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Sample records for active viral replication

  1. Cytoplasmic Viral Replication Complexes

    PubMed Central

    den Boon, Johan A.; Diaz, Arturo; Ahlquist, Paul

    2010-01-01

    Many viruses that replicate in the cytoplasm compartmentalize their genome replication and transcription in organelle-like structures that enhance replication efficiency and protection from host defenses. In particular, recent studies with diverse positive-strand RNA viruses have further elucidated the ultrastructure of membrane-bounded RNA replication complexes and their close coordination with virion assembly and budding. The structure, function and assembly of some positive-strand RNA virus replication complexes have parallels and potential evolutionary links with the replicative cores of double-strand RNA virus and retrovirus virions, and more general similarities with the replication factories of cytoplasmic DNA viruses. PMID:20638644

  2. Enhanced Viral Replication by Cellular Replicative Senescence

    PubMed Central

    Kim, Ji-Ae; Seong, Rak-Kyun

    2016-01-01

    Cellular replicative senescence is a major contributing factor to aging and to the development and progression of aging-associated diseases. In this study, we sought to determine viral replication efficiency of influenza virus (IFV) and Varicella Zoster Virus (VZV) infection in senescent cells. Primary human bronchial epithelial cells (HBE) or human dermal fibroblasts (HDF) were allowed to undergo numbers of passages to induce replicative senescence. Induction of replicative senescence in cells was validated by positive senescence-associated β-galactosidase staining. Increased susceptibility to both IFV and VZV infection was observed in senescent HBE and HDF cells, respectively, resulting in higher numbers of plaque formation, along with the upregulation of major viral antigen expression than that in the non-senescent cells. Interestingly, mRNA fold induction level of virus-induced type I interferon (IFN) was attenuated by senescence, whereas IFN-mediated antiviral effect remained robust and potent in virus-infected senescent cells. Additionally, we show that a longevity-promoting gene, sirtuin 1 (SIRT1), has antiviral role against influenza virus infection. In conclusion, our data indicate that enhanced viral replication by cellular senescence could be due to senescence-mediated reduction of virus-induced type I IFN expression. PMID:27799874

  3. Neuraminidase hemadsorption activity, conserved in avian influenza A viruses, does not influence viral replication in ducks.

    PubMed Central

    Kobasa, D; Rodgers, M E; Wells, K; Kawaoka, Y

    1997-01-01

    The N1 and N9 neuraminidase (NA) subtypes of influenza A viruses exhibit significant hemadsorption activity that localizes to a site distinct from that of the enzymatic active site. To determine the conservation of hemadsorption activity among different NAs, we have examined most of the NA subtypes from avian, swine, equine, and human virus isolates. All subtypes of avian virus NAs examined and one equine virus N8 NA possessed high levels of hemadsorption activity. A swine virus N1 NA exhibited only weak hemadsorption activity, while in human virus N1 and N2 NAs, the activity was detected at a much lower level than in avian virus NAs. NAs which possessed hemadsorption activity for chicken erythrocytes (RBCs) were similarly able to adsorb human RBCs. However, none of the hemadsorption-positive NAs could bind equine, swine, or bovine RBCs, suggesting that RBCs from these species lack molecules, recognized by the NA hemadsorption site, present on human and chicken RBCs. Mutagenesis of the putative hemadsorption site of A/duck/Hong Kong/7/75 N2 NA abolished the high level of hemadsorption activity exhibited by the wild-type protein but also resulted in a 50% reduction of the NA enzymatic activity. A transfectant virus, generated by reverse genetics, containing this mutated NA replicated 10-fold less efficiently in chicken embryo fibroblast cultures than did a transfectant virus expressing the wild-type NA. However, both viruses replicated equally well in Peking ducks. Although conservation of NA hemadsorption activity among avian virus NAs suggests the maintenance of a required function of NA, loss of the activity does not preclude the replication of the virus in an avian host. PMID:9261394

  4. MicroRNA-33a disturbs influenza A virus replication by targeting ARCN1 and inhibiting viral ribonucleoprotein activity.

    PubMed

    Hu, Yi; Jiang, Liangzhen; Lai, Wenbin; Qin, Yujie; Zhang, Tinghong; Wang, Shixiong; Ye, Xin

    2016-01-01

    In order to explore the roles of microRNA(s) [miRNA(s)] in the influenza A virus life cycle, we compared the miRNA profiles of 293T and HeLa cell lines, as influenza A virus can replicate efficiently in 293T cells but only poorly in HeLa cells. We analysed differentially expressed miRNAs and identified five, including miR-33a, that could disturb influenza A virus replication significantly. Using TargetScan analysis, we found that ARCN1 could be a potential target of miR-33a. To confirm whether miR-33a could truly target ARCN1, we generated a luciferase reporter for the ARCN1 3' untranslated region (UTR) and performed a luciferase assay. The data indicated that miR-33a could suppress the luciferase activity of the reporter for the ARCN1 3' UTR but not a reporter in which the predicted miR-33a targeting sites on ARCN1 3' UTR were mutated. We performed immunoblotting to confirm that miR-33a could downregulate the protein level of ARCN1. Consistently, the level of ARCN1 protein in HeLa cells was significantly lower than that in 293T cells. We also demonstrated that ectopic expression of ARCN1 could partially rescue the inhibitory effect of miR-33a on virus replication. Furthermore, we demonstrated that miR-33a could impede virus replication at the stage of virus internalization, which was similar to the pattern for knockdown of ARCN1, indicating that miR-33a inhibits influenza virus infection by suppressing ARCN1 expression. In addition, we found that miR-33a could also weaken the viral ribonucleoprotein activity in an ARCN1-independent manner. In conclusion, we found that miR-33a is a novel inhibitory factor for influenza A virus replication. PMID:26498766

  5. Zinc Binding Activity of Human Metapneumovirus M2-1 Protein Is Indispensable for Viral Replication and Pathogenesis In Vivo

    PubMed Central

    Cai, Hui; Zhang, Yu; Ma, Yuanmei; Sun, Jing; Liang, Xueya

    2015-01-01

    ABSTRACT Human metapneumovirus (hMPV) is a member of the Pneumovirinae subfamily in the Paramyxoviridae family that causes respiratory tract infections in humans. Unlike members of the Paramyxovirinae subfamily, the polymerase complex of pneumoviruses requires an additional cofactor, the M2-1 protein, which functions as a transcriptional antitermination factor. The M2-1 protein was found to incorporate zinc ions, although the specific role(s) of the zinc binding activity in viral replication and pathogenesis remains unknown. In this study, we found that the third cysteine (C21) and the last histidine (H25) in the zinc binding motif (CCCH) of hMPV M2-1 were essential for zinc binding activity, whereas the first two cysteines (C7 and C15) play only minor or redundant roles in zinc binding. In addition, the zinc binding motif is essential for the oligomerization of M2-1. Subsequently, recombinant hMPVs (rhMPVs) carrying mutations in the zinc binding motif were recovered. Interestingly, rhMPV-C21S and -H25L mutants, which lacked zinc binding activity, had delayed replication in cell culture and were highly attenuated in cotton rats. In contrast, rhMPV-C7S and -C15S strains, which retained 60% of the zinc binding activity, replicated as efficiently as rhMPV in cotton rats. Importantly, rhMPVs that lacked zinc binding activity triggered high levels of neutralizing antibody and provided complete protection against challenge with rhMPV. Taken together, these results demonstrate that zinc binding activity is indispensable for viral replication and pathogenesis in vivo. These results also suggest that inhibition of zinc binding activity may serve as a novel approach to rationally attenuate hMPV and perhaps other pneumoviruses for vaccine purposes. IMPORTANCE The pneumoviruses include many important human and animal pathogens, such as human respiratory syncytial virus (hRSV), hMPV, bovine RSV, and avian metapneumovirus (aMPV). Among these viruses, hRSV and hMPV are the

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

    SciTech Connect

    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 that immune activation and T cell prolifeation are key factors in AIDS pathogenesis.

  7. Direct interaction of cellular hnRNP-F and NS1 of influenza A virus accelerates viral replication by modulation of viral transcriptional activity and host gene expression

    SciTech Connect

    Lee, Jun Han; Kim, Sung-Hak; Pascua, Philippe Noriel Q.; Song, Min-Suk; Baek, Yun Hee; Jin, Xun; Choi, Joong-Kook; Kim, Chul-Joong; Kim, Hyunggee; Choi, Young Ki

    2010-02-05

    To investigate novel NS1-interacting proteins, we conducted a yeast two-hybrid analysis, followed by co-immunoprecipitation assays. We identified heterogeneous nuclear ribonucleoprotein F (hnRNP-F) as a cellular protein interacting with NS1 during influenza A virus infection. Co-precipitation assays suggest that interaction between hnRNP-F and NS1 is a common and direct event among human or avian influenza viruses. NS1 and hnRNP-F co-localize in the nucleus of host cells, and the RNA-binding domain of NS1 directly interacts with the GY-rich region of hnRNP-F determined by GST pull-down assays with truncated proteins. Importantly, hnRNP-F expression levels in host cells indicate regulatory role on virus replication. hnRNP-F depletion by small interfering RNA (siRNA) shows 10- to 100-fold increases in virus titers corresponding to enhanced viral RNA polymerase activity. Our results delineate novel mechanism of action by which NS1 accelerates influenza virus replication by modulating normal cellular mRNA processes through direct interaction with cellular hnRNP-F protein.

  8. A 3′-end structure in RNA2 of a crinivirus is essential for viral RNA synthesis and contributes to replication-associated translation activity

    PubMed Central

    Mongkolsiriwattana, Chawin; Zhou, Jaclyn S.; Ng, James C. K.

    2016-01-01

    The terminal ends in the genome of RNA viruses contain features that regulate viral replication and/or translation. We have identified a Y-shaped structure (YSS) in the 3′ terminal regions of the bipartite genome of Lettuce chlorosis virus (LCV), a member in the genus Crinivirus (family Closteroviridae). The YSS is the first in this family of viruses to be determined using Selective 2′-Hydroxyl Acylation Analyzed by Primer Extension (SHAPE). Using luciferase constructs/replicons, in vivo and in vitro assays showed that the 5′ and YSS-containing 3′ terminal regions of LCV RNA1 supported translation activity. In contrast, similar regions from LCV RNA2, including those upstream of the YSS, did not. LCV RNA2 mutants with nucleotide deletions or replacements that affected the YSS were replication deficient. In addition, the YSS of LCV RNA1 and RNA2 were interchangeable without affecting viral RNA synthesis. Translation and significant replication were observed for specific LCV RNA2 replicons only in the presence of LCV RNA1, but both processes were impaired when the YSS and/or its upstream region were incomplete or altered. These results are evidence that the YSS is essential to the viral replication machinery, and contributes to replication enhancement and replication-associated translation activity in the RNA2 replicons. PMID:27694962

  9. RIG-I and MDA-5 Detection of Viral RNA-dependent RNA Polymerase Activity Restricts Positive-Strand RNA Virus Replication

    PubMed Central

    Nikonov, Andrei; Mölder, Tarmo; Sikut, Rein; Kiiver, Kaja; Männik, Andres; Toots, Urve; Lulla, Aleksei; Lulla, Valeria; Utt, Age; Merits, Andres; Ustav, Mart

    2013-01-01

    Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 5′-triphosphate (5′-ppp) RNA and mediate IFN production. Cytosolic 5′-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-β independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 5′-ppp dsRNA and induces IFN-β through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-β induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-β production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity. PMID:24039580

  10. Activation of NF-κB by Human Papillomavirus 16 E1 Limits E1-Dependent Viral Replication through Degradation of E1

    PubMed Central

    Nakahara, Tomomi; Tanaka, Katsuyuki; Ohno, Shin-ichi; Egawa, Nagayasu; Yugawa, Takashi

    2015-01-01

    ABSTRACT NF-κB is a family of transcription factors that regulate gene expression involved in many processes, such as the inflammatory response and cancer progression. Little is known about associations of NF-κB with the human papillomavirus (HPV) life cycle. We have developed a tissue culture system to conditionally induce E1-dependent replication of the human papillomavirus 16 (HPV16) genome in human cervical keratinocytes and found that expression of HPV16 E1, a viral helicase, results in reduction of IκBα and subsequent activation of NF-κB in a manner dependent on helicase activity. Exogenous expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NF-κB, enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of phosphoinositide 3-kinases (PI3Ks), and, to a lesser extent, VE-822, an ATR kinase inhibitor, but not KU55933, an ATM kinase inhibitor, suppressed the activation of NF-κB and augmented E1-dependent replication of the HPV16 genome. Interestingly, the enhancement of E1-dependent replication of the viral genome was associated with increased stability of E1 in the presence of wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces NF-κB activation at least in part through the ATR-dependent DNA damage response and that NF-κB in turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NF-κB may constitute a negative feedback loop. IMPORTANCE A major risk factor in human papillomavirus (HPV)-associated cancers is persistent infection with high-risk HPVs. To eradicate viruses from infected tissue, it is important to understand molecular mechanisms underlying the establishment and maintenance of persistent infection. In this study, we obtained evidence that human papillomavirus 16 (HPV16) E1, a viral DNA helicase essential for amplification of the viral genomes, induces NF-κB activation and that this limits E1-dependent

  11. Behçet's disease diagnosed after acute HIV infection: viral replication activating underlying autoimmunity?

    PubMed

    Roscoe, Clay; Kinney, Rebecca; Gilles, Ryan; Blue, Sky

    2015-05-01

    Behçet's disease is an autoimmune systemic vasculitis that can occur after exposure to infectious agents. Behçet's disease also has been associated with HIV infection, including de novo development of this condition during chronic HIV infection and resolution of Behçet's disease symptoms following initiation of antiretroviral therapy. We describe a patient who presented with systemic vasculitis with skin and mucous membrane ulcerations in the setting of acute HIV infection, who was eventually diagnosed with Behçet's disease, demonstrating a possible link between acute HIV infection, immune activation and development of autoimmunity.

  12. 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

  13. Abortive infection of snakehead fish vesiculovirus in ZF4 cells was associated with the RLRs pathway activation by viral replicative intermediates.

    PubMed

    Wang, Wenwen; Asim, Muhammad; Yi, Lizhu; Hegazy, Abeer M; Hu, Xianqin; Zhou, Yang; Ai, Taoshan; Lin, Li

    2015-03-18

    Snakehead fish vesiculovirus (SHVV) is a negative strand RNA virus which can cause great economic losses in fish culture. To facilitate the study of SHVV-host interactions, the susceptibility of zebrafish embryonic fibroblast cell line (ZF4) to the SHVV was investigated in this report. The results showed that high amount of viral mRNAs and cRNAs were detected at the 3 h post-infection. However, the expressions of the viral mRNAs and cRNA were decreased dramatically after 6 h post-infection. In addition, the expressions of interferon (IFN) and interferon-induced GTP-binding protein Mx were all up regulated significantly at the late stage of the infection. Meanwhile, the expressions of Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated gene 5 (MDA5) were also all up-regulated significantly during the infection. Two isoforms of DrLGP2 from zebrafish were also cloned and analyzed. Interestingly, the expression of DrLGP2a but not DrLGP2b was significantly up-regulated at both mRNA and protein levels, indicating that the two DrLGP2 isoforms might play different roles during the SHVV infection. Transfection experiment showed that viral replicative intermediates were required for the activation of IFN-α expression. Taken together, the abortive infection of SHVV in ZF4 cells was associated with the activation of RLRs pathway, which was activated by viral replicative intermediates.

  14. In vitro replication activity of bovine viral diarrhea virus in an epithelial cell line and in bovine peripheral blood mononuclear cells.

    PubMed

    Turin, Lauretta; Lucchini, Barbara; Bronzo, Valerio; Luzzago, Camilla

    2012-11-01

    The present study focused on the in vitro infection of Madin-Darby bovine kidney (MDBK) cells and bovine peripheral blood mononuclear cells (PBMCs) from naÏve animals with non-cytopathic (ncp, BVDV-1b NY-1) and cytopathic (cp, BVDV-1a NADL) strains. Infections with 0.1 and 1 multiplicity of infections (MOI) and incubation times of 18 and 36 hr were compared. Twelve BVDV naÏve heifers were enrolled to collect PBMCs. The viral loads in MDBK cells and in PBMCs after in vitro infections were measured by real-time polymerase chain reaction (PCR) assays. The highest viral loads were measured at 1 MOI and 36 hr post infection in both cell systems and the lowest at 0.1 MOI and 18 hr with the exception of the cp strain NADL in PBMCs, for which the highest viral load was observed at 0.1 MOI and 36 hr. Viral load mean values were higher for the cp strain than the ncp strain irrespective of the extent of the infection period and MOI. The models of infection studied uncovered different replication activities respectively according to the biotype of virus, the cell substrate and the duration of infection. Replication tends to be higher in PBMCs, particularly at low MOIs and for the ncp strain.

  15. Analysis of the earliest steps of hepadnavirus replication: genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity.

