequent movement of DENV between neighboring human populations and strong local clustering of viral lineages. Long-distance migration of DENV between human population centers also occurred regularly and on short time-scales, indicating human-mediated viral invasion into northern Vietnam. Human populations in southern Vietnam were found to be the primary source of DENV circulating throughout the country, while central and northern Vietnam acted as sink populations, likely due to reduced connectedness to other populations in the case of the central regions and to the influence of temperature variability on DENV replication and vector survival and competence in the north. Finally, phylogeographic analyses suggested that viral movement follows a gravity model and indicates that population immunity and physical and economic connections between populations may play important roles in shaping patterns of DENV transmission. PMID:24340118

Lymph Node Cellular and Viral Dynamics in Natural Hosts and Impact for HIV Cure Strategies.

PubMed

Huot, Nicolas; Bosinger, Steven E; Paiardini, Mirko; Reeves, R Keith; Müller-Trutwin, Michaela

2018-01-01

Combined antiretroviral therapies (cARTs) efficiently control HIV replication leading to undetectable viremia and drastic increases in lifespan of people living with HIV. However, cART does not cure HIV infection as virus persists in cellular and anatomical reservoirs, from which the virus generally rebounds soon after cART cessation. One major anatomical reservoir are lymph node (LN) follicles, where HIV persists through replication in follicular helper T cells and is also trapped by follicular dendritic cells. Natural hosts of SIV, such as African green monkeys and sooty mangabeys, generally do not progress to disease although displaying persistently high viremia. Strikingly, these hosts mount a strong control of viral replication in LN follicles shortly after peak viremia that lasts throughout infection. Herein, we discuss the potential interplay between viral control in LNs and the resolution of inflammation, which is characteristic for natural hosts. We furthermore detail the differences that exist between non-pathogenic SIV infection in natural hosts and pathogenic HIV/SIV infection in humans and macaques regarding virus target cells and replication dynamics in LNs. Several mechanisms have been proposed to be implicated in the strong control of viral replication in natural host's LNs, such as NK cell-mediated control, that will be reviewed here, together with lessons and limitations of in vivo cell depletion studies that have been performed in natural hosts. Finally, we discuss the impact that these insights on viral dynamics and host responses in LNs of natural hosts have for the development of strategies toward HIV cure.

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.

Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence

PubMed Central

Carpentier, Alexandre; Barez, Pierre-Yves; Hamaidia, Malik; Gazon, Hélène; de Brogniez, Alix; Perike, Srikanth; Gillet, Nicolas; Willems, Luc

2015-01-01

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans. PMID:26198240

Exosome-mediated miR-146a transfer suppresses type I interferon response and facilitates EV71 infection

PubMed Central

Fu, Yuxuan; Zhang, Li; Zhang, Fang; Tang, Ting; Zhou, Qi; Feng, Chunhong; Jin, Yu

2017-01-01

Exosomes can transfer genetic materials between cells. Their roles in viral infections are beginning to be appreciated. Researches have shown that exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modulate recipient’s cellular response and result in productive infection of the recipient host. Here, we showed that EV71 infection resulted in upregulated exosome secretion and differential packaging of the viral genomic RNA and miR-146a into exosomes. We provided evidence showing that miR-146a was preferentially enriched in exosomes while the viral RNA was not in infected cells. Moreover, the exosomes contained replication-competent EV71 RNA in complex with miR-146a, Ago2, and GW182 and could mediate EV71 transmission independent of virus-specific receptor. The exosomal viral RNA could be transferred to and replicate in a new target cell while the exosomal miR-146a suppressed type I interferon response in the target cell, thus facilitating the viral replication. Additionally, we found that the IFN-stimulated gene factors (ISGs), BST-2/tetherin, were involved in regulating EV71-induced upregulation of exosome secretion. Importantly, in vivo study showed that exosomal viral RNA exhibited differential tissue accumulation as compared to the free virus particles. Together, our findings provide evidence that exosomes secreted by EV71-infected cells selectively packaged high level miR-146a that can be functionally transferred to and facilitate exosomal EV71 RNA to replicate in the recipient cells by suppressing type I interferon response. PMID:28910400

Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation.

PubMed

Pyeon, Dohun; Timani, Khalid Amine; Gulraiz, Fahad; He, Johnny J; Park, In-Woo

2016-09-02

HIV-1 Nef is necessary and may be sufficient for HIV-1-associated AIDS pathogenicity, in that knockout of Nef alone can protect HIV-infected patients from AIDS. We therefore investigated the feasibility of physical knockout of Nef, using the host ubiquitin proteasome system in HIV-1-infected cells. Our co-immunoprecipitation analysis demonstrated that Nef interacted with ubiquitin specific protease 15 (USP15), and that USP15, which is known to stabilize cellular proteins, degraded Nef. Nef could also cause decay of USP15, although Nef-mediated degradation of USP15 was weaker than USP15-mediated Nef degradation. Direct interaction between Nef and USP15 was essential for the observed reciprocal decay of the proteins. Further, USP15 degraded not only Nef but also HIV-1 structural protein, Gag, thereby substantially inhibiting HIV-1 replication. However, Gag did not degrade USP15, indicating that the Nef and USP15 complex, in distinction to other viral proteins, play an integral role in coordinating viral protein degradation and hence HIV-1 replication. Moreover, Nef and USP15 globally suppressed ubiquitylation of cellular proteins, indicating that these proteins are major determinants for the stability of cellular as well as viral proteins. Taken together, these data indicate that Nef and USP15 are vital in regulating degradation of viral and cellular proteins and thus HIV-1 replication, and specific degradation of viral, not cellular proteins, by USP15 points to USP15 as a candidate therapeutic agent to combat AIDS by eliminating viral proteins from the infected cells via USP15-mediated proteosomal degradation. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Emetine inhibits replication of RNA and DNA viruses without generating drug-resistant virus variants.

PubMed

Khandelwal, Nitin; Chander, Yogesh; Rawat, Krishan Dutt; Riyesh, Thachamvally; Nishanth, Chikkahonnaiah; Sharma, Shalini; Jindal, Naresh; Tripathi, Bhupendra N; Barua, Sanjay; Kumar, Naveen

2017-08-01

At a noncytotoxic concentration, emetine was found to inhibit replication of DNA viruses [buffalopoxvirus (BPXV) and bovine herpesvirus 1 (BHV-1)] as well as RNA viruses [peste des petits ruminants virus (PPRV) and Newcastle disease virus (NDV)]. Using the time-of-addition and virus step-specific assays, we showed that emetine treatment resulted in reduced synthesis of viral RNA (PPRV and NDV) and DNA (BPXV and BHV-1) as well as inhibiting viral entry (NDV and BHV-1). In addition, emetine treatment also resulted in decreased synthesis of viral proteins. In a cell free endogenous viral polymerase assay, emetine was found to significantly inhibit replication of NDV, but not BPXV genome, suggesting that besides directly inhibiting specific viral polymerases, emetine may also target other factors essentially required for efficient replication of the viral genome. Moreover, emetine was found to significantly inhibit BPXV-induced pock lesions on chorioallantoic membrane (CAM) along with associated mortality of embryonated chicken eggs. At a lethal dose 50 (LD 50 ) of 126.49 ng/egg and at an effective concentration 50 (EC 50 ) of 3.03 ng/egg, the therapeutic index of the emetine against BPXV was determined to be 41.74. Emetine was also found to significantly delay NDV-induced mortality in chicken embryos associated with reduced viral titers. Further, emetine-resistant mutants were not observed upon long-term (P = 25) sequential passage of BPXV and NDV in cell culture. Collectively, we have extended the effective antiviral activity of emetine against diverse groups of DNA and RNA viruses and propose that emetine could provide significant therapeutic value against some of these viruses without inducing an antiviral drug-resistant phenotype. Copyright © 2017 Elsevier B.V. All rights reserved.