    PubMed Central

    Köck, J; Schlicht, H J

    1993-01-01

    Hepadnaviruses contain a relaxed circular DNA genome (RC-DNA) with discontinuities in both strands. Upon infectious entry into a host cell, this genome is converted into a covalently closed superhelical form (CCC-DNA), which later serves as the template for transcription. Here we examined whether the viral polymerase activity is required for this repair reaction. Primary hepatocytes prepared from embryonated duck eggs were infected with the duck hepatitis B virus. Conversion of the RC-DNA into the CCC-DNA was then analyzed by a newly developed polymerase chain reaction technique. This method allows the efficient discrimination between the two DNA forms and is sensitive enough to monitor repair of the infecting viral DNA in the absence of replication and amplification. Thus, we were able to monitor this process in the presence of a potent inhibitor of the viral polymerase, the nucleoside analog 2',3'-dideoxyguanosine. The data show that inhibition of the viral polymerase activity has no influence on genome repair, suggesting that this enzymatic function is not required for conversion of the RC-DNA into the CCC-DNA. Consequently, antiviral drugs blocking the polymerase activity cannot prevent the infectious entry of the virus into a host cell. Images PMID:8331730

  16. Abortive infection of snakehead fish vesiculovirus in ZF4 cells was associated with the RLRs pathway activation by viral replicative intermediates.

    PubMed

    Wang, Wenwen; Asim, Muhammad; Yi, Lizhu; Hegazy, Abeer M; Hu, Xianqin; Zhou, Yang; Ai, Taoshan; Lin, Li

    2015-01-01

    Snakehead fish vesiculovirus (SHVV) is a negative strand RNA virus which can cause great economic losses in fish culture. To facilitate the study of SHVV-host interactions, the susceptibility of zebrafish embryonic fibroblast cell line (ZF4) to the SHVV was investigated in this report. The results showed that high amount of viral mRNAs and cRNAs were detected at the 3 h post-infection. However, the expressions of the viral mRNAs and cRNA were decreased dramatically after 6 h post-infection. In addition, the expressions of interferon (IFN) and interferon-induced GTP-binding protein Mx were all up regulated significantly at the late stage of the infection. Meanwhile, the expressions of Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated gene 5 (MDA5) were also all up-regulated significantly during the infection. Two isoforms of DrLGP2 from zebrafish were also cloned and analyzed. Interestingly, the expression of DrLGP2a but not DrLGP2b was significantly up-regulated at both mRNA and protein levels, indicating that the two DrLGP2 isoforms might play different roles during the SHVV infection. Transfection experiment showed that viral replicative intermediates were required for the activation of IFN-α expression. Taken together, the abortive infection of SHVV in ZF4 cells was associated with the activation of RLRs pathway, which was activated by viral replicative intermediates. PMID:25794284

  17. Abortive Infection of Snakehead Fish Vesiculovirus in ZF4 Cells Was Associated with the RLRs Pathway Activation by Viral Replicative Intermediates

    PubMed Central

    Wang, Wenwen; Asim, Muhammad; Yi, Lizhu; Hegazy, Abeer M.; Hu, Xianqin; Zhou, Yang; Ai, Taoshan; Lin, Li

    2015-01-01

    Snakehead fish vesiculovirus (SHVV) is a negative strand RNA virus which can cause great economic losses in fish culture. To facilitate the study of SHVV-host interactions, the susceptibility of zebrafish embryonic fibroblast cell line (ZF4) to the SHVV was investigated in this report. The results showed that high amount of viral mRNAs and cRNAs were detected at the 3 h post-infection. However, the expressions of the viral mRNAs and cRNA were decreased dramatically after 6 h post-infection. In addition, the expressions of interferon (IFN) and interferon-induced GTP-binding protein Mx were all up regulated significantly at the late stage of the infection. Meanwhile, the expressions of Retinoic acid-inducible gene I (RIG-I) and Melanoma differentiation-associated gene 5 (MDA5) were also all up-regulated significantly during the infection. Two isoforms of DrLGP2 from zebrafish were also cloned and analyzed. Interestingly, the expression of DrLGP2a but not DrLGP2b was significantly up-regulated at both mRNA and protein levels, indicating that the two DrLGP2 isoforms might play different roles during the SHVV infection. Transfection experiment showed that viral replicative intermediates were required for the activation of IFN-α expression. Taken together, the abortive infection of SHVV in ZF4 cells was associated with the activation of RLRs pathway, which was activated by viral replicative intermediates. PMID:25794284

  18. Hepatitis C Virus RNA Replication Depends on Specific Cis- and Trans-Acting Activities of Viral Nonstructural Proteins

    PubMed Central

    Kazakov, Teymur; Yang, Feng; Ramanathan, Harish N.; Kohlway, Andrew; Diamond, Michael S.; Lindenbach, Brett D.

    2015-01-01

    Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV), an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS) genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3–5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3–5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally. PMID:25875808

  19. Activity of a novel quinoxaline derivative against human immunodeficiency virus type 1 reverse transcriptase and viral replication.

    PubMed Central

    Kleim, J P; Bender, R; Billhardt, U M; Meichsner, C; Riess, G; Rösner, M; Winkler, I; Paessens, A

    1993-01-01

    S-2720 [6-chloro-3,3-dimethyl-4-(isopropenyloxycarbonyl)-3,4- dihydroquinoxalin-2(1H)-thione], a quinoxaline derivative, was found to be a very potent inhibitor of both human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) activity and HIV-1 replication in tissue culture. Like other nonnucleoside RT inhibitors, S-2720 does not affect the HIV-2 RT. A S-2720-resistant virus was selected and shown to possess a mutation within the RT-coding region that has not previously been described. Notably, this mutation gives rise to a dramatic decrease in enzyme activity. S-2720, therefore, belongs to a new class of RT inhibitors that bind differently to the RT than other known nonnucleoside RT inhibitors. As no toxic effects were observed with S-2720 in mice, these quinoxaline derivatives deserve further evaluation to prove their potency as possible therapeutic agents for HIV-1 infection. PMID:7692812

  20. Lymphoid cells in the spleens of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication.

    PubMed Central

    Korba, B E; Wells, F; Tennant, B C; Cote, P J; Gerin, J L

    1987-01-01

    Lymphoid cells were purified from the spleens of 15 woodchucks and examined for the presence of woodchuck hepatitis virus (WHV). Lymphoid cells from the spleens of eight of eight chronically infected animals contained high levels of WHV RNA and DNA. A 100-fold lower level of WHV DNA was found in the spleen from one of five animals that had recovered from acute WHV infections 2 years before this analysis. No WHV nucleic acids were observed in either of two uninfected animals. WHV DNA patterns in the lymphoid cells from the spleens of the chronically infected animals, which included the presence of single-stranded DNA and RNA-DNA hybrid molecules, were identical to those observed in WHV-infected liver. WHV DNA in these cells was present in intact, 27-nm core particles which also contained the endogenous DNA polymerase activity. These results indicate that the spleen is a site of active WHV DNA replication and is most likely a major source of WHV-infected cells in the circulating lymphoid cell population. Images PMID:3573141

  1. HSV-1 ICP0: paving the way for viral replication.

    PubMed

    Smith, Miles C; Boutell, Chris; Davido, David J

    2011-04-01

    Herpes simplex virus type 1 (HSV-1) has two distinct phases of its viral life cycle: lytic and latent. One viral immediate-early protein that is responsible for determining the balance between productive lytic replication and reactivation from latency is infected cell protein 0 (ICP0). ICP0 is a 775-amino acid really interesting new gene (RING)-finger-containing protein that possesses E3 ubiquitin ligase activity, which is required for ICP0 to activate HSV-1 gene expression, disrupt nuclear domain (ND) 10 structures, mediate the degradation of cellular proteins, and evade the host cell's intrinsic and innate antiviral defenses. This article examines our current understanding of ICP0's transactivating, E3 ubiquitin ligase, and antihost defense activities and their inter-relationships to one another. Lastly, we will discuss how these properties of ICP0 may be utilized as possible targets for HSV-1 antiviral therapies. PMID:21765858

  2. Mathematical model of the Tat-Rev regulation of HIV-1 replication in an activated cell predicts the existence of oscillatory dynamics in the synthesis of viral components

    PubMed Central

    2014-01-01

    Background The life cycle of human immunodeficiency virus type-1 (HIV-1) makes possible the realization of regulatory strategies that can lead to complex dynamical behavior of the system. We analyze the strategy which is based on two feedback mechanisms, one mediating a positive regulation of the virus replication by Tat protein via the antitermination of the genomic RNAs transcription on TAR (transactivation responsive) element of the proviral DNA and the second mechanism providing a negative regulation of the splicing of the full-length (9 kb) RNAs and incompletely spliced (4 kb) RNAs via their transport from the nucleus to the cytoplasm. Although the existence of these two regulatory feedback loops has been considered in other mathematical models, none of them examined the conditions for the emergence of complex oscillatory patterns in the intracellular dynamics of viral components. Results We developed a mechanistic mathematical model for the Tat-Rev mediated regulation of HIV-1 replication, which considers the activation of proviral DNA transcription, the Tat-specific antitermination of transcription on TAR-element, resulting in the synthesis of the full-length 9 kb RNA, the splicing of the 9 kb RNA down to the 4 kb RNA and the 4 kb RNA to 2 kb RNA, the transport of 2 kb mRNAs from the nucleus to the cytoplasm by the intracellular mechanisms, the multiple binding of the Rev protein to RRE (Rev Response Element) sites on 9 kb and 4 kb RNA resulting in their export to the cytoplasm and the synthesis of Tat and Rev proteins in the cytoplasm followed by their transport into the nucleus. The degradation of all viral proteins and RNAs both in the cytoplasm and the nucleus is described. The model parameters values were derived from the published literature data. The model was used to examine the dynamics of the synthesis of the viral proteins Tat and Rev, the mRNAs under the intracellular conditions specific for activated HIV-1 infected macrophages. In addition, we

  3. HSV-1 Remodels Host Telomeres To Facilitate Viral Replication

    PubMed Central

    Deng, Zhong; Kim, Eui Tae; Vladimirova, Olga; Dheekollu, Jayaraju; Wang, Zhuo; Newhart, Alyshia; Liu, Dongmei; Myers, Jaclyn L.; Hensley, Scott E.; Moffat, Jennifer; Janicki, Susan M.; Fraser, Nigel W.; Knipe, David M.; Weitzman, Matthew D.; Lieberman, Paul M.

    2015-01-01

    Summary Telomeres protect the ends of cellular chromosomes. We show here that infection with herpes simplex virus 1 (HSV-1) results in chromosomal structural aberrations at telomeres and the accumulation of telomere dysfunction-induced DNA damage foci (TIFs). At the molecular level, HSV-1 induces transcription of telomere repeat-containing RNA (TERRA), followed by the proteolytic degradation of the telomere protein TPP1, and loss of the telomere repeat DNA signal. The HSV-1 encoded E3 ubiquitin ligase ICP0 is required for TERRA transcription and facilitates TPP1 degradation. shRNA depletion of TPP1 increases viral replication, arguing that TPP1inhibits viral replication. Viral replication protein ICP8 forms foci that coincide with telomeric proteins and ICP8 null virus failed to degrade telomere DNA signal. These findings suggest that HSV-1 reorganizes telomeres to form ICP8-associated pre-replication foci and promotes viral genomic replication. PMID:25497088

  4. Immunoactivation induced by chronic viral infection inhibits viral replication and drives immunosuppression through sustained IFN-I responses.

    PubMed

    Honke, Nadine; Shaabani, Namir; Merches, Katja; Gassa, Asmae; Kraft, Anke; Ehrhardt, Katrin; Häussinger, Dieter; Löhning, Max; Dittmer, Ulf; Hengel, Hartmut; Recher, Mike; Lang, Philipp A; Lang, Karl S

    2016-02-01

    Acute or chronic viral infections can lead to generalized immunosuppression. Several mechanisms, such as immunopathology of CD8(+) T cells, inhibitory receptors, or regulatory T (Treg) cells, contribute to immune dysfunction. Moreover, patients with chronic viral infections usually do not respond to vaccination, a finding that has not been previously explained. Recently, we reported that CD169(+) macrophages enforce viral replication, which is essential for guaranteeing antigen synthesis and efficient adaptive immune responses. In the present study, we used a chronic lymphocytic choriomeningitis virus infection mouse model to determine whether this mechanism is affected by chronic viral infection, which may impair the activation of adaptive immunity. We found that enforced viral replication of a superinfecting virus is completely blunted in chronically infected mice. This absence of enforced viral replication in CD169(+) macrophages is not explained by CD8(+) T-cell-mediated immunopathology but rather by prolonged IFN-I responses. Consequently, the absence of viral replication impairs both antigen production and the adaptive immune response against the superinfecting virus. These findings indicate that chronic infection leads to sustained IFN-I action, which is responsible for the absence of an antiviral immune response against a secondary viral infection. PMID:26507703

  5. Immunoactivation induced by chronic viral infection inhibits viral replication and drives immunosuppression through sustained IFN‐I responses

    PubMed Central

    Honke, Nadine; Shaabani, Namir; Merches, Katja; Gassa, Asmae; Kraft, Anke; Ehrhardt, Katrin; Häussinger, Dieter; Löhning, Max; Dittmer, Ulf; Hengel, Hartmut; Recher, Mike; Lang, Philipp A.

    2015-01-01

    Acute or chronic viral infections can lead to generalized immunosuppression. Several mechanisms, such as immunopathology of CD8+ T cells, inhibitory receptors, or regulatory T (Treg) cells, contribute to immune dysfunction. Moreover, patients with chronic viral infections usually do not respond to vaccination, a finding that has not been previously explained. Recently, we reported that CD169+ macrophages enforce viral replication, which is essential for guaranteeing antigen synthesis and efficient adaptive immune responses. In the present study, we used a chronic lymphocytic choriomeningitis virus infection mouse model to determine whether this mechanism is affected by chronic viral infection, which may impair the activation of adaptive immunity. We found that enforced viral replication of a superinfecting virus is completely blunted in chronically infected mice. This absence of enforced viral replication in CD169+ macrophages is not explained by CD8+ T‐cell‐mediated immunopathology but rather by prolonged IFN‐I responses. Consequently, the absence of viral replication impairs both antigen production and the adaptive immune response against the superinfecting virus. These findings indicate that chronic infection leads to sustained IFN‐I action, which is responsible for the absence of an antiviral immune response against a secondary viral infection. PMID:26507703

  6. Aquareovirus NS80 Initiates Efficient Viral Replication by Retaining Core Proteins within Replication-Associated Viral Inclusion Bodies.

    PubMed

    Yan, Liming; Zhang, Jie; Guo, Hong; Yan, Shicui; Chen, Qingxiu; Zhang, Fuxian; Fang, Qin

    2015-01-01

    Viral inclusion bodies (VIBs) are specific intracellular compartments for reoviruses replication and assembly. Aquareovirus nonstructural protein NS80 has been identified to be the major constituent for forming globular VIBs in our previous study. In this study, we investigated the role of NS80 in viral structural proteins expression and viral replication. Immunofluorescence assays showed that NS80 could retain five core proteins or inner-capsid proteins (VP1-VP4 and VP6), but not outer-capsid proteins (VP5 and VP7), within VIBs in co-transfected or infected cells. Further co-immunoprecipitation analysis confirmed that NS80 could interact with each core protein respectively. In addition, we found that newly synthesized viral RNAs co-localized with VIBs. Furthermore, time-course analysis of viral structural proteins expression showed that the expression of NS80 was detected first, followed by the detection of inner shell protein VP3, and then of other inner-capsid proteins, suggesting that VIBs were essential for the formation of viral core frame or progeny virion. Moreover, knockdown of NS80 by shRNA not only inhibited the expression of aquareovirus structural proteins, but also inhibited viral infection. These results indicated that NS80-based VIBs were formed at earlier stage of infection, and NS80 was able to coordinate the expression of viral structural proteins and viral replication.

  7. Simian virus 40 T antigen can transcriptionally activate and mediate viral DNA replication in cells which lack the retinoblastoma susceptibility gene product.