Transcriptional Downregulation of ORF50/Rta by Methotrexate Inhibits the Switch of Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 from Latency to Lytic Replication

PubMed Central

Curreli, Francesca; Cerimele, Francesca; Muralidhar, Sumitra; Rosenthal, Leonard J.; Cesarman, Ethel; Friedman-Kien, Alvin E.; Flore, Ornella

2002-01-01

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a cellular dihydrofolate reductase (DHFR) homologue. Methotrexate (MTX), a potent anti-inflammatory agent, inhibits cellular DHFR activity. We investigated the effect of noncytotoxic doses of MTX on latency and lytic KSHV replication in two KSHV-infected primary effusion lymphoma cell lines (BC-3 and BC-1) and in MTX-resistant BC-3 cells (MTX-R-BC-3 cells). Treatment with MTX completely prevented tetradecanoyl phorbol acetate-induced viral DNA replication and strongly decreased viral lytic transcript levels, even in MTX-resistant cells. However, the same treatment had no effect on transcription of cellular genes and KSHV latent genes. One of the lytic transcripts inhibited by MTX, ORF50/Rta (open reading frame), is an immediate-early gene encoding a replication-transcription activator required for expression of other viral lytic genes. Therefore, transcription of genes downstream of ORF50/Rta was inhibited, including those encoding the viral G-protein-coupled receptor (GPCR), viral interleukin-6, and K12/kaposin, which have been shown to be transforming in vitro and oncogenic in mice. Resistance to MTX has been documented in cultured cells and also in patients treated with this drug. However, MTX showed an inhibitory activity even in MTX-R-BC-3 cells. Two currently available antiherpesvirus drugs, cidofovir and foscarnet, had no effect on the transcription of these viral oncogenes and ORF50/Rta. MTX is the first example of a compound shown to downregulate the expression of ORF50/Rta and therefore prevent viral transforming gene transcription. Given that the expression of these genes may be important for tumor development, MTX could play a role in the future management of KSHV-associated malignancies. PMID:11967335

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.

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

Integrase inhibitor reversal dynamics indicate unintegrated HIV-1 dna initiate de novo integration.

PubMed

Thierry, Sylvain; Munir, Soundasse; Thierry, Eloïse; Subra, Frédéric; Leh, Hervé; Zamborlini, Alessia; Saenz, Dyana; Levy, David N; Lesbats, Paul; Saïb, Ali; Parissi, Vincent; Poeschla, Eric; Deprez, Eric; Delelis, Olivier

2015-03-12

Genomic integration, an obligate step in the HIV-1 replication cycle, is blocked by the integrase inhibitor raltegravir. A consequence is an excess of unintegrated viral DNA genomes, which undergo intramolecular ligation and accumulate as 2-LTR circles. These circularized genomes are also reliably observed in vivo in the absence of antiviral therapy and they persist in non-dividing cells. However, they have long been considered as dead-end products that are not precursors to integration and further viral propagation. Here, we show that raltegravir action is reversible and that unintegrated viral DNA is integrated in the host cell genome after raltegravir removal leading to HIV-1 replication. Using quantitative PCR approach, we analyzed the consequences of reversing prolonged raltegravir-induced integration blocks. We observed, after RAL removal, a decrease of 2-LTR circles and a transient increase of linear DNA that is subsequently integrated in the host cell genome and fuel new cycles of viral replication. Our data highly suggest that 2-LTR circles can be used as a reserve supply of genomes for proviral integration highlighting their potential role in the overall HIV-1 replication cycle.

The host ubiquitin-dependent segregase VCP/p97 is required for the onset of human cytomegalovirus replication

PubMed Central

Lin, Yao-Tang; Grey, Finn

2017-01-01

The human cytomegalovirus major immediate early proteins IE1 and IE2 are critical drivers of virus replication and are considered pivotal in determining the balance between productive and latent infection. IE1 and IE2 are derived from the same primary transcript by alternative splicing and regulation of their expression likely involves a complex interplay between cellular and viral factors. Here we show that knockdown of the host ubiquitin-dependent segregase VCP/p97, results in loss of IE2 expression, subsequent suppression of early and late gene expression and, ultimately, failure in virus replication. RNAseq analysis showed increased levels of IE1 splicing, with a corresponding decrease in IE2 splicing following VCP knockdown. Global analysis of viral transcription showed the expression of a subset of viral genes is not reduced despite the loss of IE2 expression, including UL112/113. Furthermore, Immunofluorescence studies demonstrated that VCP strongly colocalised with the viral replication compartments in the nucleus. Finally, we show that NMS-873, a small molecule inhibitor of VCP, is a potent HCMV antiviral with potential as a novel host targeting therapeutic for HCMV infection. PMID:28494016

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

PubMed

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

2016-06-30

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

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

Kaposi’s sarcoma–associated herpesvirus stably clusters its genomes across generations to maintain itself extrachromosomally

PubMed Central

Chiu, Ya-Fang; Sugden, Arthur U.

2017-01-01

Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi’s sarcoma–associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning and viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected. PMID:28696226

Kaposi’s sarcoma–associated herpesvirus stably clusters its genomes across generations to maintain itself extrachromosomally

DOE PAGES

Chiu, Ya-Fang; Sugden, Arthur U.; Fox, Kathryn; ...

2017-07-10

Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi’s sarcoma–associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning andmore » viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected.« less

Kaposi’s sarcoma–associated herpesvirus stably clusters its genomes across generations to maintain itself extrachromosomally

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

Chiu, Ya-Fang; Sugden, Arthur U.; Fox, Kathryn

Genetic elements that replicate extrachromosomally are rare in mammals; however, several human tumor viruses, including the papillomaviruses and the gammaherpesviruses, maintain their plasmid genomes by tethering them to cellular chromosomes. We have uncovered an unprecedented mechanism of viral replication: Kaposi’s sarcoma–associated herpesvirus (KSHV) stably clusters its genomes across generations to maintain itself extrachromosomally. To identify and characterize this mechanism, we developed two complementary, independent approaches: live-cell imaging and a predictive computational model. The clustering of KSHV requires the viral protein, LANA1, to bind viral genomes to nucleosomes arrayed on both cellular and viral DNA. Clustering affects both viral partitioning andmore » viral genome numbers of KSHV. The clustering of KSHV plasmids provides it with an effective evolutionary strategy to rapidly increase copy numbers of genomes per cell at the expense of the total numbers of cells infected.« less

Mathematical models of immune effector responses to viral infections: Virus control versus the development of pathology

NASA Astrophysics Data System (ADS)

Wodarz, Dominik

2005-12-01

This article reviews mathematical models which have investigated the importance of lytic and non-lytic immune responses for the control of viral infections. Lytic immune responses fight the virus by killing infected cells, while non-lytic immune responses fight the virus by inhibiting viral replication while leaving the infected cell alive. The models suggest which types or combinations of immune responses are required to resolve infections which vary in their characteristics, such as the rate of viral replication and the rate of virus-induced target cell death. This framework is then applied to persistent infections and viral evolution. It is investigated how viral evolution and antigenic escape can influence the relative balance of lytic and non-lytic responses over time, and how this might correlate with the transition from an asymptomatic infection to pathology. This is discussed in the specific context of hepatitis C virus infection.

Multiple Restrictions of Human Immunodeficiency Virus Type 1 in Feline Cells▿

PubMed Central

Münk, Carsten; Zielonka, Jörg; Constabel, Hannelore; Kloke, Björn-Philipp; Rengstl, Benjamin; Battenberg, Marion; Bonci, Francesca; Pistello, Mauro; Löchelt, Martin; Cichutek, Klaus

2007-01-01

The productive replication of human immunodeficiency virus type 1 (HIV-1) occurs exclusively in defined cells of human or chimpanzee origin, explaining why heterologous animal models for HIV replication, pathogenesis, vaccination, and therapy are not available. This lack of an animal model for HIV-1 studies prompted us to examine the susceptibility of feline cells in order to evaluate the cat (Felis catus) as an animal model for studying HIV-1. Here, we report that feline cell lines harbor multiple restrictions with respect to HIV-1 replication. The feline CD4 receptor does not permit virus infection. Feline T-cell lines MYA-1 and FeT-1C showed postentry restrictions resulting in low HIV-1 luciferase reporter activity and low expression of viral Gag-Pol proteins when pseudotyped vectors were used. Feline fibroblastic CrFK and KE-R cells, expressing human CD4 and CCR5, were very permissive for viral entry and HIV-long terminal repeat-driven expression but failed to support spreading infection. KE-R cells displayed a profound block with respect to release of HIV-1 particles. In contrast, CrFK cells allowed very efficient particle production; however, the CrFK cell-derived HIV-1 particles had low specific infectivity. We subsequently identified feline apolipoprotein B-editing catalytic polypeptide 3 (feAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity. CrFK cells express at least three different APOBEC3s: APOBEC3C, APOBEC3H, and APOBEC3CH. While the feAPOBEC3C did not significantly inhibit HIV-1, the feAPOBEC3H and feAPOBEC3CH induced G to A hypermutations of the viral cDNA and reduced the infectivity ∼10- to ∼40-fold. PMID:17459941

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

PubMed Central

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

2017-01-01

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

Parvoviral Left-End Hairpin Ears Are Essential during Infection for Establishing a Functional Intranuclear Transcription Template and for Efficient Progeny Genome Encapsidation

PubMed Central

Li, Lei; Cotmore, Susan F.

2013-01-01

The 121-nucleotide left-end telomere of Minute Virus of Mice (MVM) can be folded into a Y-shaped hairpin with short axial ears that are highly conserved within genus Parvovirus. To explore their potential role(s) during infection, we constructed infectious plasmid clones that lacked one or other ear. Although these were nonviable when transfected into A9 cells, excision of the viral genome and DNA amplification appeared normal, and viral transcripts and proteins were expressed, but progeny virion production was minimal, supporting the idea of a potential role for the ears in genome packaging. To circumvent the absence of progeny that confounded further analysis of these mutants, plasmids were transfected into 293T cells both with and without an adenovirus helper construct, generating single bursts of progeny. These virions bound to A9 cells and were internalized but failed to initiate viral transcription, protein expression, or DNA replication. No defects in mutant virion stability or function could be detected in vitro. Significantly, mutant capsid gene expression and DNA replication could be rescued by coinfection with wild-type virions carrying a replication-competent, capsid-gene-replacement vector. To pinpoint where such complementation occurred, prior transfection of plasmids expressing only MVM nonstructural proteins was explored. NS1 alone, but not NS2, rescued transcription and protein expression from both P4 and P38 promoters, whereas NS1 molecules deleted for their C-terminal transactivation domain did not. These results suggest that the mutant virions reach the nucleus, uncoat, and are converted to duplex DNA but require an intact left-end hairpin structure to form the initiating transcription complex. PMID:23903839

Infection cycles of large DNA viruses: Emerging themes and underlying questions

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

Mutsafi, Yael, E-mail: yael.mutsafi@weizmann.ac.il; Fridmann-Sirkis, Yael; Milrot, Elad

The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in themore » crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these ‘nuclear-like’ organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the ‘stargate’ portal that is used for genome release. Such a ‘division of labor’ is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed. - Highlights: • The discovery of giant DNA viruses blurs the distinction between viruses and cells. • Mimivirus and Vaccinia replicate exclusively in their host cytoplasm. • Mimivirus genome is delivered through a unique portal coined the Stargate. • Generation of Mimivirus internal membrane proceeds through a novel pathway.« less

A viral deubiquitylating enzyme targets viral RNA-dependent RNA polymerase and affects viral infectivity

PubMed Central

Chenon, Mélanie; Camborde, Laurent; Cheminant, Soizic; Jupin, Isabelle

2012-01-01

Selective protein degradation via the ubiquitin-proteasome system (UPS) plays an essential role in many major cellular processes, including host–pathogen interactions. We previously reported that the tightly regulated viral RNA-dependent RNA polymerase (RdRp) of the positive-strand RNA virus Turnip yellow mosaic virus (TYMV) is degraded by the UPS in infected cells, a process that affects viral infectivity. Here, we show that the TYMV 98K replication protein can counteract this degradation process thanks to its proteinase domain. In-vitro assays revealed that the recombinant proteinase domain is a functional ovarian tumour (OTU)-like deubiquitylating enzyme (DUB), as is the 98K produced during viral infection. We also demonstrate that 98K mediates in-vivo deubiquitylation of TYMV RdRp protein—its binding partner within replication complexes—leading to its stabilization. Finally, we show that this DUB activity contributes to viral infectivity in plant cells. The identification of viral RdRp as a specific substrate of the viral DUB enzyme thus reveals the intricate interplay between ubiquitylation, deubiquitylation and the interaction between viral proteins in controlling levels of RdRp and viral infectivity. PMID:22117220

High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

PubMed Central

Janovitz, Tyler; Klein, Isaac A.; Oliveira, Thiago; Mukherjee, Piali; Nussenzweig, Michel C.; Sadelain, Michel

2013-01-01

Viral integrations are important in human biology, yet genome-wide integration profiles have not been determined for many viruses. Adeno-associated virus (AAV) infects most of the human population and is a prevalent gene therapy vector. AAV integrates into the human genome with preference for a single locus, termed AAVS1. However, the genome-wide integration of AAV has not been defined, and the principles underlying this recombination remain unclear. Using a novel high-throughput approach, integrant capture sequencing, nearly 12 million AAV junctions were recovered from a human cell line, providing five orders of magnitude more data than were previously available. Forty-five percent of integrations occurred near AAVS1, and several thousand novel integration hotspots were identified computationally. Most of these occurred in genes, with dozens of hotspots targeting known oncogenes. Viral replication protein binding sites (RBS) and transcriptional activity were major factors favoring integration. In a first for eukaryotic viruses, the data reveal a unique asymmetric integration profile with distinctive directional orientation of viral genomes. These studies provide a new understanding of AAV integration biology through the use of unbiased high-throughput data acquisition and bioinformatics. PMID:23720718

Analysis of BZLF1 mRNA detection in saliva as a marker for active replication of Epstein-Barr virus.

PubMed

Fagin, Ursula; Nerbas, Linda; Vogl, Bastian; Jabs, Wolfram J

2017-06-01

Monitoring replicative Epstein-Barr virus (EBV) infection still remains a challenge in modern laboratory routine. The immediate-early protein BZLF1 mediates the switch between latent and replicate forms of EBV infection. The aim of this study was to analyze the feasibility of BZLF1 mRNA detection in saliva as a marker for active replication of the virus. Various specimens (saliva, plasma, PBMC) from 17 patients with EBV-induced infectious mononucleosis (IM) and 4 control patients were examined for expression of viral BZLF1 mRNA by means of real-time PCR. BZLF1 expression was correlated to the amount of viral DNA in either compartment. Digestion of plasma and saliva samples with DNase I allowed distinguishing between encapsidated and naked viral DNA. BZLF1 transcripts were found in all different types of specimens in varying frequencies. BZLF1 expression in saliva, PBMC, and plasma correlated with viral load in each compartment. Interestingly, those patients with detectable BZLF1 expression in saliva had a more severe course of infection with longer duration of hospitalization. In conclusion, this study demonstrates the feasibility of BZLF1 mRNA detection in saliva specimens during replicative EBV infection. Its significance for the diagnosis of reactivated EBV infection, particularly under immunosuppression, has to be elucidated in further studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Genome Cyclization as Strategy for Flavivirus RNA Replication

PubMed Central

Villordo, Sergio M.; Gamarnik, Andrea V.