    PubMed Central

    Trifillis, P; Picardi, J; Alwine, J C

    1990-01-01

    Simian virus 40 T antigen is a multifunctional protein which has recently been shown to form a complex with the retinoblastoma susceptibility gene product (Rb protein) (J.A. DeCaprio, J.W. Ludlow, J. Figge, J.-Y. Shaw, C.-M. Huang, W.-H. Lee, E. Marsilio, E. Paucha, and D.M. Livingston, Cell 54:275-283, 1988; P. Whyte, K.J. Buchkovich, J.M. Horowitz, S.H. Friend, M. Raybuck, R.A. Weinberg, and E. Harlow, Nature (London) 334:124-129, 1988). This interaction may facilitate some of the functions of T antigen. The ability of simian virus 40 T antigen to mediate transcriptional activation and viral DNA replication was tested in human osteosarcoma cell lines U-2OS and Saos-2, which are Rb positive and Rb negative, respectively. Both functions of T antigen were efficient in both cell lines. Hence, these functions can occur in the absence of Rb protein. Images PMID:2154611

  8. SV40 replication in human mesothelial cells induces HGF/Met receptor activation: A model for viral-related carcinogenesis of human malignant mesothelioma

    PubMed Central

    Cacciotti, Paola; Libener, Roberta; Betta, Piergiacomo; Martini, Fernanda; Porta, Camillo; Procopio, Antonio; Strizzi, Luigi; Penengo, Lorenza; Tognon, Mauro; Mutti, Luciano; Gaudino, Giovanni

    2001-01-01

    Recent studies suggested that simian virus 40 (SV40) may cause malignant mesothelioma, although the pathogenic mechanism is unclear. We found that in SV40-positive malignant mesothelioma cells, the hepatocyte growth factor (HGF) receptor (Met) was activated. In human mesothelial cells (HMC) transfected with full-length SV40 DNA (SV40-HMC), Met receptor activation was associated with S-phase entry, acquisition of a fibroblastoid morphology, and the assembly of viral particles. Coculture experiments revealed the ability of SV40-HMC to infect permissive monkey cells (CV-1), HMC, and murine BNL CL cells. Cocultured human and murine SV40-positive cells expressed HGF, showed Met tyrosine phosphorylation and S-phase entry, and acquired a spindle-shaped morphology (spBNL), whereas CV-1 cells were lysed. Cocultured HMC inherited from SV40-HMC the infectivity, as they induced lysis in cocultured CV-1 cells. Treatment with suramin or HGF-blocking antibodies inhibited Met tyrosine phosphorylation in all large T antigen (Tag)-positive cells and reverted the spindle-shaped morphology of spBNL. This finding indicated that Met activation and subsequent biological effects were mediated by an autocrine HGF circuit. This, in turn, was causally related to Tag expression, being induced by transfection with the SV40 early region alone. Our findings suggest that when SV40 infects HMC it causes Met activation via an autocrine loop. Furthermore, SV40 replicates in HMC and infects the adjacent HMC, inducing an HGF-dependent Met activation and cell-cycle progression into S phase. This may explain how a limited number of SV40-positive cells may be sufficient to direct noninfected HMC toward malignant transformation. PMID:11572935

  9. Cyclophilins as Modulators of Viral Replication

    PubMed Central

    Frausto, Stephen D.; Lee, Emily; Tang, Hengli

    2013-01-01

    Cyclophilins are peptidyl‐prolyl cis/trans isomerases important in the proper folding of certain proteins. Mounting evidence supports varied roles of cyclophilins, either positive or negative, in the life cycles of diverse viruses, but the nature and mechanisms of these roles are yet to be defined. The potential for cyclophilins to serve as a drug target for antiviral therapy is evidenced by the success of non-immunosuppressive cyclophilin inhibitors (CPIs), including Alisporivir, in clinical trials targeting hepatitis C virus infection. In addition, as cyclophilins are implicated in the predisposition to, or severity of, various diseases, the ability to specifically and effectively modulate their function will prove increasingly useful for disease intervention. In this review, we will summarize the evidence of cyclophilins as key mediators of viral infection and prospective drug targets. PMID:23852270

  10. Morphological, Biochemical, and Functional Study of Viral Replication Compartments Isolated from Adenovirus-Infected Cells

    PubMed Central

    Hidalgo, Paloma; Anzures, Lourdes; Hernández-Mendoza, Armando; Guerrero, Adán; Wood, Christopher D.; Valdés, Margarita; Dobner, Thomas

    2016-01-01

    ABSTRACT Adenovirus (Ad) replication compartments (RC) are nuclear microenvironments where the viral genome is replicated and a coordinated program of late gene expression is established. These virus-induced nuclear sites seem to behave as central hubs for the regulation of virus-host cell interactions, since proteins that promote efficient viral replication as well as factors that participate in the antiviral response are coopted and concentrated there. To gain further insight into the activities of viral RC, here we report, for the first time, the morphology, composition, and activities of RC isolated from Ad-infected cells. Morphological analyses of isolated RC particles by superresolution microscopy showed that they were indistinguishable from RC within infected cells and that they displayed a dynamic compartmentalization. Furthermore, the RC-containing fractions (RCf) proved to be functional, as they directed de novo synthesis of viral DNA and RNA as well as RNA splicing, activities that are associated with RC in vivo. A detailed analysis of the production of viral late mRNA from RCf at different times postinfection revealed that viral mRNA splicing occurs in RC and that the synthesis, posttranscriptional processing, and release from RC to the nucleoplasm of individual viral late transcripts are spatiotemporally separate events. The results presented here demonstrate that RCf are a powerful system for detailed study into RC structure, composition, and activities and, as a result, the determination of the molecular mechanisms that induce the formation of these viral sites of adenoviruses and other nuclear-replicating viruses. IMPORTANCE RC may represent molecular hubs where many aspects of virus-host cell interaction are controlled. Here, we show by superresolution microscopy that RCf have morphologies similar to those of RC within Ad-infected cells and that they appear to be compartmentalized, as nucleolin and DBP display different localization in the

  11. Macrophage activation and human immunodeficiency virus infection: HIV replication directs macrophages towards a pro-inflammatory phenotype while previous activation modulates macrophage susceptibility to infection and viral production.

    PubMed

    Porcheray, Fabrice; Samah, Boubekeur; Léone, Cathie; Dereuddre-Bosquet, Nathalie; Gras, Gabriel

    2006-05-25

    Macrophages are pivotal for the regulation of immune and inflammatory responses, but whether their role in HIV infection is protective or deleterious remains unclear. In this study, we investigated the effect of pro- and anti-inflammatory stimuli on macrophage sensitivity to two different aspects of HIV infection: their susceptibility to infection stricto sensu, which we measured by endpoint titration method, and their ability to support virus spread, which we measured by using an RT activity assay in infection kinetics. We show a partially protective role for pro-inflammatory agents as well as for IL-4. We also illustrate that various different stimuli display differential effects on macrophage susceptibility to HIV and on virus replication that occurs thereafter. On the other hand, HIV replication strongly repressed CD206 and CD163 expression, thus clearly orientating macrophages towards a pro-inflammatory phenotype, but independently of TNF. Taken together, our results emphasize that HIV infection of macrophages sets up inflammation at the cell level but through unexpected mechanisms. This may limit target susceptibility and participate in virus clearance but may also result in tissue damage.

  12. Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation

    PubMed Central

    Wong, Mun-Teng

    2016-01-01

    ABSTRACT Hepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the membranous web (MW). The role that human choline kinase-α (hCKα) plays in HCV replication remains elusive. Here, we first showed that hCKα activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCKα colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER) and that HCV infection increased hCKα localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCKα on the ER, and hCKα activity was required for effective trafficking of hCKα and NS5A to the ER. Coimmunoprecipitation showed that hCKα was recruited onto the viral RC presumably through its binding to NS5A domain 1 (D1). hCKα silencing or treatment with CK37, an hCKα activity inhibitor, abolished HCV-induced MW formation. In addition, hCKα depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCKα activity was not essential for the binding of NS5A to hCKα or NS5B. These findings demonstrate that hCKα forms a complex with NS5A and that hCKα activity enhances the targeting of the complex to the ER, where hCKα protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication. IMPORTANCE HCV infection reorganizes the cellular membrane to create an active viral replication site named the membranous web (MW). Here, we report that human choline kinase-α (hCKα) acts as an essential host factor for HCV RNA replication. A fraction of hCKα colocalizes with the viral replication complex (RC) on the endoplasmic reticulum

  13. Differential host response, rather than early viral replication efficiency, correlates with pathogenicity caused by influenza viruses.

    PubMed

    Askovich, Peter S; Sanders, Catherine J; Rosenberger, Carrie M; Diercks, Alan H; Dash, Pradyot; Navarro, Garnet; Vogel, Peter; Doherty, Peter C; Thomas, Paul G; Aderem, Alan

    2013-01-01

    Influenza viruses exhibit large, strain-dependent differences in pathogenicity in mammalian hosts. Although the characteristics of severe disease, including uncontrolled viral replication, infection of the lower airway, and highly inflammatory cytokine responses have been extensively documented, the specific virulence mechanisms that distinguish highly pathogenic strains remain elusive. In this study, we focused on the early events in influenza infection, measuring the growth rate of three strains of varying pathogenicity in the mouse airway epithelium and simultaneously examining the global host transcriptional response over the first 24 hours. Although all strains replicated equally rapidly over the first viral life-cycle, their growth rates in both lung and tracheal tissue strongly diverged at later times, resulting in nearly 10-fold differences in viral load by 24 hours following infection. We identified separate networks of genes in both the lung and tracheal tissues whose rapid up-regulation at early time points by specific strains correlated with a reduced viral replication rate of those strains. The set of early-induced genes in the lung that led to viral growth restriction is enriched for both NF-κB binding site motifs and members of the TREM1 and IL-17 signaling pathways, suggesting that rapid, NF-κB -mediated activation of these pathways may contribute to control of viral replication. Because influenza infection extending into the lung generally results in severe disease, early activation of these pathways may be one factor distinguishing high- and low-pathogenicity strains.

  14. Herpesvirus Replication Compartments Originate with Single Incoming Viral Genomes

    PubMed Central

    Kobiler, O.; Brodersen, P.; Taylor, M. P.; Ludmir, E. B.; Enquist, L. W.

    2011-01-01

    ABSTRACT Previously we described a method to estimate the average number of virus genomes expressed in an infected cell. By analyzing the color spectrum of cells infected with a mixture of isogenic pseudorabies virus (PRV) recombinants expressing three fluorophores, we estimated that fewer than seven incoming genomes are expressed, replicated, and packaged into progeny per cell. In this report, we expand this work and describe experiments demonstrating the generality of the method, as well as providing more insight into herpesvirus replication. We used three isogenic PRV recombinants, each expressing a fluorescently tagged VP26 fusion protein (VP26 is a capsid protein) under the viral VP26 late promoter. We calculated a similar finite limit on the number of expressed viral genomes, indicating that this method is independent of the promoter used to transcribe the fluorophore genes, the time of expression of the fluorophore (early versus late), and the insertion site of the fluorophore gene in the PRV genome (UL versus US). Importantly, these VP26 fusion proteins are distributed equally in punctate virion assembly structures in each nucleus, which improves the signal-to-noise ratio when determining the color spectrum of each cell. To understand how the small number of genomes are distributed among the replication compartments, we used a two-color fluorescent in situ hybridization assay. Most viral replication compartments in the nucleus occupy unique nuclear territories, implying that they arose from single genomes. Our experiments suggest a correlation between the small number of expressed viral genomes and the limited number of replication compartments. PMID:22186611

  15. Viral Replication Protein Inhibits Cellular Cofilin Actin Depolymerization Factor to Regulate the Actin Network and Promote Viral Replicase Assembly

    PubMed Central

    Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Barajas, Daniel; Risco, Cristina; Nagy, Peter D.

    2016-01-01

    RNA viruses exploit host cells by co-opting host factors and lipids and escaping host antiviral responses. Previous genome-wide screens with Tomato bushy stunt virus (TBSV) in the model host yeast have identified 18 cellular genes that are part of the actin network. In this paper, we show that the p33 viral replication factor interacts with the cellular cofilin (Cof1p), which is an actin depolymerization factor. Using temperature-sensitive (ts) Cof1p or actin (Act1p) mutants at a semi-permissive temperature, we find an increased level of TBSV RNA accumulation in yeast cells and elevated in vitro activity of the tombusvirus replicase. We show that the large p33 containing replication organelle-like structures are located in the close vicinity of actin patches in yeast cells or around actin cable hubs in infected plant cells. Therefore, the actin filaments could be involved in VRC assembly and the formation of large viral replication compartments containing many individual VRCs. Moreover, we show that the actin network affects the recruitment of viral and cellular components, including oxysterol binding proteins and VAP proteins to form membrane contact sites for efficient transfer of sterols to the sites of replication. Altogether, the emerging picture is that TBSV, via direct interaction between the p33 replication protein and Cof1p, controls cofilin activities to obstruct the dynamic actin network that leads to efficient subversion of cellular factors for pro-viral functions. In summary, the discovery that TBSV interacts with cellular cofilin and blocks the severing of existing filaments and the formation of new actin filaments in infected cells opens a new window to unravel the way by which viruses could subvert/co-opt cellular proteins and lipids. By regulating the functions of cofilin and the actin network, which are central nodes in cellular pathways, viruses could gain supremacy in subversion of cellular factors for pro-viral functions. PMID:26863541

  16. SIV antigen immunization induces transient antigen-specific T cell responses and selectively activates viral replication in draining lymph nodes in retroviral suppressed rhesus macaques

    PubMed Central

    2011-01-01

    Background HIV infection causes a qualitative and quantitative loss of CD4+ T cell immunity. The institution of anti-retroviral therapy (ART) restores CD4+ T cell responses to many pathogens, but HIV-specific responses remain deficient. Similarly, therapeutic immunization with HIV antigens of chronically infected, ART treated subjects results in poor induction of HIV-specific CD4 responses. In this study, we used a macaque model of ART treatment during chronic infection to study the virologic consequences of SIV antigen stimulation in lymph nodes early after immunization. Rhesus CMV (RhCMV) seropositive, Mamu A*01 positive rhesus macaques were chronically infected with SIVmac251 and treated with ART. The immune and viral responses to SIV gag and RhCMV pp65 antigen immunization in draining lymph nodes and peripheral blood were analyzed. Animals were immunized on contralateral sides with SIV gag and RhCMV pp65 encoding plasmids, which allowed lymph nodes draining each antigen to be obtained at the same time from the same animal for direct comparison. Results We observed that both SIV and RhCMV immunizations stimulated transient antigen-specific T cell responses in draining lymph nodes. The RhCMV-specific responses were potent and sustained (50 days post-immunization) in the periphery, while the SIV-specific responses were transient and extinguished quickly. The SIV antigen stimulation selectively induced transient SIV replication in draining lymph nodes. Conclusions The data are consistent with a model whereby viral replication in response to SIV antigen stimulation limits the generation of SIV antigen-specific responses and suggests a potential mechanism for the early loss and poor HIV-specific CD4+ T cell response observed in HIV-infected individuals. PMID:21752277

  17. Effects of Adenovirus Type 5 E1A Isoforms on Viral Replication in Arrested Human Cells

    PubMed Central

    Radko, Sandi; Jung, Richard; Olanubi, Oladunni; Pelka, Peter

    2015-01-01

    Human adenovirus has evolved to infect and replicate in terminally differentiated human epithelial cells, predominantly those within the airway, the gut, or the eye. To overcome the block to viral DNA replication present in these cells, the virus expresses the Early 1A proteins (E1A). These immediate early proteins drive cells into S-phase and induce expression of all other viral early genes. During infection, several E1A isoforms are expressed with proteins of 289, 243, 217, 171, and 55 residues being present for human adenovirus type 5. Here we examine the contribution that the two largest E1A isoforms make to the viral life cycle in growth-arrested normal human fibroblasts. Viruses that express E1A289R were found to replicate better than those that do not express this isoform. Importantly, induction of several viral genes was delayed in a virus expressing E1A243R, with several viral structural proteins undetectable by western blot. We also highlight the changes in E1A isoforms detected during the course of viral infection. Furthermore, we show that viral DNA replication occurs more efficiently, leading to higher number of viral genomes in cells infected with viruses that express E1A289R. Finally, induction of S-phase specific genes differs between viruses expressing different E1A isoforms, with those having E1A289R leading to, generally, earlier activation of these genes. Overall, we provide an overview of adenovirus replication using modern molecular biology approaches and further insights into the contribution that E1A isoforms make to the life cycle of human adenovirus in arrested human fibroblasts. PMID:26448631

  18. Variant upstream regulatory region sequences differentially regulate human papillomavirus type 16 DNA replication throughout the viral life cycle.

    PubMed

    Hubert, Walter G

    2005-05-01

    While the central role of the viral upstream regulatory region (URR) in the human papillomavirus (HPV) life cycle has been well established, its effects on viral replication factor expression and plasmid replication of HPV type 16 (HPV16) remain unclear. Some nonprototypic variants of HPV16 contain altered URR sequences and are considered to increase the oncogenic risk of infections. To determine the relationship between viral replication and variant URRs, hybrid viral genomes were constructed with the replication-competent HPV16 prototype W12 and analyzed in assays which recapitulate the different phases of normal viral replication. The establishment efficiencies of hybrid HPV16 genomes differed about 20-fold among European prototypes and variants from Africa and America. Generally, European and African genomes exhibited the lowest replication efficiencies. The high replication levels observed with American variants were primarily attributable to their efficient expression of the replication factors E1 and E2. The maintenance levels of these viral genomes varied about fivefold, which correlated with their respective establishment phenotypes and published P(97) activities. Vegetative DNA amplification could also be observed with replicating HPV16 genomes. These results indicate that efficient E1/E2 expression and elevated plasmid replication levels during the persistent stage of infection may comprise a risk factor in HPV16-mediated oncogenesis.