2017-01-01

Long-range and local RNA-RNA contacts in viral RNA genomes result in tertiary structures that modulate the function of enhancers, promoters, and silencers during translation, RNA replication, and encapsidation. In the case of flaviviruses, the presence of inverted complementary sequences at the 5′ and 3′ ends of the genome mediate long-range RNA interactions and RNA cyclization. The circular conformation of flavivirus genomes was demonstrated to be essential for RNA amplification. New ideas about the mechanisms by which circular genomes participate in flavivirus replication have emerged in the last few years. Here, we will describe the latest information about cis-acting elements involved in flavivirus genome cyclization, RNA promoter elements required for viral polymerase recognition, and how these elements together coordinate viral RNA synthesis. PMID:18703097

Clinical significance of hepatitis B virion and SVP productivity: relationships between intrahepatic and serum markers in chronic hepatitis B patients

PubMed Central

Jackson, Kathy; Lim, Seng Gee; Sulaiman, Ali; Pakasi, Levina S; Gani, Rino A; Hasan, Irsan; Sulaiman, Andri Sanityoso; Lesmana, Laurentius A; Hammond, Rachel; Revill, Peter; Locarnini, Stephen; Bowden, Scott David

2014-01-01

Background Clinical use of hepatitis B viral (HBV) quantitative seromarker\\s remains questionable since it is not precisely known whether they represent intrahepatic viral replication. Covalently closed circular DNA (cccDNA), relaxed circular DNA (rcDNA), and pregenomic RNA (pgRNA) are more likely to represent active HBV replication and their measurement can be used to derive virion productivity (VP; rcDNA/cccDNA), subviral particle (SVP) productivity (quantitative HBsAg/cccDNA), and replicative activity (RA; pgRNA/cccDNA). These can be used to compare relative HBV replication between HBeAg-negative and -positive patients. Objective To study the clinical significance of intrahepatic HBV replication phenomenon between HBeAg-negative and -positive patients and its correlation with quantitative HBV seromarkers. Method This was a prospective study between January 2010 and December 2011. Study subjects were naive chronic hepatitis B patients from Cipto Mangunkusumo and Medistra Hospitals. All patient samples underwent liver biochemistry and HBV seromarkers testing (HBeAg, quantitative HBsAg and HBV DNA levels), and patients underwent liver biopsy. Stored liver specimens were analysed for intrahepatic rcDNA, cccDNA, and pgRNA with quantification performed by real-time PCR. Comparison of HBV markers between HBsAg-positive and -negative patients was carried out using the Mann–Whitney U-test. Pearson’s correlation test was performed among HBV intrahepatic and seromarkers using their log-transformed values. Results A total of 104 patients were enrolled in this study; 54 (51.9%) were male. Patients’ mean age was 41.9 ± 11.63 years (range 19–70 years). Sixty-one patients (58.7%) were HBeAg-negative. All HBV markers were significantly higher in HBeAg-positive than HBeAg-negative patients, except for SVP productivity and RA. Serum HBV DNA was strongly correlated with intrahepatic total HBV DNA (r = 0.771), cccDNA (r = 0.774), and rcDNA (r = 0.780) while serum quantitative HBsAg showed only moderate correlation with intrahepatic total DNA (r = 0.671), cccDNA (r = 0.632), rcDNA (r = 0.675), and SVP productivity (r = 0.557). Conclusions Serum HBV DNA concentration and quantitative HBsAg might not accurately predict intrahepatic viral activity. Virion and SVP production do not occur in parallel with replicative activity. PMID:24918014

Escape is a more common mechanism than avidity reduction for evasion of CD8+ T cell responses in primary human immunodeficiency virus type 1 infection

PubMed Central

2011-01-01

Background CD8+ T cells play an important role in control of viral replication during acute and early human immunodeficiency virus type 1 (HIV-1) infection, contributing to containment of the acute viral burst and establishment of the prognostically-important persisting viral load. Understanding mechanisms that impair CD8+ T cell-mediated control of HIV replication in primary infection is thus of importance. This study addressed the relative extent to which HIV-specific T cell responses are impacted by viral mutational escape versus reduction in response avidity during the first year of infection. Results 18 patients presenting with symptomatic primary HIV-1 infection, most of whom subsequently established moderate-high persisting viral loads, were studied. HIV-specific T cell responses were mapped in each individual and responses to a subset of optimally-defined CD8+ T cell epitopes were followed from acute infection onwards to determine whether they were escaped or declined in avidity over time. During the first year of infection, sequence variation occurred in/around 26/33 epitopes studied (79%). In 82% of cases of intra-epitopic sequence variation, the mutation was confirmed to confer escape, although T cell responses were subsequently expanded to variant sequences in some cases. In contrast, < 10% of responses to index sequence epitopes declined in functional avidity over the same time-frame, and a similar proportion of responses actually exhibited an increase in functional avidity during this period. Conclusions Escape appears to constitute a much more important means of viral evasion of CD8+ T cell responses in acute and early HIV infection than decline in functional avidity of epitope-specific T cells. These findings support the design of vaccines to elicit T cell responses that are difficult for the virus to escape. PMID:21635736

Adverse events post smallpox-vaccination: insights from tail scarification infection in mice with Vaccinia virus.

PubMed

Mota, Bruno E F; Gallardo-Romero, Nadia; Trindade, Giliane; Keckler, M Shannon; Karem, Kevin; Carroll, Darin; Campos, Marco A; Vieira, Leda Q; da Fonseca, Flávio G; Ferreira, Paulo C P; Bonjardim, Cláudio A; Damon, Inger K; Kroon, Erna G

2011-04-15

Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1(-/-)) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (µMT) produced no mortality. However, viral clearance in µMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1(-/-) with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1(-/-), and passive transfer of WT T cells to Rag1(-/-) animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify individuals with higher risk of complications after infection with poxvirus.

Recent Insights into the HIV/AIDS Pandemic

PubMed Central

Becerra, Juan C.; Bildstein, Lukas S.; Gach, Johannes S.

2016-01-01

Etiology, transmission and protection: Transmission of HIV, the causative agent of AIDS, occurs predominantly through bodily fluids. Factors that significantly alter the risk of HIV transmission include male circumcision, condom use, high viral load, and the presence of other sexually transmitted diseases. Pathology/Symptomatology: HIV infects preferentially CD4+ T lymphocytes, and Monocytes. Because of their central role in regulating the immune response, depletion of CD4+ T cells renders the infected individual incapable of adequately responding to microorganisms otherwise inconsequential. Epidemiology, incidence and prevalence: New HIV infections affect predominantly young heterosexual women and homosexual men. While the mortality rates of AIDS related causes have decreased globally in recent years due to the use of highly active antiretroviral therapy (HAART) treatment, a vaccine remains an elusive goal. Treatment and curability: For those afflicted HIV infection remains a serious illness. Nonetheless, the use of advanced therapeutics have transformed a dire scenario into a chronic condition with near average life spans. When to apply those remedies appears to be as important as the remedies themselves. The high rate of HIV replication and the ability to generate variants are central to the viral survival strategy and major barriers to be overcome. Molecular mechanisms of infection: In this review, we assemble new details on the molecular events from the attachment of the virus, to the assembly and release of the viral progeny. Yet, much remains to be learned as understanding of the molecular mechanisms used in viral replication and the measures engaged in the evasion of immune surveillance will be important to develop effective interventions to address the global HIV pandemic. PMID:28357381

HIV-1 Pathogenesis: The Virus

PubMed Central

Swanstrom, Ronald; Coffin, John

2012-01-01

Transmission of HIV-1 results in the establishment of a new infection, typically starting from a single virus particle. That virion replicates to generate viremia and persistent infection in all of the lymphoid tissue in the body. HIV-1 preferentially infects T cells with high levels of CD4 and those subsets of T cells that express CCR5, particularly memory T cells. Most of the replicating virus is in the lymphoid tissue, yet most of samples studied are from blood. For the most part the tissue and blood viruses represent a well-mixed population. With the onset of immunodeficiency, the virus evolves to infect new cell types. The tropism switch involves switching from using CCR5 to CXCR4 and corresponds to an expansion of infected cells to include naïve CD4+ T cells. Similarly, the virus evolves the ability to enter cells with low levels of CD4 on the surface and this potentiates the ability to infect macrophages, although the scope of sites where infection of macrophages occurs and the link to pathogenesis is only partly known and is clear only for infection of the central nervous system. A model linking viral evolution to these two pathways has been proposed. Finally, other disease states related to immunodeficiency may be the result of viral infection of additional tissues, although the evidence for a direct role for the virus is less strong. Advancing immunodeficiency creates an environment in which viral evolution results in viral variants that can target new cell types to generate yet another class of opportunistic infections (i.e., HIV-1 with altered tropism). PMID:23143844

A furoviral replicase recruits host HSP70 to membranes for viral RNA replication

PubMed Central

Yang, Jian; Zhang, Fen; Cai, Nian-Jun; Wu, Ne; Chen, Xuan; Li, Jing; Meng, Xiang-Feng; Zhu, Tong-Quan; Chen, Jian-Ping; Zhang, Heng-Mu

2017-01-01

Many host factors have been identified to be involved in viral infection. However, although furoviruses cause important diseases of cereals worldwide, no host factors have yet been identified that interact with furoviral genes or participate in the viral infection cycle. In this study, both TaHSP70 and NbHSP70 were up-regulated in Chinese wheat mosaic furovirus (CWMV)-infected plants. Their overexpression and inhibition were correlated with the accumulation of viral genomic RNAs, suggesting that the HSP70 genes could be necessary for CWMV infection. The subcellular distributions of TaHSP70 and NbHSP70 were significantly affected by CWMV infection or by infiltration of RNA1 alone. Further assays showed that the viral replicase encoded by CWMV RNA1 interacts with both TaHSP70 and NbHSP70 in vivo and vitro and that its region aa167–333 was responsible for the interaction. Subcellular assays showed that the viral replicase could recruit both TaHSP70 and NbHSP70 from the cytoplasm or nucleus to the granular aggregations or inclusion-like structures on the intracellular membrane system, suggesting that both HSP70s may be recruited into the viral replication complex (VRC) to promote furoviral replication. This is the first host factor identified to be involved in furoviral infection, which extends the list and functional scope of HSP70 chaperones. PMID:28367995

Early innate immune responses to Sin Nombre hantavirus occur independently of IFN regulatory factor 3, characterized pattern recognition receptors, and viral entry.

PubMed

Prescott, Joseph B; Hall, Pamela R; Bondu-Hawkins, Virginie S; Ye, Chunyan; Hjelle, Brian

2007-08-01

Sin Nombre virus (SNV) is a highly pathogenic New World virus and etiologic agent of hantavirus cardiopulmonary syndrome. We have previously shown that replication-defective virus particles are able to induce a strong IFN-stimulated gene (ISG) response in human primary cells. RNA viruses often stimulate the innate immune response by interactions between viral nucleic acids, acting as a pathogen-associated molecular pattern, and cellular pattern-recognition receptors (PRRs). Ligand binding to PRRs activates transcription factors which regulate the expression of antiviral genes, and in all systems examined thus far, IFN regulatory factor 3 (IRF3) has been described as an essential intermediate for induction of ISG expression. However, we now describe a model in which IRF3 is dispensable for the induction of ISG transcription in response to viral particles. IRF3-independent ISG transcription in human hepatoma cell lines is initiated early after exposure to SNV virus particles in an entry- and replication-independent fashion. Furthermore, using gene knockdown, we discovered that this activation is independent of the best-characterized RNA- and protein-sensing PRRs including the cytoplasmic caspase recruitment domain-containing RNA helicases and the TLRs. SNV particles engage a heretofore unrecognized PRR, likely located at the cell surface, and engage a novel IRF3-independent pathway that activates the innate immune response.

Experimental transmission of vesicular stomatitis New Jersey virus from Simulium vittatum to cattle: clinical outcome is influenced by site of insect feeding.

PubMed

Mead, D G; Lovett, K Rainwater; Murphy, M D; Pauszek, S J; Smoliga, G; Gray, E W; Noblet, R; Overmyer, J; Rodriguez, L L

2009-07-01

Vesicular stomatitis New Jersey virus (VSNJV) is an insect-transmitted Rhabdovirus causing vesicular disease in domestic livestock including cattle, horses, and pigs. Natural transmission during epidemics remains poorly understood, particularly in cattle, one of the most affected species during outbreaks. This study reports the first successful transmission of VSNJV to cattle by insect bite resulting in clinical disease. When infected black flies (Simulium vittatum Zetterstedt) fed at sites where VS lesions are usually observed (mouth, nostrils, and foot coronary band), infection occurred, characterized by local viral replication, vesicular lesions, and high neutralizing antibody titers (> 1: 256). Viral RNA was detected up to 9 d postinfection in tissues collected during necropsy from lesion sites and lymph nodes draining those sites. Interestingly, when flies were allowed to feed on flank or neck skin, viral replication was poor, lesions were not observed, and low levels of neutralizing antibodies (range, 1:8-1:32) developed. Viremia was never observed in any of the animals and infectious virus was not recovered from tissues on necropsies performed between 8 and 27 d postinfection. Demonstration that VSNJV transmission to cattle by infected black flies can result in clinical disease contributes to a better understanding of the epidemiology and potential prevention and control methods for this important disease.

Human immunodeficiency viruses appear compartmentalized to the female genital tract in cross-sectional analyses but genital lineages do not persist over time.

PubMed

Bull, Marta E; Heath, Laura M; McKernan-Mullin, Jennifer L; Kraft, Kelli M; Acevedo, Luis; Hitti, Jane E; Cohn, Susan E; Tapia, Kenneth A; Holte, Sarah E; Dragavon, Joan A; Coombs, Robert W; Mullins, James I; Frenkel, Lisa M

2013-04-15

Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viruses mix between genital tract and blood. Eight women with ongoing HIV replication were studied during a period of 1.5 to 4.5 years. Multiple viral sequences were derived by single-genome amplification of the HIV C2-V5 region of env from genital secretions and blood plasma. Maximum likelihood phylogenies were evaluated for compartmentalization using 4 statistical tests. In cross-sectional analyses compartmentalization of genital from blood viruses was detected in three of eight women by all tests; this was associated with tissue specific clades containing multiple monotypic sequences. In longitudinal analysis, the tissues-specific clades did not persist to form viral lineages. Rather, across women, HIV lineages were comprised of both genital tract and blood sequences. The observation of genital-specific HIV clades only in cross-sectional analysis and an absence of genital-specific lineages in longitudinal analyses suggest a dynamic interchange of HIV variants between the female genital tract and blood.

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.

Cis-acting RNA elements in the Hepatitis C virus RNA genome

PubMed Central

Sagan, Selena M.; Chahal, Jasmin; Sarnow, Peter

2017-01-01

Hepatitis C virus (HCV) infection is a rapidly increasing global health problem with an estimated 170 million people infected worldwide. HCV is a hepatotropic, positive-sense RNA virus of the family Flaviviridae. As a positive-sense RNA virus, the HCV genome itself must serve as a template for translation, replication and packaging. The viral RNA must therefore be a dynamic structure that is able to readily accommodate structural changes to expose different regions of the genome to viral and cellular proteins to carry out the HCV life cycle. The ∼9600 nucleotide viral genome contains a single long open reading frame flanked by 5′ and 3′ non-coding regions that contain cis-acting RNA elements important for viral translation, replication and stability. Additional cis-acting RNA elements have also been identified in the coding sequences as well as in the 3′ end of the negative-strand replicative intermediate. Herein, we provide an overview of the importance of these cis-acting RNA elements in the HCV life cycle. PMID:25576644

Ebola virus: the role of macrophages and dendritic cells in the pathogenesis of Ebola hemorrhagic fever.