  19. Going viral: a review of replication-selective oncolytic adenoviruses

    PubMed Central

    Larson, Christopher; Oronsky, Bryan; Scicinski, Jan; Fanger, Gary R.; Stirn, Meaghan; Oronsky, Arnold; Reid, Tony R.

    2015-01-01

    Oncolytic viruses have had a tumultuous course, from the initial anecdotal reports of patients having antineoplastic effects after natural viral infections a century ago to the development of current cutting-edge therapies in clinical trials. Adenoviruses have long been the workhorse of virotherapy, and we review both the scientific and the not-so-scientific forces that have shaped the development of these therapeutics from wild-type viral pathogens, turning an old foe into a new friend. After a brief review of the mechanics of viral replication and how it has been modified to engineer tumor selectivity, we give particular attention to ONYX-015, the forerunner of virotherapy with extensive clinical testing that pioneered the field. The findings from those as well as other oncolytic trials have shaped how we now view these viruses, which our immune system has evolved to vigorously attack, as promising immunotherapy agents. PMID:26280277

  20. Multiple effects of mutations in human immunodeficiency virus type 1 integrase on viral replication.

    PubMed Central

    Engelman, A; Englund, G; Orenstein, J M; Martin, M A; Craigie, R

    1995-01-01

    The integration of a DNA copy of the human immunodeficiency virus type 1 (HIV-1) genome into a chromosome of an infected cell is a pivotal step in virus replication. Integration requires the activity of the virus-encoded integrase, which enters the cell as a component of the virion. Results of numerous mutagenesis studies have identified amino acid residues and protein domains of HIV-1 integrase critical for in vitro activity, but only a few of these mutants have been studied for their effects on HIV replication. We have introduced site-directed changes into an infectious DNA clone of HIV-1 and show that integrase mutations can affect virus replication at a variety of steps. We identified mutations that altered virion morphology, levels of particle-associated integrase and reverse transcriptase, and viral DNA synthesis. One replication-defective mutant virus which had normal morphology and protein composition displayed increased levels of circular viral DNA following infection of a T-cell line. This virus also had a significant titer in a CD4-positive indicator cell assay, which requires the viral Tat protein. Although unintegrated viral DNA can serve as a template for Tat expression in infected indicator cells, this level of expression is insufficient to support a spreading viral infection in CD4-positive lymphocytes. PMID:7535863

  1. 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…

  2. Histone deacetylase 6 inhibition enhances oncolytic viral replication in glioma

    PubMed Central

    Nakashima, Hiroshi; Kaufmann, Johanna K.; Wang, Pin-Yi; Nguyen, Tran; Speranza, Maria-Carmela; Kasai, Kazue; Okemoto, Kazuo; Otsuki, Akihiro; Nakano, Ichiro; Fernandez, Soledad; Goins, William F.; Grandi, Paola; Glorioso, Joseph C.; Lawler, Sean; Cripe, Timothy P.; Chiocca, E. Antonio

    2015-01-01

    Oncolytic viral (OV) therapy, which uses genetically engineered tumor-targeting viruses, is being increasingly used in cancer clinical trials due to the direct cytolytic effects of this treatment that appear to provoke a robust immune response against the tumor. As OVs enter tumor cells, intrinsic host defenses have the potential to hinder viral replication and spread within the tumor mass. In this report, we show that histone deacetylase 6 (HDAC6) in tumor cells appears to alter the trafficking of post-entry OVs from the nucleus toward lysosomes. In glioma cell lines and glioma-stem–like cells, HDAC6 inhibition (HDAC6i) by either pharmacologic or genetic means substantially increased replication of oncolytic herpes simplex virus type 1 (oHSV). Moreover, HDAC6i increased shuttling of post-entry oHSV to the nucleus. In addition, electron microscopic analysis revealed that post-entry oHSVs are preferentially taken up into glioma cells through the endosomal pathway rather than via fusion at the cell surface. Together, these findings illustrate a mechanism of glioma cell defense against an incoming infection by oHSV and identify possible approaches to enhance oHSV replication and subsequent lysis of tumor cells. PMID:26524593

  3. Histone deacetylase 6 inhibition enhances oncolytic viral replication in glioma.

    PubMed

    Nakashima, Hiroshi; Kaufmann, Johanna K; Wang, Pin-Yi; Nguyen, Tran; Speranza, Maria-Carmela; Kasai, Kazue; Okemoto, Kazuo; Otsuki, Akihiro; Nakano, Ichiro; Fernandez, Soledad; Goins, William F; Grandi, Paola; Glorioso, Joseph C; Lawler, Sean; Cripe, Timothy P; Chiocca, E Antonio

    2015-11-01

    Oncolytic viral (OV) therapy, which uses genetically engineered tumor-targeting viruses, is being increasingly used in cancer clinical trials due to the direct cytolytic effects of this treatment that appear to provoke a robust immune response against the tumor. As OVs enter tumor cells, intrinsic host defenses have the potential to hinder viral replication and spread within the tumor mass. In this report, we show that histone deacetylase 6 (HDAC6) in tumor cells appears to alter the trafficking of post-entry OVs from the nucleus toward lysosomes. In glioma cell lines and glioma-stem-like cells, HDAC6 inhibition (HDAC6i) by either pharmacologic or genetic means substantially increased replication of oncolytic herpes simplex virus type 1 (oHSV). Moreover, HDAC6i increased shuttling of post-entry oHSV to the nucleus. In addition, electron microscopic analysis revealed that post-entry oHSVs are preferentially taken up into glioma cells through the endosomal pathway rather than via fusion at the cell surface. Together, these findings illustrate a mechanism of glioma cell defense against an incoming infection by oHSV and identify possible approaches to enhance oHSV replication and subsequent lysis of tumor cells. PMID:26524593

  4. The Viral E8^E2C Repressor Limits Productive Replication of Human Papillomavirus 16

    PubMed Central

    Straub, Elke; Dreer, Marcel; Fertey, Jasmin; Iftner, Thomas

    2014-01-01

    Productive replication of human papillomavirus type 16 (HPV16) occurs only in differentiated keratinocyte cells. In addition to the viral E2 activator protein, HPV16 and related HPV types express transcripts coding for an E8^E2C fusion protein, which limits genome replication in undifferentiated keratinocytes. To address E8^E2C's role in productive replication of HPV16, stable keratinocyte cell lines containing wild-type (wt), E8^E2C knockout (E8−), or E8 KWK mutant (mt) genomes, in which conserved E8 residues were inactivated, were established. Copy numbers of E8− and E8 KWK mt genomes and amounts of early and late viral transcripts were greatly increased compared to those for the wt in undifferentiated keratinocytes, suggesting that HPV16 E8^E2C activities are highly dependent upon the E8 part. Upon differentiation in organotypic cultures, E8 mt genomes displayed higher early viral transcript levels, but no changes in cellular differentiation or virus-induced cellular DNA replication in suprabasal cells were observed. E8 mt genomes were amplified to higher copy numbers and showed increased L1 transcripts compared to wt genomes. Furthermore, the number of cells expressing the viral late protein E4 or L1 or amplifying viral genomes was greatly increased in E8 mt cell lines. In wild-type cells, E8^E2C transcript levels did not decrease by differentiation. Our data indicate that the E8^E2C repressor limits viral transcription and replication throughout the complete life cycle of HPV16. PMID:24198405

  5. 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

  6. Inactivation of the Human Cytomegalovirus US20 Gene Hampers Productive Viral Replication in Endothelial Cells

    PubMed Central

    Cavaletto, Noemi; Luganini, Anna

    2015-01-01

    a key target for viral dissemination and replication in the host, and to contribute to both viral persistence and associated inflammation and vascular diseases. Replication in endothelial cells depends on the activities of a set of viral proteins that regulate different stages of the HCMV replication cycle in an endothelial cell type-specific manner and thereby act as determinants of viral tropism. Here, we report the requirement of a HCMV protein as a postentry tropism factor in endothelial cells. The identification and characterization of HCMV endotheliotropism-regulating proteins will advance our understanding of the molecular mechanisms of HCMV-related pathogenesis and help lead to the design of new antiviral strategies able to exploit these functions. PMID:26311874

  7. Control of viral replication after cessation of HAART

    PubMed Central

    2011-01-01

    We describe two patients who did not experience a viral rebound after cessation of HAART which was initiated for progressive disease. CD4 T-cell count remained stable in one patient and progressively declined in the other, despite apparent viral control. We failed to identify any immune activation or genetic markers that could offer an explanation for this unusual "secondary controller" status. But their viruses are clearly less fit compared to viruses from rebounders. PMID:21314914

  8. Drug targeting of HIV-1 RNA.DNA hybrid structures: thermodynamics of recognition and impact on reverse transcriptase-mediated ribonuclease H activity and viral replication.

    PubMed

    Li, Tsai-Kun; Barbieri, Christopher M; Lin, Hsin-Chin; Rabson, Arnold B; Yang, Gengcheng; Fan, Yupeng; Gaffney, Barbara L; Jones, Roger A; Pilch, Daniel S

    2004-08-01

    RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1.

  9. Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication

    PubMed Central

    Taneja, Poonam; Nasheuer, Heinz-Peter; Hartmann, Hella; Grosse, Frank; Fanning, Ellen; Weisshart, Klaus

    2007-01-01

    The initiation of SV40 (simian virus 40) DNA replication requires the co-operative interactions between the viral Tag (large T-antigen), RPA (replication protein A) and Pol (DNA polymerase α-primase) on the template DNA. Binding interfaces mapped on these enzymes and expressed as peptides competed with the mutual interactions of the native proteins. Prevention of the genuine interactions was accomplished only prior to the primer synthesis step and blocked the assembly of a productive initiation complex. Once the complex was engaged in the synthesis of an RNA primer and its extension, the interfering effects of the peptides ceased, suggesting a stable association of the replication factors during the initiation phase. Specific antibodies were still able to disrupt preformed interactions and inhibited primer synthesis and extension activities, underlining the crucial role of specific protein–protein contacts during the entire initiation process. PMID:17666013

  10. Methods for monitoring dynamics of pulmonary RSV replication by viral culture and by real-time reverse transcription-PCR in vivo: Detection of abortive viral replication.

    PubMed

    Boukhvalova, Marina S; Yim, Kevin C; Prince, Gregory A; Blanco, Jorge C G

    2010-03-01

    Viral infection is normally detected either by viral culture or by PCR methods. Rarely is a combination of the two techniques used in the same study. Yet, when applied simultaneously, viral culture and PCR may reveal important features of viral biology, such as an abortive replication, as in the case of respiratory syncytial virus (RSV) infection. In this unit, we describe methods for detecting abortive RSV replication in a cotton rat model by using the plaque-forming unit assay and the real-time reverse-transcription PCR (qRT-PCR) assay. All steps of the process of monitoring viral replication in vivo are described, starting from the design of animal infection protocols. We continue on to the methods for extracting and processing lung samples for viral culture and RNA extraction, and finish with the actual methods of viral titration by the qRT-PCR and the plaque-forming unit assays.

  11. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins

    PubMed Central

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-01-01

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%–99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy. PMID:26114473

  12. RNase P Ribozymes Inhibit the Replication of Human Cytomegalovirus by Targeting Essential Viral Capsid Proteins.

    PubMed

    Yang, Zhu; Reeves, Michael; Ye, Jun; Trang, Phong; Zhu, Li; Sheng, Jingxue; Wang, Yu; Zen, Ke; Wu, Jianguo; Liu, Fenyong

    2015-06-24

    An engineered RNase P-based ribozyme variant, which was generated using the in vitro selection procedure, was used to target the overlapping mRNA region of two proteins essential for human cytomegalovirus (HCMV) replication: capsid assembly protein (AP) and protease (PR). In vitro studies showed that the generated variant, V718-A, cleaved the target AP mRNA sequence efficiently and its activity was about 60-fold higher than that of wild type ribozyme M1-A. Furthermore, we observed a reduction of 98%-99% in AP/PR expression and an inhibition of 50,000 fold in viral growth in cells with V718-A, while a 75% reduction in AP/PR expression and a 500-fold inhibition in viral growth was found in cells with M1-A. Examination of the antiviral effects of the generated ribozyme on the HCMV replication cycle suggested that viral DNA encapsidation was inhibited and as a consequence, viral capsid assembly was blocked when the expression of AP and PR was inhibited by the ribozyme. Thus, our study indicates that the generated ribozyme variant is highly effective in inhibiting HCMV gene expression and blocking viral replication, and suggests that engineered RNase P ribozyme can be potentially developed as a promising gene-targeting agent for anti-HCMV therapy.

  13. 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

  14. A cysteine-rich metal-binding domain from rubella virus non-structural protein is essential for viral protease activity and virus replication.

    PubMed

    Zhou, Yubin; Tzeng, Wen-Pin; Ye, Yiming; Huang, Yun; Li, Shunyi; Chen, Yanyi; Frey, Teryl K; Yang, Jenny J

    2009-01-15

    The protease domain within the RUBV (rubella virus) NS (non-structural) replicase proteins functions in the self-cleavage of the polyprotein precursor into the two mature proteins which form the replication complex. This domain has previously been shown to require both zinc and calcium ions for optimal activity. In the present study we carried out metal-binding and conformational experiments on a purified cysteine-rich minidomain of the RUBV NS protease containing the putative Zn(2+)-binding ligands. This minidomain bound to Zn(2+) with a stoichiometry of approximately 0.7 and an apparent dissociation constant of <500 nM. Fluorescence quenching and 8-anilinonaphthalene-1-sulfonic acid fluorescence methods revealed that Zn(2+) binding resulted in conformational changes characterized by shielding of hydrophobic regions from the solvent. Mutational analyses using the minidomain identified residues Cys(1175), Cys(1178), Cys(1225) and Cys(1227) were required for the binding of Zn(2+). Corresponding mutational analyses using a RUBV replicon confirmed that these residues were necessary for both proteolytic activity of the NS protease and viability. The present study demonstrates that the CXXC(X)(48)CXC Zn(2+)-binding motif in the RUBV NS protease is critical for maintaining the structural integrity of the protease domain and essential for proteolysis and virus replication. PMID:18795894

  15. The N-Terminal of Aquareovirus NS80 Is Required for Interacting with Viral Proteins and Viral Replication

    PubMed Central

    Zhang, Jie; Guo, Hong; Chen, Qingxiu; Zhang, Fuxian; Fang, Qin

    2016-01-01

    Reovirus replication and assembly occurs within viral inclusion bodies that formed in specific intracellular compartments of cytoplasm in infected cells. Previous study indicated that aquareovirus NS80 is able to form inclusion bodies, and also can retain viral proteins within its inclusions. To better understand how NS80 performed in viral replication and assembly, the functional regions of NS80 associated with other viral proteins in aquareovirus replication were investigated in this study. Deletion mutational analysis and rotavirus NSP5-based protein association platform were used to detect association regions. Immunofluorescence images indicated that different N-terminal regions of NS80 could associate with viral proteins VP1, VP4, VP6 and NS38. Further co-immunoprecipitation analysis confirmed the interaction between VP1, VP4, VP6 or NS38 with different regions covering the N-terminal amino acid (aa, 1–471) of NS80, respectively. Moreover, removal of NS80 N-terminal sequences required for interaction with proteins VP1, VP4, VP6 or NS38 not only prevented the capacity of NS80 to support viral replication in NS80 shRNA-based replication complementation assays, but also inhibited the expression of aquareovirus proteins, suggesting that N-terminal regions of NS80 are necessary for viral replication. These results provided a foundational basis for further understanding the role of NS80 in viral replication and assembly during aquareovirus infection. PMID:26871941

  16. Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus

    PubMed Central

    Xu, Kai; Nagy, Peter D.