PubMed

Bray, Mike; Geisbert, Thomas W

2005-08-01

Ebola hemorrhagic fever is a severe viral infection characterized by fever, shock and coagulation defects. Recent studies in macaques show that major features of illness are caused by effects of viral replication on macrophages and dendritic cells. Infected macrophages produce proinflammatory cytokines, chemokines and tissue factor, attracting additional target cells and inducing vasodilatation, increased vascular permeability and disseminated intravascular coagulation. However, they cannot restrict viral replication, possibly because of suppression of interferon responses. Infected dendritic cells also secrete proinflammatory mediators, but cannot initiate antigen-specific responses. In consequence, virus disseminates to these and other cell types throughout the body, causing multifocal necrosis and a syndrome resembling septic shock. Massive "bystander" apoptosis of natural killer and T cells further impairs immunity. These findings suggest that modifying host responses would be an effective therapeutic strategy, and treatment of infected macaques with a tissue-factor inhibitor reduced both inflammation and viral replication and improved survival.

Analysis of JC virus DNA replication using a quantitative and high-throughput assay

PubMed Central

Shin, Jong; Phelan, Paul J.; Chhum, Panharith; Bashkenova, Nazym; Yim, Sung; Parker, Robert; Gagnon, David; Gjoerup, Ole; Archambault, Jacques; Bullock, Peter A.

2015-01-01

Progressive Multifocal Leukoencephalopathy (PML) is caused by lytic replication of JC virus (JCV) in specific cells of the central nervous system. Like other polyomaviruses, JCV encodes a large T-antigen helicase needed for replication of the viral DNA. Here, we report the development of a luciferase-based, quantitative and high-throughput assay of JCV DNA replication in C33A cells, which, unlike the glial cell lines Hs 683 and U87, accumulate high levels of nuclear T-ag needed for robust replication. Using this assay, we investigated the requirement for different domains of T-ag, and for specific sequences within and flanking the viral origin, in JCV DNA replication. Beyond providing validation of the assay, these studies revealed an important stimulatory role of the transcription factor NF1 in JCV DNA replication. Finally, we show that the assay can be used for inhibitor testing, highlighting its value for the identification of antiviral drugs targeting JCV DNA replication. PMID:25155200

Tombusvirus RNA replication depends on the TOR pathway in yeast and plants.

PubMed

Inaba, Jun-Ichi; Nagy, Peter D

2018-06-01

Similar to other (+)RNA viruses, tomato bushy stunt virus (TBSV) utilizes metabolites, lipids, membranes, and co-opted host factors during replication. The coordination of cell metabolism and growth with environmental cues is performed by the target of rapamycin (TOR) kinase in eukaryotic cells. In this paper, we find that TBSV replication partially inhibits TOR activity, likely due to recruitment of glycolytic enzymes to the viral replication compartment, which results in reduced ATP levels in the cytosol. Complete inhibition of TOR activity with rapamycin in yeast or AZD8055 inhibitor in plants reduces tombusvirus replication. We find that high glucose concentration, which stimulates TOR activity, enhanced tombusvirus replication in yeast. Depletion of yeast Sch9 or plant S6K1 kinase, a downstream effector of TOR, also inhibited tombusvirus replication in yeast and plant or the assembly of the viral replicase in vitro. Altogether, the TOR pathway is crucial for TBSV to replicate efficiently in hosts. Copyright © 2018 Elsevier Inc. All rights reserved.

RNA genetics

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

Domingo, E.; Holland, J.J.; Ahlquist, P.

1988-01-01

This book contains the proceedings on RNA genetics: RNA-directed virus replication Volume 1. Topics covered include: Replication of the poliovirus genome; Influenza viral RNA transcription and replication; and Relication of the reoviridal: Information derived from gene cloning and expression.

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

Viral Metagenomics on Animals as a Tool for the Detection of Zoonoses Prior to Human Infection?

PubMed Central

Temmam, Sarah; Davoust, Bernard; Berenger, Jean-Michel; Raoult, Didier; Desnues, Christelle

2014-01-01

Many human viral infections have a zoonotic, i.e., wild or domestic animal, origin. Several zoonotic viruses are transmitted to humans directly via contact with an animal or indirectly via exposure to the urine or feces of infected animals or the bite of a bloodsucking arthropod. If a virus is able to adapt and replicate in its new human host, human-to-human transmissions may occur, possibly resulting in an epidemic, such as the A/H1N1 flu pandemic in 2009. Thus, predicting emerging zoonotic infections is an important challenge for public health officials in the coming decades. The recent development of viral metagenomics, i.e., the characterization of the complete viral diversity isolated from an organism or an environment using high-throughput sequencing technologies, is promising for the surveillance of such diseases and can be accomplished by analyzing the viromes of selected animals and arthropods that are closely in contact with humans. In this review, we summarize our current knowledge of viral diversity within such animals (in particular blood-feeding arthropods, wildlife and domestic animals) using metagenomics and present its possible future application for the surveillance of zoonotic and arboviral diseases. PMID:24918293

Host factor SPCS1 regulates the replication of Japanese encephalitis virus through interactions with transmembrane domains of NS2B.

PubMed

Ma, Le; Li, Fang; Zhang, Jing-Wei; Li, Wei; Zhao, Dong-Ming; Wang, Han; Hua, Rong-Hong; Bu, Zhi-Gao

2018-03-28

Signal peptidase complex subunit 1 (SPCS1) is a newly identified host factor that regulates flavivirus replication, but the molecular mechanism is not fully understood. Herein, using Japanese encephalitis virus (JEV) as a model, we investigated the mechanism through which host factor SPCS1 regulates the replication of flaviviruses. We first validated the regulatory function of SPCS1 in JEV propagation by knocking down and knocking out endogenous SPCS1. Loss of SPCS1 function markedly reduced intracellular virion assembly and production of infectious JEV particles, but did not affect virus cell entry, RNA replication, or translation. SPCS1 was found to interact with NS2B, which is involved in post-translational protein processing and viral assembly. Serial deletion mutation of the JEV NS2B protein revealed that two transmembrane domains, NS2B (1-49) and NS2B (84-131), interact with SPCS1. Further mutagenesis analysis of conserved flavivirus residues in two SPCS1 interaction domains of NS2B demonstrated that G12A, G37A, and G47A in NS2B (1-49), and P112A in NS2B (84-131), weakened the interaction with SPCS1. Deletion mutation of SPCS1 revealed that SPCS1 (91-169) which containing two transmembrane domains was involved in the interaction with both NS2B (1-49) and NS2B (84-131). Taken together, the results demonstrate that SPCS1 affects viral replication by interacting with NS2B, thereby influencing post-translational processing of JEV proteins and the assembly of virions. IMPORTANCE Understanding viral-host interactions is important for elucidating the molecular mechanisms of viral propagation, and identifying potential anti-viral targets. Previous reports demonstrated that SPCS1 is involved in the flavivirus life cycle, but the mechanism remains unknown. In this study, we confirmed that SPCS1 participates in the post-translational protein processing and viral assembly stages of the JEV lifecycle, but not in the cell entry, genome RNA replication, or translation stages. Furthermore, we found that SPCS1 interacts with two independent transmembrane domains of the Flavivirus NS2B protein. NS2B also interacts with NS2A, which is proposed to mediate viral assembly. Therefore, we propose a protein-protein interaction model showing how SPCS1 participates in the assembly of JEV particles. The findings expand our understanding of how host factors participate in the flavivirus replication lifecycle, and identify potential anti-viral targets for combatting flavivirus infection. Copyright © 2018 American Society for Microbiology.

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

PubMed Central

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

2015-01-01

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

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

PubMed

Kolber, Michael A

2007-01-01

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

Dexamethasone treatment decreases replication of viral hemorrhagic septicemia virus in Epithelioma papulosum cyprini cells.

PubMed

Kim, Min Sun; Lee, Su Jin; Choi, Seung Hyuk; Kang, Yue Jai; Kim, Ki Hong

2017-05-01

The expression of Mx1 in EPC cells after treatment with poly(I:C) or infection with viral hemorrhagic septicemia virus (VHSV) was significantly suppressed by treatment with dexamethasone. However, the titer of VHSV did not increase but instead decreased after dexamethasone treatment. This suggests that dexamethasone not only downregulates type I IFN but also affects certain factors that are necessary for VHSV replication. An important effect of HSP90 on replication of RNA viruses and downregulation of HSP90 by glucocorticoids have been reported. In this study, dexamethasone downregulated HSP90α expression in EPC cells that were stimulated with poly(I:C) or infected with VHSV. Furthermore, cells treated with an HSP90 inhibitor, geldanamycin, showed significantly decreased titers of VHSV, suggesting that HSP90 may be an important host component involved in VHSV replication, and HSP90 inhibition might be one of the causes for the observed reduction in viral titer caused by dexamethasone treatment.