    2016-01-01

    Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs). The VRCs built by Tomato bushy stunt virus (TBSV) are enriched with phosphatidylethanolamine (PE) through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP)-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5–positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment. PMID:27760128

  17. Dissecting the role of the ϕ29 terminal protein DNA binding residues in viral DNA replication

    PubMed Central

    Holguera, Isabel; Muñoz-Espín, Daniel; Salas, Margarita

    2015-01-01

    Phage ϕ29 DNA replication takes place by a protein-priming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the ϕ29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo. In the present work we have studied the involvement of the TP N-terminal domain residues responsible for DNA binding in the different stages of viral DNA replication by assaying the in vitro activity of purified TP N-terminal mutant proteins. The results show that mutation of TP residues involved in DNA binding affects the catalytic activity of the DNA polymerase in initiation, as the Km for the initiating nucleotide is increased when these mutant proteins are used as primers. Importantly, this initiation defect was relieved by using the ϕ29 double-stranded DNA binding protein p6 in the reaction, which decreased the Km of the DNA polymerase for dATP about 130–190 fold. Furthermore, the TP N-terminal domain was shown to be required both for a proper interaction with the DNA polymerase and for an efficient viral DNA amplification. PMID:25722367

  18. Increased amounts of the influenza virus nucleoprotein do not promote higher levels of viral genome replication.

    PubMed

    Mullin, Anne E; Dalton, Rosa M; Amorim, Maria Joao; Elton, Debra; Digard, Paul

    2004-12-01

    Influenza virus genome replication requires the virus-encoded nucleoprotein (NP), partly because it is necessary to encapsidate the viral genomic RNA (vRNA) and antigenomic cRNA segments into ribonucleoproteins (RNPs). However, there is also evidence that NP actively regulates viral RNA synthesis and there is a long-standing hypothesis that increased concentrations of NP in the cell are responsible for a switch from genome transcription to replication. Here, this hypothesis is tested in a recombinant setting and in the context of virus infection. In a plasmid-based system for reconstituting active viral RNPs in cells, titration of increasing amounts of NP did not promote higher levels of genome replication relative to transcription, but in fact caused the opposite effect. An approximately fourfold reduction in the ratio of genomic and antigenomic RNAs to mRNA was seen across an 80-fold range of NP plasmid concentrations. When cells were transfected with the same amounts of NP plasmid to establish a concentration gradient of NP prior to virus superinfection, no change in the ratio of cRNA to mRNA was seen for segments 5 and 7, or for the ratio of segment 5 vRNA to mRNA. A slight reduction in the ratio of segment 7 vRNA to mRNA was seen. These findings do not support the simple hypothesis that increased intracellular concentrations of NP promote influenza virus genome replication.

  19. Influenza A Viral Replication Is Blocked by Inhibition of the Inositol-requiring Enzyme 1 (IRE1) Stress Pathway*

    PubMed Central

    Hassan, Ihab H.; Zhang, Michael S.; Powers, Linda S.; Shao, Jian Q.; Baltrusaitis, Jonas; Rutkowski, D. Thomas; Legge, Kevin; Monick, Martha M.

    2012-01-01

    Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications. PMID:22194594

  20. Viral hijacking of a replicative helicase loader and its implications for helicase loading control and phage replication

    PubMed Central

    Hood, Iris V; Berger, James M

    2016-01-01

    Replisome assembly requires the loading of replicative hexameric helicases onto origins by AAA+ ATPases. How loader activity is appropriately controlled remains unclear. Here, we use structural and biochemical analyses to establish how an antimicrobial phage protein interferes with the function of the Staphylococcus aureus replicative helicase loader, DnaI. The viral protein binds to the loader’s AAA+ ATPase domain, allowing binding of the host replicative helicase but impeding loader self-assembly and ATPase activity. Close inspection of the complex highlights an unexpected locus for the binding of an interdomain linker element in DnaI/DnaC-family proteins. We find that the inhibitor protein is genetically coupled to a phage-encoded homolog of the bacterial helicase loader, which we show binds to the host helicase but not to the inhibitor itself. These findings establish a new approach by which viruses can hijack host replication processes and explain how loader activity is internally regulated to prevent aberrant auto-association. DOI: http://dx.doi.org/10.7554/eLife.14158.001 PMID:27244442

  1. Peptide Aptamers That Bind to a Geminivirus Replication Protein Interfere with Viral Replication in Plant Cells †

    PubMed Central

    Lopez-Ochoa, Luisa; Ramirez-Prado, Jorge; Hanley-Bowdoin, Linda

    2006-01-01

    The AL1 protein of tomato golden mosaic virus (TGMV), a member of the geminivirus family, is essential for viral replication in plants. Its N terminus contains three conserved motifs that mediate origin recognition and DNA cleavage during the initiation of rolling-circle replication. We used the N-terminal domain of TGMV AL1 as bait in a yeast two-hybrid screen of a random peptide aptamer library constrained in the active site of the thioredoxin A (TrxA) gene. The screen selected 88 TrxA peptides that also bind to the full-length TGMV AL1 protein. Plant expression cassettes corresponding to the TrxA peptides and a TGMV A replicon encoding AL1 were cotransfected into tobacco protoplasts, and viral DNA replication was monitored by semiquantitative PCR. In these assays, 31 TrxA peptides negatively impacted TGMV DNA accumulation, reducing viral DNA levels to 13 to 64% of those of the wild type. All of the interfering aptamers also bound to the AL1 protein of cabbage leaf curl virus. A comparison of the 20-mer peptides revealed that their sequences are not random. The alignments detected seven potential binding motifs, five of which are more highly represented among the interfering peptides. One motif was present in 18 peptides, suggesting that these peptides interact with a hot spot in the AL1 N terminus. The peptide aptamers characterized in these studies represent new tools for studying AL1 function and can serve as the basis for the development of crops with broad-based resistance to single-stranded DNA viruses. PMID:16731923

  2. Foot-and-Mouth Disease Virus Utilizes an Autophagic Pathway During Viral Replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Infection with positive-strand RNA viruses results in the rearrangement of intracellular membranes into viral replication complexes (VRC) which are the sites of viral RNA replication. Cellular autophagy has been proposed to be a mechanism of VRC formation for a number of positive-stranded RNA viruse...

  3. Tonsillar crypt epithelium is an important extra-central nervous system site for viral replication in EV71 encephalomyelitis.

    PubMed

    He, Yaoxin; Ong, Kien Chai; Gao, Zifen; Zhao, Xishun; Anderson, Virginia M; McNutt, Michael A; Wong, Kum Thong; Lu, Min

    2014-03-01

    Enterovirus 71 (EV71; family Picornaviridae, species human Enterovirus A) usually causes hand, foot, and mouth disease, which may rarely be complicated by fatal encephalomyelitis. We investigated extra-central nervous system (extra-CNS) tissues capable of supporting EV71 infection and replication, and have correlated tissue infection with expression of putative viral entry receptors, scavenger receptor B2 (SCARB2), and P-selectin glycoprotein ligand-1 (PSGL-1). Formalin-fixed, paraffin-embedded CNS and extra-CNS tissues from seven autopsy cases were examined by IHC and in situ hybridization to evaluate viral antigens and RNA. Viral receptors were identified with IHC. In all seven cases, the CNS showed stereotypical distribution of inflammation and neuronal localization of viral antigens and RNA, confirming the clinical diagnosis of EV71 encephalomyelitis. In six cases in which tonsillar tissues were available, viral antigens and/or RNA were localized to squamous epithelium lining the tonsillar crypts. Tissues from the gastrointestinal tract, pancreas, mesenteric nodes, spleen, and skin were all negative for viral antigens/RNA. Our novel findings strongly suggest that tonsillar crypt squamous epithelium supports active viral replication and represents an important source of viral shedding that facilitates person-to-person transmission by both the fecal-oral or oral-oral routes. It may also be a portal for viral entry. A correlation between viral infection and SCARB2 expression appears to be more significant than for PSGL-1 expression.

  4. Impact of HLA-B*81-associated mutations in HIV-1 Gag on viral replication capacity.

    PubMed

    Wright, Jaclyn K; Naidoo, Vanessa L; Brumme, Zabrina L; Prince, Jessica L; Claiborne, Daniel T; Goulder, Philip J R; Brockman, Mark A; Hunter, Eric; Ndung'u, Thumbi

    2012-03-01

    HIV-1 attenuation resulting from immune escape mutations selected in Gag may contribute to slower disease progression in HIV-1-infected individuals expressing certain HLA class I alleles. We previously showed that the protective allele HLA-B*81 and the HLA-B*81-selected Gag T186S mutation are strongly associated with a lower viral replication capacity of recombinant viruses encoding Gag-protease derived from individuals chronically infected with HIV-1 subtype C. In the present study, we directly tested the effect of this mutation on viral replication capacity. In addition, we investigated potential compensatory effects of various polymorphisms, including other HLA-B*81-associated mutations that significantly covary with the T186S mutation. Mutations were introduced into a reference subtype B backbone and into patient-derived subtype C sequences in subtype B and C backbones by site-directed mutagenesis. The exponential-phase growth of mutant and wild-type viruses was assayed by flow cytometry of a green fluorescent protein reporter T cell line or by measurement of HIV-1 reverse transcriptase activity in culture supernatants. Engineering of the T186S mutation alone into all patient-derived subtype C sequences failed to yield replication-competent viruses, while in the subtype B sequence, the T186S mutation resulted in impaired replication capacity. Only the T186S mutation in combination with the T190I mutation yielded replication-competent viruses for all virus backbones tested; however, these constructs replicated slower than the wild type, suggesting that only partial compensation is mediated by the T190I mutation. Constructs encoding the T186S mutation in combination with other putative compensatory mutations were attenuated or defective. These results suggest that the T186S mutation is deleterious to HIV-1 subtype C replication and likely requires complex compensatory pathways, which may contribute to the clinical benefit associated with HLA-B*81. PMID:22238317

  5. Detecting the ability of viral, bacterial and eukaryotic replication proteins to track along DNA.

    PubMed

    Tinker, R L; Kassavetis, G A; Geiduschek, E P

    1994-11-15

    The phage T4 gene 45 protein (gp45), Escherichia coli beta and the eukaryotic proliferating cell nuclear antigen (PCNA) function in replication as processivity factors of their corresponding DNA polymerases. The T4 gp45 also functions as the transcriptional activator that connects expression of viral late genes to DNA replication. DNA tracking is an essential component of the replication and transcription regulatory functions of T4 gp45. The ability of gp45, beta and PCNA to track along DNA has been analyzed by photocrosslinking. Each of these proteins must be loaded onto DNA by a species-specific assembly factor. For gp45 and beta, the density of traffic along DNA is determined by a dynamic balance between continuous protein loading and unloading, and is also dependent on interaction with the conjugate single-stranded DNA binding protein.

  6. TRIM52 inhibits Japanese Encephalitis Virus replication by degrading the viral NS2A

    PubMed Central

    Fan, Wenchun; Wu, Mengge; Qian, Suhong; Zhou, Yun; Chen, Huanchun; Li, Xiangmin; Qian, Ping

    2016-01-01

    The members of tripartite-motif containing (TRIM) protein participate in various cellular processes and play an important role in host antiviral function. TRIM proteins exert their antiviral activity either directly by degrading viral proteins through their E3 ligase activity, or indirectly by promoting host innate immunity. This study demonstrated for the first time that TRIM52 is a novel antiviral TRIM protein against Japanese encephalitis virus (JEV) infection. Overexpression of TRIM52 restricted JEV replication in BHK-21 and 293T cells. In addition, JEV nonstructural protein 2A (NS2A) is a protein that interacts with TRIM52. Their interaction degraded NS2A in a proteasome-dependent manner via the E3 ligase activity of TRIM52. Thus, TRIM52 is a novel antiviral TRIM protein, and it exerted antiviral activity against JEV infection by targeting and degrading viral NS2A. PMID:27667714

  7. Nbs1-dependent binding of Mre11 to adenovirus E4 mutant viral DNA is important for inhibiting DNA replication

    SciTech Connect

    Mathew, Shomita S.; Bridge, Eileen

    2008-04-25

    Adenovirus (Ad) infections stimulate the activation of cellular DNA damage response and repair pathways. Ad early regulatory proteins prevent activation of DNA damage responses by targeting the MRN complex, composed of the Mre11, Rad50 and Nbs1 proteins, for relocalization and degradation. In the absence of these viral proteins, Mre11 colocalizes with viral DNA replication foci. Mre11 foci formation at DNA damage induced by ionizing radiation depends on the Nbs1 component of the MRN complex and is stabilized by the mediator of DNA damage checkpoint protein 1 (Mdc1). We find that Nbs1 is required for Mre11 localization at DNA replication foci in Ad E4 mutant infections. Mre11 is important for Mdc1 foci formation in infected cells, consistent with its role as a sensor of DNA damage. Chromatin immunoprecipitation assays indicate that both Mre11 and Mdc1 are physically bound to viral DNA, which could account for their localization in viral DNA containing foci. Efficient binding of Mre11 to E4 mutant DNA depends on the presence of Nbs1, and is correlated with a significant E4 mutant DNA replication defect. Our results are consistent with a model in which physical interaction of Mre11 with viral DNA is mediated by Nbs1, and interferes with viral DNA replication.

  8. Nucleolin interacts with influenza A nucleoprotein and contributes to viral ribonucleoprotein complexes nuclear trafficking and efficient influenza viral replication.

    PubMed

    Terrier, Olivier; Carron, Coralie; De Chassey, Benoît; Dubois, Julia; Traversier, Aurélien; Julien, Thomas; Cartet, Gaëlle; Proust, Anaïs; Hacot, Sabine; Ressnikoff, Denis; Lotteau, Vincent; Lina, Bruno; Diaz, Jean-Jacques; Moules, Vincent; Rosa-Calatrava, Manuel

    2016-01-01

    Influenza viruses replicate their single-stranded RNA genomes in the nucleus of infected cells and these replicated genomes (vRNPs) are then exported from the nucleus to the cytoplasm and plasma membrane before budding. To achieve this export, influenza viruses hijack the host cell export machinery. However, the complete mechanisms underlying this hijacking remain not fully understood. We have previously shown that influenza viruses induce a marked alteration of the nucleus during the time-course of infection and notably in the nucleolar compartment. In this study, we discovered that a major nucleolar component, called nucleolin, is required for an efficient export of vRNPs and viral replication. We have notably shown that nucleolin interacts with the viral nucleoprotein (NP) that mainly constitutes vRNPs. Our results suggest that this interaction could allow vRNPs to "catch" the host cell export machinery, a necessary step for viral replication. PMID:27373907

  9. Nucleolin interacts with influenza A nucleoprotein and contributes to viral ribonucleoprotein complexes nuclear trafficking and efficient influenza viral replication

    PubMed Central

    Terrier, Olivier; Carron, Coralie; De Chassey, Benoît; Dubois, Julia; Traversier, Aurélien; Julien, Thomas; Cartet, Gaëlle; Proust, Anaïs; Hacot, Sabine; Ressnikoff, Denis; Lotteau, Vincent; Lina, Bruno; Diaz, Jean-Jacques; Moules, Vincent; Rosa-Calatrava, Manuel

    2016-01-01

    Influenza viruses replicate their single-stranded RNA genomes in the nucleus of infected cells and these replicated genomes (vRNPs) are then exported from the nucleus to the cytoplasm and plasma membrane before budding. To achieve this export, influenza viruses hijack the host cell export machinery. However, the complete mechanisms underlying this hijacking remain not fully understood. We have previously shown that influenza viruses induce a marked alteration of the nucleus during the time-course of infection and notably in the nucleolar compartment. In this study, we discovered that a major nucleolar component, called nucleolin, is required for an efficient export of vRNPs and viral replication. We have notably shown that nucleolin interacts with the viral nucleoprotein (NP) that mainly constitutes vRNPs. Our results suggest that this interaction could allow vRNPs to “catch” the host cell export machinery, a necessary step for viral replication. PMID:27373907

  10. Engineered retargeting of viral RNA replication complexes to an alternative intracellular membrane.

    PubMed

    Miller, David J; Schwartz, Michael D; Dye, Billy T; Ahlquist, Paul

    2003-11-01

    Positive-strand RNA virus replication complexes are universally associated with intracellular membranes, although different viruses use membranes derived from diverse and sometimes multiple organelles. We investigated whether unique intracellular membranes are required for viral RNA replication complex formation and function in yeast by retargeting protein A, the Flock House virus (FHV) RNA-dependent RNA polymerase. Protein A, the only viral protein required for FHV RNA replication, targets and anchors replication complexes to outer mitochondrial membranes in part via an N-proximal sequence that contains a transmembrane domain. We replaced the FHV protein A mitochondrial outer membrane-targeting sequence with the N-terminal endoplasmic reticulum (ER)-targeting sequence from the yeast NADP cytochrome P450 oxidoreductase or inverted C-terminal ER-targeting sequences from the hepatitis C virus NS5B polymerase or the yeast t-SNARE Ufe1p. Confocal immunofluorescence microscopy confirmed that protein A chimeras retargeted to the ER. FHV subgenomic and genomic RNA accumulation in yeast expressing ER-targeted protein A increased 2- to 13-fold over that in yeast expressing wild-type protein A, despite similar protein A levels. Density gradient flotation assays demonstrated that ER-targeted protein A remained membrane associated, and in vitro RNA-dependent RNA polymerase assays demonstrated an eightfold increase in the in vitro RNA synthesis activity of the ER-targeted FHV RNA replication complexes. Electron microscopy showed a change in the intracellular membrane alterations from a clustered mitochondrial distribution with wild-type protein A to the formation of perinuclear layers with ER-targeted protein A. We conclude that specific intracellular membranes are not required for FHV RNA replication complex formation and function.

  11. Structure-Function Relationships Underlying the Replication Fidelity of Viral RNA-Dependent RNA Polymerases

    PubMed Central

    Campagnola, Grace; McDonald, Seth; Beaucourt, Stéphanie; Vignuzzi, Marco

    2014-01-01

    ABSTRACT Viral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity. IMPORTANCE Positive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating

  12. Activation of BPV-1 replication in vitro by the transcription factor E2

    NASA Astrophysics Data System (ADS)

    Yang, Liu; Li, Rong; Mohr, Ian J.; Clark, Robin; Botchan, Michael R.

    1991-10-01

    Soluble extracts from uninfected murine cells supplemented with purified viral E1 and E2 proteins support the replication of exogenously added papilloma virus DNA. The E2 transactivator stimulates the binding of the E1 replication protein to the minimal origin of replication and activates DNA replication. These results support the concept that transcription factors have a direct role in the initiation of DNA replication in eukaryotes by participating in the assembly of a complex at the origin of replication.

  13. 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.

  14. The cis-acting replication element of the Hepatitis C virus genome recruits host factors that influence viral replication and translation

    PubMed Central

    Ríos-Marco, Pablo; Romero-López, Cristina; Berzal-Herranz, Alfredo

    2016-01-01

    The cis-acting replication element (CRE) of the hepatitis C virus (HCV) RNA genome is a region of conserved sequence and structure at the 3′ end of the open reading frame. It participates in a complex and dynamic RNA-RNA interaction network involving, among others, essential functional domains of the 3′ untranslated region and the internal ribosome entry site located at the 5′ terminus of the viral genome. A proper balance between all these contacts is critical for the control of viral replication and translation, and is likely dependent on host factors. Proteomic analyses identified a collection of proteins from a hepatoma cell line as CRE-interacting candidates. A large fraction of these were RNA-binding proteins sharing highly conserved RNA recognition motifs. The vast majority of these proteins were validated by bioinformatics tools that consider RNA-protein secondary structure. Further characterization of representative proteins indicated that hnRNPA1 and HMGB1 exerted negative effects on viral replication in a subgenomic HCV replication system. Furthermore DDX5 and PARP1 knockdown reduced the HCV IRES activity, suggesting an involvement of these proteins in HCV translation. The identification of all these host factors provides new clues regarding the function of the CRE during viral cycle progression. PMID:27165399

  15. The cis-acting replication element of the Hepatitis C virus genome recruits host factors that influence viral replication and translation.

    PubMed

    Ríos-Marco, Pablo; Romero-López, Cristina; Berzal-Herranz, Alfredo

    2016-01-01

    The cis-acting replication element (CRE) of the hepatitis C virus (HCV) RNA genome is a region of conserved sequence and structure at the 3' end of the open reading frame. It participates in a complex and dynamic RNA-RNA interaction network involving, among others, essential functional domains of the 3' untranslated region and the internal ribosome entry site located at the 5' terminus of the viral genome. A proper balance between all these contacts is critical for the control of viral replication and translation, and is likely dependent on host factors. Proteomic analyses identified a collection of proteins from a hepatoma cell line as CRE-interacting candidates. A large fraction of these were RNA-binding proteins sharing highly conserved RNA recognition motifs. The vast majority of these proteins were validated by bioinformatics tools that consider RNA-protein secondary structure. Further characterization of representative proteins indicated that hnRNPA1 and HMGB1 exerted negative effects on viral replication in a subgenomic HCV replication system. Furthermore DDX5 and PARP1 knockdown reduced the HCV IRES activity, suggesting an involvement of these proteins in HCV translation. The identification of all these host factors provides new clues regarding the function of the CRE during viral cycle progression. PMID:27165399

  16. Autographa californica Multiple Nucleopolyhedrovirus DNA Polymerase C Terminus Is Required for Nuclear Localization and Viral DNA Replication

    PubMed Central

    Feng, Guozhong

    2014-01-01

    insufficient for viral DNA synthesis and virus replication. Rather, we identified three features, including two nuclear localization signals and a highly conserved 10-amino-acid sequence in the AcMNPV DNApol C terminus, all three of which are important for both nuclear localization of DNApol and for DNApol activity, as measured by viral DNA synthesis and virus replication. PMID:25008932

  17. Cyclophilin E Functions as a Negative Regulator to Influenza Virus Replication by Impairing the Formation of the Viral Ribonucleoprotein Complex

    PubMed Central

    Wang, Zengfu; Liu, Xiaoling; Zhao, Zhendong; Xu, Chongfeng; Zhang, Ke; Chen, Caiwei; Sun, Lei; Gao, George F.; Ye, Xin; Liu, Wenjun

    2011-01-01

    Background The nucleoprotein (NP) of influenza A virus is a multifunctional protein that plays a critical role in the replication and transcription of the viral genome. Therefore, examining host factors that interact with NP may shed light on the mechanism of host restriction barriers and the tissue tropism of influenza A virus. Here, Cyclophilin E (CypE), a member of the peptidyl-propyl cis-trans isomerase (PPIase) family, was found to bind to NP and inhibit viral replication and transcription. Methodology/Principal Findings In the present study, CypE was found to interact with NP but not with the other components of the viral ribonucleoprotein complex (vRNP): PB1, PB2, and PA. Mutagenesis data revealed that the CypE domain comprised of residues 137–186 is responsible for its binding to NP. Functional analysis results indicated that CypE is a negative regulator in the influenza virus life cycle. Furthermore, knock-down of CypE resulted in increased levels of three types of viral RNA, suggesting that CypE negatively affects viral replication and transcription. Moreover, up-regulation of CypE inhibited the activity of influenza viral polymerase. We determined that the molecular mechanism by which CypE negatively regulates influenza virus replication and transcription is by interfering with NP self-association and the NP-PB1 and NP-PB2 interactions. Conclusions/Significance CypE is a host restriction factor that inhibits the functions of NP, as well as viral replication and transcription, by impairing the formation of the vRNP. The data presented here will help us to better understand the molecular mechanisms of host restriction barriers, host adaptation, and tissue tropism of influenza A virus. PMID:21887220

  18. A Dimeric Rep Protein Initiates Replication of a Linear Archaeal Virus Genome: Implications for the Rep Mechanism and Viral Replication ▿ †

    PubMed Central

    Oke, Muse; Kerou, Melina; Liu, Huanting; Peng, Xu; Garrett, Roger A.; Prangishvili, David; Naismith, James H.; White, Malcolm F.

    2011-01-01

    The Rudiviridae are a family of rod-shaped archaeal viruses with covalently closed, linear double-stranded DNA (dsDNA) genomes. Their replication mechanisms remain obscure, although parallels have been drawn to the Poxviridae and other large cytoplasmic eukaryotic viruses. Here we report that a protein encoded in the 34-kbp genome of the rudivirus SIRV1 is a member of the replication initiator (Rep) superfamily of proteins, which initiate rolling-circle replication (RCR) of diverse viruses and plasmids. We show that SIRV Rep nicks the viral hairpin terminus, forming a covalent adduct between an active-site tyrosine and the 5′ end of the DNA, releasing a 3′ DNA end as a primer for DNA synthesis. The enzyme can also catalyze the joining reaction that is necessary to reseal the DNA hairpin and terminate replication. The dimeric structure points to a simple mechanism through which two closely positioned active sites, each with a single tyrosine residue, work in tandem to catalyze DNA nicking and joining. We propose a novel mechanism for rudivirus DNA replication, incorporating the first known example of a Rep protein that is not linked to RCR. The implications for Rep protein function and viral replication are discussed. PMID:21068244

  19. KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription

    NASA Astrophysics Data System (ADS)

    Gupta, Namrata; Thakker, Suhani; Verma, Subhash C.

    2016-09-01

    The establishment of latency is an essential for lifelong persistence and pathogenesis of Kaposi’s sarcoma-associated herpesvirus (KSHV). Latency-associated nuclear antigen (LANA) is the most abundantly expressed protein during latency and is important for viral genome replication and transcription. Replication-coupled nucleosome assembly is a major step in packaging the newly synthesized DNA into chromatin, but the mechanism of KSHV genome chromatinization post-replication is not understood. Here, we show that nucleosome assembly protein 1-like protein 1 (NAP1L1) associates with LANA. Our binding assays revealed an association of LANA with NAP1L1 in KSHV-infected cells, which binds through its amino terminal domain. Association of these proteins confirmed their localization in specific nuclear compartments of the infected cells. Chromatin immunoprecipitation assays from NAP1L1-depleted cells showed LANA-mediated recruitment of NAP1L1 at the terminal repeat (TR) region of the viral genome. Presence of NAP1L1 stimulated LANA-mediated DNA replication and persistence of a TR-containing plasmid. Depletion of NAP1L1 led to a reduced nucleosome positioning on the viral genome. Furthermore, depletion of NAP1L1 increased the transcription of viral lytic genes and overexpression decreased the promoter activities of LANA-regulated genes. These results confirmed that LANA recruitment of NAP1L1 helps in assembling nucleosome for the chromatinization of newly synthesized viral DNA.

  20. KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription

    PubMed Central

    Gupta, Namrata; Thakker, Suhani; Verma, Subhash C.

    2016-01-01

    The establishment of latency is an essential for lifelong persistence and pathogenesis of Kaposi’s sarcoma-associated herpesvirus (KSHV). Latency-associated nuclear antigen (LANA) is the most abundantly expressed protein during latency and is important for viral genome replication and transcription. Replication-coupled nucleosome assembly is a major step in packaging the newly synthesized DNA into chromatin, but the mechanism of KSHV genome chromatinization post-replication is not understood. Here, we show that nucleosome assembly protein 1-like protein 1 (NAP1L1) associates with LANA. Our binding assays revealed an association of LANA with NAP1L1 in KSHV-infected cells, which binds through its amino terminal domain. Association of these proteins confirmed their localization in specific nuclear compartments of the infected cells. Chromatin immunoprecipitation assays from NAP1L1-depleted cells showed LANA-mediated recruitment of NAP1L1 at the terminal repeat (TR) region of the viral genome. Presence of NAP1L1 stimulated LANA-mediated DNA replication and persistence of a TR-containing plasmid. Depletion of NAP1L1 led to a reduced nucleosome positioning on the viral genome. Furthermore, depletion of NAP1L1 increased the transcription of viral lytic genes and overexpression decreased the promoter activities of LANA-regulated genes. These results confirmed that LANA recruitment of NAP1L1 helps in assembling nucleosome for the chromatinization of newly synthesized viral DNA. PMID:27599637

  1. KSHV encoded LANA recruits Nucleosome Assembly Protein NAP1L1 for regulating viral DNA replication and transcription.

    PubMed

    Gupta, Namrata; Thakker, Suhani; Verma, Subhash C

    2016-01-01

    The establishment of latency is an essential for lifelong persistence and pathogenesis of Kaposi's sarcoma-associated herpesvirus (KSHV). Latency-associated nuclear antigen (LANA) is the most abundantly expressed protein during latency and is important for viral genome replication and transcription. Replication-coupled nucleosome assembly is a major step in packaging the newly synthesized DNA into chromatin, but the mechanism of KSHV genome chromatinization post-replication is not understood. Here, we show that nucleosome assembly protein 1-like protein 1 (NAP1L1) associates with LANA. Our binding assays revealed an association of LANA with NAP1L1 in KSHV-infected cells, which binds through its amino terminal domain. Association of these proteins confirmed their localization in specific nuclear compartments of the infected cells. Chromatin immunoprecipitation assays from NAP1L1-depleted cells showed LANA-mediated recruitment of NAP1L1 at the terminal repeat (TR) region of the viral genome. Presence of NAP1L1 stimulated LANA-mediated DNA replication and persistence of a TR-containing plasmid. Depletion of NAP1L1 led to a reduced nucleosome positioning on the viral genome. Furthermore, depletion of NAP1L1 increased the transcription of viral lytic genes and overexpression decreased the promoter activities of LANA-regulated genes. These results confirmed that LANA recruitment of NAP1L1 helps in assembling nucleosome for the chromatinization of newly synthesized viral DNA. PMID:27599637

  2. Mechanisms of inhibition of viral replication in plants

    SciTech Connect

    Not Available

    1990-01-01

    We have made a number of interesting observations of importance to the fields of virology and plant molecular biology. Topics include the genome of cucumber mosaic virus (CMV), recombination of the CMV genome, transgenic plants and viral movement genes, mapping resistance breakage sequences in the tomato mosaic virus (TMV) genome, and mapping pathogeneticity domains and viral RNA heterogeneity. 1 fig., 1 tab.

  3. Positive-strand RNA viruses stimulate host phosphatidylcholine synthesis at viral replication sites

    PubMed Central

    Zhang, Jiantao; Zhang, Zhenlu; Chukkapalli, Vineela; Nchoutmboube, Jules A.; Li, Jianhui; Randall, Glenn; Belov, George A.; Wang, Xiaofeng

    2016-01-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their viral replication complexes (VRCs); however, how these viruses modulate host lipid metabolism to accommodate such membrane proliferation and rearrangements is not well defined. We show that a significantly increased phosphatidylcholine (PC) content is associated with brome mosaic virus (BMV) replication in both natural host barley and alternate host yeast based on a lipidomic analysis. Enhanced PC levels are primarily associated with the perinuclear ER membrane, where BMV replication takes place. More specifically, BMV replication protein 1a interacts with and recruits Cho2p (choline requiring 2), a host enzyme involved in PC synthesis, to the site of viral replication. These results suggest that PC synthesized at the site of VRC assembly, not the transport of existing PC, is responsible for the enhanced accumulation. Blocking PC synthesis by deleting the CHO2 gene resulted in VRCs with wider diameters than those in wild-type cells; however, BMV replication was significantly inhibited, highlighting the critical role of PC in VRC formation and viral replication. We further show that enhanced PC levels also accumulate at the replication sites of hepatitis C virus and poliovirus, revealing a conserved feature among a group of positive-strand RNA viruses. Our work also highlights a potential broad-spectrum antiviral strategy that would disrupt PC synthesis at the sites of viral replication but would not alter cellular processes. PMID:26858414

  4. Viral and Cellular Genomes Activate Distinct DNA Damage Responses

    PubMed Central

    Shah, Govind A.; O’Shea, Clodagh C.

    2015-01-01

    Summary In response to cellular genome breaks, MRE11/RAD50/NBS1 (MRN) activates a global ATM DNA damage response (DDR) that prevents cellular replication. Here we show that MRN-ATM also has critical functions in defending the cell against DNA viruses. We reveal temporally distinct responses to adenovirus genomes: a critical MRN-ATM DDR that must be inactivated by E1B-55K/E4-ORF3 viral oncoproteins and a global MRN independent ATM DDR to viral nuclear domains that does not impact viral replication. We show that MRN binds to adenovirus genomes and activates a localized ATM response that specifically prevents viral DNA replication. In contrast to chromosomal breaks, ATM activation is not amplified by H2AX across megabases of chromatin to induce global signaling and replicative arrest. Thus, γH2AX foci discriminate ‘self’ and ‘non-self’ genomes and determine if a localized anti-viral or global ATM response is appropriate. This provides an elegant mechanism to neutralize viral genomes without jeopardizing cellular viability. PMID:26317467

  5. HTLV-1 Rex Tunes the Cellular Environment Favorable for Viral Replication.

    PubMed

    Nakano, Kazumi; Watanabe, Toshiki

    2016-02-24

    Human T-cell leukemia virus type-1 (HTLV-1) Rex is a viral RNA binding protein. The most important and well-known function of Rex is stabilizing and exporting viral mRNAs from the nucleus, particularly for unspliced/partially-spliced mRNAs encoding the structural proteins essential for viral replication. Without Rex, these unspliced viral mRNAs would otherwise be completely spliced. Therefore, Rex is vital for the translation of structural proteins and the stabilization of viral genomic RNA and, thus, for viral replication. Rex schedules the period of extensive viral replication and suppression to enter latency. Although the importance of Rex in the viral life-cycle is well understood, the underlying molecular mechanism of how Rex achieves its function has not been clarified. For example, how does Rex protect unspliced/partially-spliced viral mRNAs from the host cellular splicing machinery? How does Rex protect viral mRNAs, antigenic to eukaryotic cells, from cellular mRNA surveillance mechanisms? Here we will discuss these mechanisms, which explain the function of Rex as an organizer of HTLV-1 expression based on previously and recently discovered aspects of Rex. We also focus on the potential influence of Rex on the homeostasis of the infected cell and how it can exert its function.