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

The length of an internal poly(A) tract of hibiscus latent Singapore virus is crucial for its replication.

PubMed

Niu, Shengniao; Cao, Shishu; Huang, Li-Jing; Tan, Kelvin Chee-Leong; Wong, Sek-Man

2015-01-01

Hibiscus latent Singapore virus (HLSV) mutants were constructed to study roles of its internal poly(A) tract (IPAT) in viral replication and coat protein (CP) expression. Shortening of the IPAT resulted in reduced HLSV RNA accumulation and its minimal length required for HLSV CP expression in plants was 24 nt. Disruption of a putative long range RNA-RNA interacting structure between 5' and 3' untranslated regions of HLSV-22A and -24A resulted in reduced viral RNA and undetectable CP accumulation in inoculated leaves. Replacement of the IPAT in HLSV with an upstream pseudoknot domain (UPD) of Tobacco mosaic virus (TMV) or insertion of the UPD to the immediate downstream of a 24 nt IPAT in HLSV resulted in drastically reduced viral RNA replication. Plants infected with a TMV mutant by replacement of the UPD with 43 nt IPAT exhibited milder mosaic symptoms without necrosis. We have proposed a model for HLSV replication. Copyright © 2014 Elsevier Inc. All rights reserved.

Human importin alpha and RNA do not compete for binding to influenza A virus nucleoprotein

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

Boulo, Sebastien; UJF-EMBL-CNRS UMI 3265, Unit of Virus Host-Cell Interactions, 6 rue Jules Horowitz, 38042 Grenoble Cedex 9; Akarsu, Hatice

2011-01-05

Influenza virus has a segmented genome composed of eight negative stranded RNA segments. Each segment is covered with NP forming ribonucleoproteins (vRNPs) and carries a copy of the heterotrimeric polymerase complex. As a rare phenomenon among the RNA viruses, the viral replication occurs in the nucleus and therefore implies interactions between host and viral factors, such as between importin alpha and nucleoprotein. In the present study we report that through binding with the human nuclear receptor importin {alpha}5 (Imp{alpha}5), the viral NP is no longer oligomeric but maintained as a monomer inside the complex. In this regard, Imp{alpha}5 acts asmore » a chaperone until NP is delivered in the nucleus for viral RNA encapsidation. Moreover, we show that the association of NP with the host transporter does not impair the binding of NP to RNA. The complex human Imp{alpha}5-NP binds RNA with the same affinity as wt NP alone, whereas engineered monomeric NP through point mutations binds RNA with a strongly reduced affinity.« less

Innate immune restriction and antagonism of viral RNA lacking 2'-O methylation

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

Hyde, Jennifer L.; Diamond, Michael S., E-mail: diamond@borcim.wustl.edu; Molecular Microbiology, Washington University School of Medicine, St Louis., MO 63110

N-7 and 2′-O methylation of host cell mRNA occurs in the nucleus and results in the generation of cap structures (cap 0, m{sup 7}GpppN; cap 1, m{sup 7}GpppNm) that control gene expression by modulating nuclear export, splicing, turnover, and protein synthesis. Remarkably, RNA cap modification also contributes to mammalian cell host defense as viral RNA lacking 2′-O methylation is sensed and inhibited by IFIT1, an interferon (IFN) stimulated gene (ISG). Accordingly, pathogenic viruses that replicate in the cytoplasm have evolved mechanisms to circumvent IFIT1 restriction and facilitate infection of mammalian cells. These include: (a) generating cap 1 structures on theirmore » RNA through cap-snatching or virally-encoded 2′-O methyltransferases, (b) using cap-independent means of translation, or (c) using RNA secondary structural motifs to antagonize IFIT1 binding. This review will discuss new insights as to how specific modifications at the 5′-end of viral RNA modulate host pathogen recognition responses to promote infection and disease.« less

A universal expression/silencing vector in plants.

PubMed

Peretz, Yuval; Mozes-Koch, Rita; Akad, Fuad; Tanne, Edna; Czosnek, Henryk; Sela, Ilan

2007-12-01

A universal vector (IL-60 and auxiliary constructs), expressing or silencing genes in every plant tested to date, is described. Plants that have been successfully manipulated by the IL-60 system include hard-to-manipulate species such as wheat (Triticum duram), pepper (Capsicum annuum), grapevine (Vitis vinifera), citrus, and olive (Olea europaea). Expression or silencing develops within a few days in tomato (Solanum lycopersicum), wheat, and most herbaceous plants and in up to 3 weeks in woody trees. Expression, as tested in tomato, is durable and persists throughout the life span of the plant. The vector is, in fact, a disarmed form of Tomato yellow leaf curl virus, which is applied as a double-stranded DNA and replicates as such. However, the disarmed virus does not support rolling-circle replication, and therefore viral progeny single-stranded DNA is not produced. IL-60 does not integrate into the plant's genome, and the construct, including the expressed gene, is not heritable. IL-60 is not transmitted by the Tomato yellow leaf curl virus's natural insect vector. In addition, artificial satellites were constructed that require a helper virus for replication, movement, and expression. With IL-60 as the disarmed helper "virus," transactivation occurs, resulting in an inducible expressing/silencing system. The system's potential is demonstrated by IL-60-derived suppression of a viral-silencing suppressor of Grapevine virus A, resulting in Grapevine virus A-resistant/tolerant plants.

Flavivirus RNA Replication: Essential Viral Functions as Targets for Antiviral Therapeutics

DTIC Science & Technology

1991-12-31

We established yellow fever virus ( YFV ) as our study system and assembled all the necessary virologic, molecular biologic, and immunologic reagents for...the detailed study of viral RNA replication. Methodologies for detection, identification, and analysis of YFV polypeptides and RNA species were in...place. Several YFV protesn coding regions were engineered into various surrogate expression systems, and the recombinant proteins thereby produced would

Development of a luciferase based viral inhibition assay to evaluate vaccine induced CD8 T-cell responses

PubMed Central

Naarding, Marloes A.; Fernandez-Fernandez, Natalia; Kappes, John C.; Hayes, Peter; Ahmed, Tina; Icyuz, Mert; Edmonds, Tara G.; Bergin, Philip; Anzala, Omu; Hanke, Tomas; Clark, Lorna; Cox, Josephine H.; Cormier, Emmanuel; Ochsenbauer, Christina; Gilmour, Jill

2014-01-01

Emergence of SIV and HIV specific CD8 T cells has been shown to correlate with control of in vivo replication. Poor correlation between IFN-γ ELISPOT responses and in vivo control of the virus has triggered the development of more relevant assays to assess functional HIV-1 specific CD8 T-cell responses for the evaluation and prioritization of new HIV-1 vaccine candidates. We previously established a viral inhibition assay (VIA) that measures the ability of vaccine-induced CD8 T-cell responses to inhibit viral replication in autologous CD4 T cells. In this assay, viral replication is determined by measuring p24 in the culture supernatant. Here we describe the development of a novel VIA, referred to as IMC LucR VIA that exploits replication-competent HIV-1 infectious molecular clones (IMCs) in which the complete proviral genome is strain-specific and which express the Renilla luciferase (LucR) gene to determine viral growth and inhibition. The introduction of the luciferase readout does provide significant improvement of the read out time. In addition to switching to the LucR read out, changes made to the overall protocol resulted in the miniaturization of the assay from a 48 to a 96-well plate format, which preserved sample and allowed for the introduction of replicates. The overall assay time was reduced from 13 to 8 days. The assay has a high degree of specificity, and the previously observed non-specific background inhibition in cells from HIV-1 negative volunteers has been reduced dramatically. Importantly, we observed an increase in positive responses, indicating an improvement in sensitivity compared to the original VIA. Currently, only a limited number of “whole-genome” IMC-LucR viruses are available and our efforts will focus on expanding the panel to better evaluate anti-viral breadth. Overall, we believe the IMC LucR VIA provides a platform to assess functional CD8 T-cell responses in large-scale clinical trial testing, which will enhance the ability to select the most promising HIV-1 vaccine candidates capable of controlling HIV-1 replication in vivo. PMID:24291126

Evidence that viral RNAs have evolved for efficient, two-stage packaging.