  6. HTLV-1 Rex Tunes the Cellular Environment Favorable for Viral Replication.

    PubMed

    Nakano, Kazumi; Watanabe, Toshiki

    2016-03-01

    Human T-cell leukemia virus type-1 (HTLV-1) Rex is a viral RNA binding protein. The most important and well-known function of Rex is stabilizing and exporting viral mRNAs from the nucleus, particularly for unspliced/partially-spliced mRNAs encoding the structural proteins essential for viral replication. Without Rex, these unspliced viral mRNAs would otherwise be completely spliced. Therefore, Rex is vital for the translation of structural proteins and the stabilization of viral genomic RNA and, thus, for viral replication. Rex schedules the period of extensive viral replication and suppression to enter latency. Although the importance of Rex in the viral life-cycle is well understood, the underlying molecular mechanism of how Rex achieves its function has not been clarified. For example, how does Rex protect unspliced/partially-spliced viral mRNAs from the host cellular splicing machinery? How does Rex protect viral mRNAs, antigenic to eukaryotic cells, from cellular mRNA surveillance mechanisms? Here we will discuss these mechanisms, which explain the function of Rex as an organizer of HTLV-1 expression based on previously and recently discovered aspects of Rex. We also focus on the potential influence of Rex on the homeostasis of the infected cell and how it can exert its function. PMID:26927155

  7. The TPR domain in the host Cyp40-like cyclophilin binds to the viral replication protein and inhibits the assembly of the tombusviral replicase.

    PubMed

    Lin, Jing-Yi; Mendu, Venugopal; Pogany, Judit; Qin, Jun; Nagy, Peter D

    2012-02-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-coded proteins acting either as susceptibility or resistance factors. Previous genome-wide screens and global proteomics approaches with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host revealed the involvement of cyclophilins, which are a large family of host prolyl isomerases, in TBSV replication. In this paper, we identified those members of the large cyclophilin family that interacted with the viral replication proteins and inhibited TBSV replication. Further characterization of the most effective cyclophilin, the Cyp40-like Cpr7p, revealed that it strongly inhibits many steps during TBSV replication in a cell-free replication assay. These steps include viral RNA recruitment inhibited via binding of Cpr7p to the RNA-binding region of the viral replication protein; the assembly of the viral replicase complex and viral RNA synthesis. Since the TPR (tetratricopeptide repeats) domain, but not the catalytic domain of Cpr7p is needed for the inhibitory effect on TBSV replication, it seems that the chaperone activity of Cpr7p provides the negative regulatory function. We also show that three Cyp40-like proteins from plants can inhibit TBSV replication in vitro and Cpr7p is also effective against Nodamura virus, an insect pathogen. Overall, the current work revealed a role for Cyp40-like proteins and their TPR domains as regulators of RNA virus replication.

  8. Inhibition of RNase H activity and viral replication by single mutations in the 3' region of Moloney murine leukemia virus reverse transcriptase.

    PubMed Central

    Repaske, R; Hartley, J W; Kavlick, M F; O'Neill, R R; Austin, J B

    1989-01-01

    Selected conserved amino acids in the putative RNase H domain of reverse transcriptase (RT) were modified in a molecularly cloned infectious provirus and in a Moloney murine leukemia virus RT expression vector by site-directed mutagenesis. Substitution of either of two conserved aspartic acid residues in proviral DNA prevented production of infectious particles in transfected NIH 3T3 cells, and the same modifications depressed RT-associated RNase H activity by more than 25-fold with little or no effect on polymerase activity. PMID:2464706

  9. Transcriptional activation of homologous and heterologous genes by the hepatitis B virus X gene product in cells permissive for viral replication.

    PubMed Central

    Colgrove, R; Simon, G; Ganem, D

    1989-01-01

    The potential of the hepadnavirus X gene product to activate gene expression in trans was tested through a series of cotransfections of X expression vectors with a variety of potential targets for transactivation. The X gene products from human hepatitis B virus (HBV), woodchuck hepatitis virus, and ground squirrel hepatitis virus are all equally active in augmenting the expression of a wide array of target promoters in both permissive and nonpermissive cells. Using the HBV genome itself as the source of X protein, we demonstrate that transactivation of HBV and heterologous genes occurs when X protein is expressed in its native state during productive infection of permissive cells. Run-on transcription analysis indicates that this transactivation occurs at the level of primary transcription. Images PMID:2788226

  10. Viral and Cellular Components of AAV2 Replication Compartments

    PubMed Central

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

    2013-01-01

    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

  11. Viral and Cellular Components of AAV2 Replication Compartments.

    PubMed

    Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

    2013-01-01

    Adeno-associated virus 2 (AAV2) is a helpervirus-dependent parvovirus with a bi-phasic life cycle comprising latency in absence and lytic replication in presence of a helpervirus, such as adenovirus (Ad) or herpes simplex virus type 1 (HSV-1). Helpervirus-supported AAV2 replication takes place in replication compartments (RCs) in the cell nucleus where virus DNA replication and transcription occur. RCs consist of a defined set of helper virus-, AAV2-, and cellular proteins. Here we compare the profile of cellular proteins recruited into AAV2 RCs or identified in Rep78-associated complexes when either Ad or HSV-1 is the helpervirus, and we discuss the potential roles of some of these proteins in AAV2 and helpervirus infection. PMID:24222808

  12. Deformed wing virus: replication and viral load in mites (Varroa destructor).

    PubMed

    Gisder, Sebastian; Aumeier, Pia; Genersch, Elke

    2009-02-01

    Deformed wing virus (DWV) normally causes covert infections but can have devastating effects on bees by inducing morphological deformity or even death when transmitted by the ectoparasitic mite Varroa destructor. In order to determine the role of V. destructor in the development of crippled wings, we analysed individual mites for the presence and replication of DWV. The results supported the correlation between viral replication in mites and morphologically deformed bees. Quantification of viral genome equivalents revealed that mites capable of inducing an overt DWV infection contained 10(10)-10(12) genome equivalents per mite. In contrast, mites which could not induce crippled wings contained a maximum of only 10(8) viral genome equivalents per mite. We conclude that the development of crippled wings not only depends on DWV transmission by V. destructor but also on viral replication in V. destructor and on the DWV titre in the parasitizing mites.

  13. Annexin V Incorporated into Influenza Virus Particles Inhibits Gamma Interferon Signaling and Promotes Viral Replication

    PubMed Central

    Berri, Fatma; Haffar, Ghina; Lê, Vuong Ba; Sadewasser, Anne; Paki, Katharina; Lina, Bruno; Wolff, Thorsten

    2014-01-01

    ABSTRACT During the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-γ)-induced Stat phosphorylation and IFN-γ-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-γ signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-γ antiviral immune response by incorporating A5 into their envelope during the budding process. IMPORTANCE Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes. PMID

  14. Interaction of NCOR/SMRT Repressor Complexes with Papillomavirus E8^E2C Proteins Inhibits Viral Replication.

    PubMed

    Dreer, Marcel; Fertey, Jasmin; van de Poel, Saskia; Straub, Elke; Madlung, Johannes; Macek, Boris; Iftner, Thomas; Stubenrauch, Frank

    2016-04-01

    Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins

  15. Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways.

    PubMed

    Rainey, Stephanie M; Martinez, Julien; McFarlane, Melanie; Juneja, Punita; Sarkies, Peter; Lulla, Aleksei; Schnettler, Esther; Varjak, Margus; Merits, Andres; Miska, Eric A; Jiggins, Francis M; Kohl, Alain

    2016-04-01

    The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3' open reading frame than the 5' non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia's antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced

  16. Wolbachia Blocks Viral Genome Replication Early in Infection without a Transcriptional Response by the Endosymbiont or Host Small RNA Pathways

    PubMed Central

    McFarlane, Melanie; Juneja, Punita; Sarkies, Peter; Lulla, Aleksei; Schnettler, Esther; Varjak, Margus; Merits, Andres; Miska, Eric A.; Jiggins, Francis M.; Kohl, Alain

    2016-01-01

    The intracellular endosymbiotic bacterium Wolbachia can protect insects against viral infection, and is being introduced into mosquito populations in the wild to block the transmission of arboviruses that infect humans and are a major public health concern. To investigate the mechanisms underlying this antiviral protection, we have developed a new model system combining Wolbachia-infected Drosophila melanogaster cell culture with the model mosquito-borne Semliki Forest virus (SFV; Togaviridae, Alphavirus). Wolbachia provides strong antiviral protection rapidly after infection, suggesting that an early stage post-infection is being blocked. Wolbachia does appear to have major effects on events distinct from entry, assembly or exit as it inhibits the replication of an SFV replicon transfected into the cells. Furthermore, it causes a far greater reduction in the expression of proteins from the 3´ open reading frame than the 5´ non-structural protein open reading frame, indicating that it is blocking the replication of viral RNA. Further to this separation of the replicase proteins and viral RNA in transreplication assays shows that uncoupling of viral RNA and replicase proteins does not overcome Wolbachia’s antiviral activity. This further suggests that replicative processes are disrupted, such as translation or replication, by Wolbachia infection. This may occur by Wolbachia mounting an active antiviral response, but the virus did not cause any transcriptional response by the bacterium, suggesting that this is not the case. Host microRNAs (miRNAs) have been implicated in protection, but again we found that host cell miRNA expression was unaffected by the bacterium and neither do our findings suggest any involvement of the antiviral siRNA pathway. We conclude that Wolbachia may directly interfere with early events in virus replication such as translation of incoming viral RNA or RNA transcription, and this likely involves an intrinsic (as opposed to an induced

  17. Polyoma Viral DNA Replicated as a Nucleoprotein Complex in Close Association with the Host Cell Chromatin

    PubMed Central

    Seebeck, Thomas; Weil, Roger

    1974-01-01

    Polyoma viral DNA is shown to be replicated in close association with the mouse cell chromatin. Two virus-specific nucleoprotein complexes, designated complex A and B, can be dissociated from the isolated chromatin by gentle homogenization in 0.5 M NaCl. Complex A contains only replicating polyoma (Py) DNA whereas complex B contains only mature Py DNA I. The results show, furthermore, that complex A, containing viral DNA in different stages of replication, and complex B are both nucleoproteins with the same buoyant density. The data presently available suggest that newly synthesized stretches of Py DNA are immediately complexed with mouse cell histones and that complex B becomes the “core” of progeny Py virions. These results suggested that Py-induced replication of the mouse cell chromatin may be necessary to provide replicating Py DNA with histones. PMID:4362862

  18. 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.

  19. Hepatitis C Virus Translation Preferentially Depends on Active RNA Replication

    PubMed Central

    Liu, Helene Minyi; Aizaki, Hideki; Machida, Keigo; Ou, J.-H. James; Lai, Michael M. C.

    2012-01-01

    Hepatitis C virus (HCV) RNA initiates its replication on a detergent-resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication-defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome. PMID:22937067

  20. Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells.

    PubMed

    Saji, Chiaki; Higashi, Chizuka; Niinaka, Yasufumi; Yamada, Koji; Noguchi, Kohji; Fujimuro, Masahiro

    2011-12-01

    Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-κB inhibitory molecule (IκBα) and suppressed the transcriptional activity of NF-κB in PEL cells. The NF-κB specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-κB signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-κB signaling is upregulated by proteasome-dependent degradation of IκBα. The suppression of NF-κB signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome inhibitors may represent a novel strategy for the treatment of KSHV infection and KSHV-associated lymphomas.

  1. Oncolytic Herpes simplex virus expressing yeast cytosine deaminase: relationship between viral replication, transgene expression, prodrug bioactivation

    PubMed Central

    Yamada, Suguru; Kuroda, Toshihiko; Fuchs, Bryan C.; He, Xiaoying; Supko, Jeffrey G.; Schmitt, Anthony; McGinn, Christopher M.; Lanuti, Michael; Tanabe, Kenneth K.

    2011-01-01

    Yeast cytosine deaminase (yCD) is a well-characterized prodrug/enzyme system that converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), and has been combined with oncolytic viruses. However, in vivo studies of the interactions between 5-FC bioactivation and viral replication have not been previously reported, nor have the kinetics of transgene expression and the pharmacokinetics of 5-FC and 5-FU. We constructed a replication-conditional HSV-1 expressing yCD and examined cytotoxicity when 5-FC was initiated at different times after viral infection, and observed that earlier 5-FC administration led to greater cytotoxicity than later 5-FC administration in vitro and in vivo. Twelve days of 5-FC administration was superior to 6 days in animal models, but dosing beyond 12 days did not further enhance efficacy. Consistent with the dosing schedule results, both viral genomic DNA copy number and viral titers were observed to peak on Day 3 after viral injection and gradually decrease thereafter. The virus is replication-conditional and was detected in tumors for as long as 2 weeks after viral injection. The maximum relative extent of yCD conversion of 5-FC to 5-FU in tumors was observed on Day 6 after viral injection and it decreased progressively thereafter. The observation that 5-FU generation within tumors did not lead to appreciable levels of systemic 5-FU (<10 ng/ml) is important and has not been previously reported. The approaches used in these studies of the relationship between the viral replication kinetics, transgene expression, prodrug administration and anti-tumor efficacy are useful in the design of clinical trials of armed, oncolytic viruses. PMID:22076044

  2. (Mechanisms of inhibition of viral replication in plants)

    SciTech Connect

    Not Available

    1991-01-01

    During the last year we have made a number of important observations in the fields of virology and plant molecular biology. By directly sequencing Tomato Mosaic Virus (ToMV) movement genes, previously undetected sequence alterations common to specific viral strains were found. The difficulty in regenerating transgenic tomato plants containing the Tm-2 gene was overcome. Tobacco plants transformed with Cucumber Mosaic Virus (CMV) are being characterized. Analysis of transgenic tobacco plants expressing CMV coat protein have shown no correlation between coat protein expression and level of resistance. Specific amino acid changes have been found to correlate with CMV resistance breaking and degree of pathogenicity. Satellite RNAs are shown to be too unstable for use as a biological control agent. The aphid transmission domain CMV has been localized to one (or more) of three amino acids; constructs have been made to determine the exact amino acids involved. 15 refs.

  3. Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model

    PubMed Central

    2014-01-01

    Background We previously reported that Enterovirus 71 (EV71) infection activates autophagy, which promotes viral replication both in vitro and in vivo. In the present study we further investigated whether EV71 infection of neuronal SK-N-SH cells induces an autophagic flux. Furthermore, the effects of autophagy on EV71-related pathogenesis and viral load were evaluated after intracranial inoculation of mouse-adapted EV71 (MP4 strain) into 6-day-old ICR suckling mice. Results We demonstrated that in EV71-infected SK-N-SH cells, EV71 structural protein VP1 and nonstructural protein 2C co-localized with LC3 and mannose-6-phosphate receptor (MPR, endosome marker) proteins by immunofluorescence staining, indicating amphisome formation. Together with amphisome formation, EV71 induced an autophagic flux, which could be blocked by NH4Cl (inhibitor of acidification) and vinblastine (inhibitor of fusion), as demonstrated by Western blotting. Suckling mice intracranially inoculated with EV71 showed EV71 VP1 protein expression (representing EV71 infection) in the cerebellum, medulla, and pons by immunohistochemical staining. Accompanied with these infected brain tissues, increased expression of LC3-II protein as well as formation of LC3 aggregates, autophagosomes and amphisomes were detected. Amphisome formation, which was confirmed by colocalization of EV71-VP1 protein or LC3 puncta and the endosome marker protein MPR. Thus, EV71-infected suckling mice (similar to EV71-infected SK-N-SH cells) also show an autophagic flux. The physiopathological parameters of EV71-MP4 infected mice, including body weight loss, disease symptoms, and mortality were increased compared to those of the uninfected mice. We further blocked EV71-induced autophagy with the inhibitor 3-methyladenine (3-MA), which attenuated the disease symptoms and decreased the viral load in the brain tissues of the infected mice. Conclusions In this study, we reveal that EV71 infection of suckling mice induces an

  4. 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

  5. Hepatitis B Virus Stimulated Fibronectin Facilitates Viral Maintenance and Replication through Two Distinct Mechanisms.