PubMed

Borodavka, Alexander; Tuma, Roman; Stockley, Peter G

2012-09-25

Genome packaging is an essential step in virus replication and a potential drug target. Single-stranded RNA viruses have been thought to encapsidate their genomes by gradual co-assembly with capsid subunits. In contrast, using a single molecule fluorescence assay to monitor RNA conformation and virus assembly in real time, with two viruses from differing structural families, we have discovered that packaging is a two-stage process. Initially, the genomic RNAs undergo rapid and dramatic (approximately 20-30%) collapse of their solution conformations upon addition of cognate coat proteins. The collapse occurs with a substoichiometric ratio of coat protein subunits and is followed by a gradual increase in particle size, consistent with the recruitment of additional subunits to complete a growing capsid. Equivalently sized nonviral RNAs, including high copy potential in vivo competitor mRNAs, do not collapse. They do support particle assembly, however, but yield many aberrant structures in contrast to viral RNAs that make only capsids of the correct size. The collapse is specific to viral RNA fragments, implying that it depends on a series of specific RNA-protein interactions. For bacteriophage MS2, we have shown that collapse is driven by subsequent protein-protein interactions, consistent with the RNA-protein contacts occurring in defined spatial locations. Conformational collapse appears to be a distinct feature of viral RNA that has evolved to facilitate assembly. Aspects of this process mimic those seen in ribosome assembly.

Intersubtype Differences in the Effect of a Rare p24 Gag Mutation on HIV-1 Replicative Fitness

PubMed Central

Chopera, Denis R.; Cotton, Laura A.; Zawaira, Alexander; Mann, Jaclyn K.; Ngandu, Nobubelo K.; Ntale, Roman; Carlson, Jonathan M.; Mlisana, Koleka; Woodman, Zenda; de Assis Rosa, Debra; Martin, Eric; Miura, Toshiyuki; Pereyra, Florencia; Walker, Bruce D.; Gray, Clive M.; Martin, Darren P.; Ndung'u, Thumbi; Brockman, Mark A.; Karim, Salim Abdool

2012-01-01

Certain immune-driven mutations in HIV-1, such as those arising in p24Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P = 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P < 0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness. PMID:23015721

Evaluation of chimeric yellow fever 17D/dengue viral replication in ticks.

PubMed

Kazimírová, Mária; Mantel, Nathalie; Raynaud, Sandrine; Slovák, Mirko; Ustaniková, Katarína; Lang, Jean; Guy, Bruno; Barban, Veronique; Labuda, Milan

2012-11-01

Chimeric yellow fever 17D/DENV-1-4 viruses (CYD-1-4) have been developed as a tetravalent dengue vaccine candidate which is currently being evaluated in efficacy trials in Asia and America. While YF 17D and DENV are mosquito-borne flaviviruses, it has been shown that CYD-1-4 do not replicate after oral infection in mosquitoes and are not transmitted to new hosts. To further document the risk of environmental dissemination of these viruses, we evaluated the replication of CYD-1-4 in ticks, the vector of tick-borne encephalitis virus (TBEV), another member of the flavivirus family. Females of two hard tick species, Ixodes ricinus and Rhipicephalus appendiculatus, were inoculated intracoelomically with CYD-1-4 viruses and parent viruses (DENV-1-4 and YF 17D). Virus persistence and replication was assessed 2, 16, and 44 days post-inoculation by plaque titration and qRT-PCR. CYD-1-4 viruses were detected in I. ricinus ticks at early time points post-inoculation, but with infectious titers at least 100-fold lower than those observed in TBEV-infected ticks. Unlike TBEV, complete viral clearance occurred by day 44 in most ticks except for CYD-2, which had a tendency to decline. In addition, while about 70% of TBEV-infected I. ricinus nymphs acquired infection by co-feeding with infected tick females on non-viremic hosts, no co-feeding transmission of CYD-2 virus was detected. Based on these results, we conclude that the risk of dissemination of the candidate vaccine viruses by tick bite is highly unlikely.

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

Inhibition of Human Papillomavirus DNA Replication by an E1-Derived p80/UAF1-Binding Peptide

PubMed Central

Lehoux, Michaël; Fradet-Turcotte, Amélie; Lussier-Price, Mathieu; Omichinski, James G.

2012-01-01

The papillomavirus E1 helicase is recruited by E2 to the viral origin, where it assembles into a double hexamer that orchestrates replication of the viral genome. We previously identified the cellular WD40 repeat-containing protein p80/UAF1 as a novel interaction partner of E1 from anogenital human papillomavirus (HPV) types. p80 was found to interact with the first 40 residues of HPV type 31 (HPV31) E1, and amino acid substitutions within this domain abrogated the maintenance of the viral episome in keratinocytes. In this study, we report that these p80-binding substitutions reduce by 70% the ability of E1 to support transient viral DNA replication without affecting its interaction with E2 and assembly at the origin in vivo. Microscopy studies revealed that p80 is relocalized from the cytoplasm to discrete subnuclear foci by E1 and E2. Chromatin immunoprecipitation assays further revealed that p80 is recruited to the viral origin in an E1- and E2-dependent manner. Interestingly, overexpression of a 40-amino-acid-long p80-binding peptide, derived from HPV31 E1, was found to inhibit viral DNA replication by preventing the recruitment of endogenous p80 to the origin. Mutant peptides defective for p80 interaction were not inhibitory, demonstrating the specificity of this effect. Characterization of this E1 peptide by nuclear magnetic resonance (NMR) showed that it is intrinsically disordered in solution, while mapping studies indicated that the WD repeats of p80 are required for E1 interaction. These results provide additional evidence for the requirement for p80 in anogenital HPV DNA replication and highlight the potential of E1-p80 interaction as a novel antiviral target. PMID:22278251

Cyclooxygenase‐2 facilitates dengue virus replication and serves as a potential target for developing antiviral agents

PubMed Central

Lin, Chun-Kuang; Tseng, Chin-Kai; Wu, Yu-Hsuan; Liaw, Chih-Chuang; Lin, Chun-Yu; Huang, Chung-Hao; Chen, Yen-Hsu; Lee, Jin-Ching

2017-01-01

Cyclooxygenase-2 (COX-2) is one of the important mediators of inflammation in response to viral infection, and it contributes to viral replication, for example, cytomegalovirus or hepatitis C virus replication. The role of COX-2 in dengue virus (DENV) replication remains unclear. In the present study, we observed an increased level of COX-2 in patients with dengue fever compared with healthy donors. Consistent with the clinical data, an elevated level of COX-2 expression was also observed in DENV-infected ICR suckling mice. Using cell-based experiments, we revealed that DENV-2 infection significantly induced COX-2 expression and prostaglandin E2 (PGE2) production in human hepatoma Huh-7 cells. The exogenous expression of COX-2 or PGE2 treatment dose-dependently enhanced DENV-2 replication. In contrast, COX-2 gene silencing and catalytic inhibition sufficiently suppressed DENV-2 replication. In an ICR suckling mouse model, we identified that the COX-2 inhibitor NS398 protected mice from succumbing to life-threatening DENV-2 infection. By using COX-2 promoter-based analysis and specific inhibitors against signaling molecules, we identified that NF-κB and MAPK/JNK are critical factors for DENV-2-induced COX-2 expression and viral replication. Altogether, our results reveal that COX-2 is an important factor for DENV replication and can serve as a potential target for developing therapeutic agents against DENV infection. PMID:28317866