    PubMed

    Ren, Sheng; Wang, Jun; Chen, Tie-Long; Li, Hao-Yu; Wan, Yu-Shun; Peng, Nan-Fang; Gui, Xi-En; Zhu, Ying

    2016-01-01

    Fibronectin (FN) is a high molecular weight extracellular matrix protein that functions in cell adhesion, growth, migration, and embryonic development. However, little is known about the role of FN during viral infection. In the present study, we found significantly higher levels of FN in sera, and liver tissues from hepatitis B virus (HBV) patients relative to healthy individuals. HBV expression enhanced FN mRNA and protein levels in the hepatic cell lines Huh7 and HepG2. HBV infection of susceptible HepG2-sodium taurocholate co-transporting polypeptide cells also increased FN expression. We also found that transcriptional factor specificity protein 1 was involved in the induction of FN by HBV. Knockdown of FN expression significantly inhibited HBV DNA replication and protein synthesis through activating endogenous IFN-α production. In addition, FN interacted with the transforming growth factor β-activated protein kinase 1 (TAK1) and TAK1-binding protein complex and attenuated interferon signaling by inhibiting TAK1 phosphorylation. Furthermore, the nuclear translocation of NF-κB/p65 was found to be inhibited by FN. We also observed that FN promoted HBV enhancers to support HBV expression. These results suggest novel functions of endogenous FN involved in immune evasion and maintenance of HBV replication. PMID:27023403

  6. Thyroid Hormone-dependent Epigenetic Suppression of Herpes Simplex Virus-1 Gene Expression and Viral Replication in Differentiated Neuroendocrine Cells

    PubMed Central

    Figliozzi, Robert W.; Chen, Feng; Balish, Matthew; Ajavon, Amakoe; Hsia, S. Victor

    2014-01-01

    A global HSV-1 gene repression occurs during latency in sensory neurons where most viral gene transcriptions are suppressed. The molecular mechanisms of gene silencing and how stress factors trigger the reactivation are not well understood. Thyroid hormones are known to be altered due to stress, and with its nuclear receptor impart transcriptional repression or activation depending upon the hormone level. Therefore we hypothesized that triiodothyronine (T3) treatment of infected differentiated neuron like cells would reduce the ability of HSV-1 to produce viral progeny compared to untreated infected cells. Previously we identified putative thyroid hormone receptor elements (TREs) within the promoter regions of HSV-1 thymidine kinase (TK) and other key genes. Searching for a human cell line that can model neuronal HSV-1 infection, we performed HSV-1 infection experiments on differentiated human neuroendocrine cells, LNCaP. Upon androgen deprivation these cells undergo complete differentiation and exhibit neuronal-like morphology and physiology. These cells were readily infected by our HSV-1 recombinant virus, expressing GFP and maintaining many processes iconic of dendritic morphology. Our results demonstrated that differentiated LNCaP cells produced suppressive effects on HSV-1 gene expression and replication compared to its undifferentiated counterpart and T3 treatment have further decreased the viral plaque counts compared to untreated cells. Upon washout of the T3 viral plaque counts were restored, indicating an increase of viral replication. The qRT-PCR experiments using primers for TK showed reduced expression under T3 treatment. ChIP assays using a panel of antibodies for H3 lysine 9 epigenetic marks showed increased repressive marks on the promoter regions of TK. In conclusion we have demonstrated a T3 mediated quiescent infection in differentiated LNCaP cells that has potential to mimic latent infection. In this HSV-1 infection model thyroid hormone

  7. Proteasome inhibitors induce apoptosis and reduce viral replication in primary effusion lymphoma cells

    SciTech Connect

    Saji, Chiaki; Higashi, Chizuka; Niinaka, Yasufumi; Yamada, Koji; Noguchi, Kohji; Fujimuro, Masahiro

    2011-12-02

    Highlights: Black-Right-Pointing-Pointer Constitutive NF-{kappa}B signaling is essential for the survival and growth of PEL cells. Black-Right-Pointing-Pointer NF-{kappa}B signaling is upregulated by the proteasome-dependent degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress NF-{kappa}B signaling and induce apoptosis in PEL cells through stabilization of I{kappa}B{alpha}. Black-Right-Pointing-Pointer Proteasome inhibitors suppress viral replication in PEL cells during lytic KSHV infection. -- Abstract: Primary effusion lymphoma (PEL) is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV). This study provides evidence that proteasomal activity is required for both survival of PEL cells stably harboring the KSHV genome and viral replication of KSHV. We evaluated the cytotoxic effects of proteasome inhibitors on PEL cells. The proteasome inhibitors MG132, lactacystin, and proteasome inhibitor I dramatically inhibited cell proliferation and induced apoptosis of PEL cells through the accumulation of p21 and p27. Furthermore, proteasome inhibitors induced the stabilization of NF-{kappa}B inhibitory molecule (I{kappa}B{alpha}) and suppressed the transcriptional activity of NF-{kappa}B in PEL cells. The NF-{kappa}B specific inhibitor BAY11-7082 also induced apoptosis in PEL cells. The constitutive activation of NF-{kappa}B signaling is essential for the survival and growth of B cell lymphoma cells, including PEL cells. NF-{kappa}B signaling is upregulated by proteasome-dependent degradation of I{kappa}B{alpha}. The suppression of NF-{kappa}B signaling by proteasome inhibitors may contribute to the induction of apoptosis in PEL cells. In addition, proteasome activity is required for KSHV replication in KSHV latently infected PEL cells. MG132 reduced the production of progeny virus from PEL cells at low concentrations, which do not affect PEL cell growth. These findings suggest that proteasome inhibitors

  8. 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

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

    PubMed

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

    2016-02-01

    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

  10. Identification of a new dengue virus inhibitor that targets the viral NS4B protein and restricts genomic RNA replication.

    PubMed

    van Cleef, Koen W R; Overheul, Gijs J; Thomassen, Michael C; Kaptein, Suzanne J F; Davidson, Andrew D; Jacobs, Michael; Neyts, Johan; van Kuppeveld, Frank J M; van Rij, Ronald P

    2013-08-01

    Dengue virus (DENV) is an important human arthropod-borne virus with a major impact on public health. Nevertheless, a licensed vaccine or specific treatment is still lacking. We therefore screened the NIH Clinical Collection (NCC), a library of drug-like small molecules, for inhibitors of DENV replication using a cell line that contains a stably replicating DENV serotype 2 (DENV2) subgenomic replicon. The most potent DENV inhibitor in the NCC was δ opioid receptor antagonist SDM25N. This compound showed antiviral activity against wild-type DENV2 in both Hela and BHK-21 cells, but not in the C6/36 cell line derived from the mosquito Aedes albopictus. The structurally related compound naltrindole also inhibited DENV replication, albeit less potently. Using a transient subgenomic replicon, we demonstrate that SDM25N restricts genomic RNA replication rather than translation of the viral genome. We identified a single amino acid substitution (F164L) in the NS4B protein that confers resistance to SDM25N. Remarkably, an NS4B amino acid substitution (P104L), which was previously shown to confer resistance to the DENV inhibitor NITD-618, also provided resistance to SDM25N. In conclusion, we have identified a new DENV inhibitor, SDM25N, which restricts genomic RNA replication by - directly or indirectly - targeting the viral NS4B protein. PMID:23735301

  11. Differential human immunodeficiency virus expression in CD4+ cloned lymphocytes: from viral latency to replication.

    PubMed Central

    Chapel, A; Bensussan, A; Vilmer, E; Dormont, D

    1992-01-01

    By using cloning methodology, 13 CD4+, CD8-, CD45RO+, and CD29+ clones, isolated from human immunodeficiency virus (HIV)-negative donors, have been characterized and tested regarding their susceptibility to two strains of HIV type 1 (HIV-1). Infected clones possess integrated provirus. Only six are able to replicate HIV-1, while seven may normally grow without cytopathic effect and without viral replication. These results argue that all CD4+ lymphocyte clones may be infectable but that a heterogeneity exists regarding their abilities to replicate HIV-1. Images PMID:1374814

  12. Differential human immunodeficiency virus expression in CD4+ cloned lymphocytes: from viral latency to replication.

    PubMed

    Chapel, A; Bensussan, A; Vilmer, E; Dormont, D

    1992-06-01

    By using cloning methodology, 13 CD4+, CD8-, CD45RO+, and CD29+ clones, isolated from human immunodeficiency virus (HIV)-negative donors, have been characterized and tested regarding their susceptibility to two strains of HIV type 1 (HIV-1). Infected clones possess integrated provirus. Only six are able to replicate HIV-1, while seven may normally grow without cytopathic effect and without viral replication. These results argue that all CD4+ lymphocyte clones may be infectable but that a heterogeneity exists regarding their abilities to replicate HIV-1.

  13. Amphotericin B Inhibits Enterovirus 71 Replication by Impeding Viral Entry.

    PubMed

    Xu, Fengwen; Zhao, Xiaoxiao; Hu, Siqi; Li, Jian; Yin, Lijuan; Mei, Shan; Liu, Tingting; Wang, Ying; Ren, Lili; Cen, Shan; Zhao, Zhendong; Wang, Jianwei; Jin, Qi; Liang, Chen; Ai, Bin; Guo, Fei

    2016-01-01

    Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease that leads to cardiopulmonary complications and death in young children. There is thus an urgent need to find new treatments to control EV71 infection. In this study, we report potent inhibition of EV71 by a polyene antibiotic Amphotericin B. Amphotericin B profoundly diminished the expression of EV71 RNA and viral proteins in the RD cells and the HEK293 cells. As a result, EV71 production was inhibited by Amphotericin B with an EC50 (50% effective concentration) of 1.75 μM in RD cells and 0.32 μM in 293 cells. In addition to EV71, EV68 was also strongly inhibited by Amphotericin B. Results of mechanistic studies revealed that Amphotericin B targeted the early stage of EV71 infection through impairing the attachment and internalization of EV71 by host cells. As an effective anti-fungi drug, Amphotericin B thus holds the promise of formulating a novel therapeutic to treat EV71 infection.

  14. Amphotericin B Inhibits Enterovirus 71 Replication by Impeding Viral Entry.

    PubMed

    Xu, Fengwen; Zhao, Xiaoxiao; Hu, Siqi; Li, Jian; Yin, Lijuan; Mei, Shan; Liu, Tingting; Wang, Ying; Ren, Lili; Cen, Shan; Zhao, Zhendong; Wang, Jianwei; Jin, Qi; Liang, Chen; Ai, Bin; Guo, Fei

    2016-01-01

    Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease that leads to cardiopulmonary complications and death in young children. There is thus an urgent need to find new treatments to control EV71 infection. In this study, we report potent inhibition of EV71 by a polyene antibiotic Amphotericin B. Amphotericin B profoundly diminished the expression of EV71 RNA and viral proteins in the RD cells and the HEK293 cells. As a result, EV71 production was inhibited by Amphotericin B with an EC50 (50% effective concentration) of 1.75 μM in RD cells and 0.32 μM in 293 cells. In addition to EV71, EV68 was also strongly inhibited by Amphotericin B. Results of mechanistic studies revealed that Amphotericin B targeted the early stage of EV71 infection through impairing the attachment and internalization of EV71 by host cells. As an effective anti-fungi drug, Amphotericin B thus holds the promise of formulating a novel therapeutic to treat EV71 infection. PMID:27608771

  15. Amphotericin B Inhibits Enterovirus 71 Replication by Impeding Viral Entry

    PubMed Central

    Xu, Fengwen; Zhao, Xiaoxiao; Hu, Siqi; Li, Jian; Yin, Lijuan; Mei, Shan; Liu, Tingting; Wang, Ying; Ren, Lili; Cen, Shan; Zhao, Zhendong; Wang, Jianwei; Jin, Qi; Liang, Chen; Ai, Bin; Guo, Fei

    2016-01-01

    Enterovirus 71 (EV71) infection causes hand-foot-and-mouth disease that leads to cardiopulmonary complications and death in young children. There is thus an urgent need to find new treatments to control EV71 infection. In this study, we report potent inhibition of EV71 by a polyene antibiotic Amphotericin B. Amphotericin B profoundly diminished the expression of EV71 RNA and viral proteins in the RD cells and the HEK293 cells. As a result, EV71 production was inhibited by Amphotericin B with an EC50 (50% effective concentration) of 1.75 μM in RD cells and 0.32 μM in 293 cells. In addition to EV71, EV68 was also strongly inhibited by Amphotericin B. Results of mechanistic studies revealed that Amphotericin B targeted the early stage of EV71 infection through impairing the attachment and internalization of EV71 by host cells. As an effective anti-fungi drug, Amphotericin B thus holds the promise of formulating a novel therapeutic to treat EV71 infection. PMID:27608771

  16. Antisense Oligonucleotides Targeting Influenza A Segment 8 Genomic RNA Inhibit Viral Replication

    PubMed Central

    Lenartowicz, Elzbieta; Nogales, Aitor; Kierzek, Elzbieta; Kierzek, Ryszard; Martínez-Sobrido, Luis

    2016-01-01

    Influenza A virus (IAV) affects 5%–10% of the world's population every year. Through genome changes, many IAV strains develop resistance to currently available anti-influenza therapeutics. Therefore, there is an urgent need to find new targets for therapeutics against this important human respiratory pathogen. In this study, 2′-O-methyl and locked nucleic acid antisense oligonucleotides (ASOs) were designed to target internal regions of influenza A/California/04/2009 (H1N1) genomic viral RNA segment 8 (vRNA8) based on a base-pairing model of vRNA8. Ten of 14 tested ASOs showed inhibition of viral replication in Madin-Darby canine kidney cells. The best five ASOs were 11–15 nucleotides long and showed inhibition ranging from 5- to 25-fold. In a cell viability assay they showed no cytotoxicity. The same five ASOs also showed no inhibition of influenza B/Brisbane/60/2008 (Victoria lineage), indicating that they are sequence specific for IAV. Moreover, combinations of ASOs slightly improved anti-influenza activity. These studies establish the accessibility of IAV vRNA for ASOs in regions other than the panhandle formed between the 5′ and 3′ ends. Thus, these regions can provide targets for the development of novel IAV antiviral approaches. PMID:27463680

  17. KSHV targeted therapy: an update on inhibitors of viral lytic replication.

    PubMed

    Coen, Natacha; Duraffour, Sophie; Snoeck, Robert; Andrei, Graciela

    2014-11-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Since the discovery of KSHV 20 years ago, there is still no standard treatment and the management of virus-associated malignancies remains toxic and incompletely efficacious. As the majority of tumor cells are latently infected with KSHV, currently marketed antivirals that target the virus lytic cycle have shown inconsistent results in clinic. Nevertheless, lytic replication plays a major role in disease progression and virus dissemination. Case reports and retrospective studies have pointed out the benefit of antiviral therapy in the treatment and prevention of KSHV-associated diseases. As a consequence, potent and selective antivirals are needed. This review focuses on the anti-KSHV activity, mode of action and current status of antiviral drugs targeting KSHV lytic cycle. Among these drugs, different subclasses of viral DNA polymerase inhibitors and compounds that do not target the viral DNA polymerase are being discussed. We also cover molecules that target cellular kinases, as well as the potential of new drug targets and animal models for antiviral testing.

  18. Recruitment of Arabidopsis RNA Helicase AtRH9 to the Viral Replication Complex by Viral Replicase to Promote Turnip Mosaic Virus Replication

    PubMed Central

    Li, Yinzi; Xiong, Ruyi; Bernards, Mark; Wang, Aiming

    2016-01-01

    Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly dependent on host components to fulfill their life cycle. Recent studies have suggested that DEAD-box RNA helicases play vital roles in many aspects of RNA metabolism. To explore the possible role of the RNA helicases in viral infection, we used the Turnip mosaic virus (TuMV)-Arabidopsis pathosystem. The Arabidopsis genome encodes more than 100 putative RNA helicases (AtRH). Over 41 Arabidopsis T-DNA insertion mutants carrying genetic lesions in the corresponding 26 AtRH genes were screened for their requirement in TuMV infection. TuMV infection assays revealed that virus accumulation significantly decreased in the Arabidopsis mutants of three genes, AtRH9, AtRH26, and PRH75. In the present work, AtRH9 was further characterized. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that AtRH9 interacted with the TuMV NIb protein, the viral RNA-dependent RNA polymerase. Moreover, the subcellular distribution of AtRH9 was altered in the virus-infected cells, and AtRH9 was recruited to the viral replication complex. These results suggest that Arabidopsis AtRH9 is an important component of the TuMV replication complex, possibly recruited via its interaction with NIb. PMID:27456972

  19. Recruitment of Arabidopsis RNA Helicase AtRH9 to the Viral Replication Complex by Viral Replicase to Promote Turnip Mosaic Virus Replication.

    PubMed

    Li, Yinzi; Xiong, Ruyi; Bernards, Mark; Wang, Aiming

    2016-01-01

    Positive-sense RNA viruses have a small genome with very limited coding capacity and are highly dependent on host components to fulfill their life cycle. Recent studies have suggested that DEAD-box RNA helicases play vital roles in many aspects of RNA metabolism. To explore the possible role of the RNA helicases in viral infection, we used the Turnip mosaic virus (TuMV)-Arabidopsis pathosystem. The Arabidopsis genome encodes more than 100 putative RNA helicases (AtRH). Over 41 Arabidopsis T-DNA insertion mutants carrying genetic lesions in the corresponding 26 AtRH genes were screened for their requirement in TuMV infection. TuMV infection assays revealed that virus accumulation significantly decreased in the Arabidopsis mutants of three genes, AtRH9, AtRH26, and PRH75. In the present work, AtRH9 was further characterized. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays showed that AtRH9 interacted with the TuMV NIb protein, the viral RNA-dependent RNA polymerase. Moreover, the subcellular distribution of AtRH9 was altered in the virus-infected cells, and AtRH9 was recruited to the viral replication complex. These results suggest that Arabidopsis AtRH9 is an important component of the TuMV replication complex, possibly recruited via its interaction with NIb. PMID:27456972

  20. Epstein-Barr Virus BZLF1-Mediated Downregulation of Proinflammatory Factors Is Essential for Optimal Lytic Viral Replication

    PubMed Central

    Li, Yuqing; Long, Xubing; Huang, Lu; Yang, Mengtian; Yuan, Yan; Wang, Yan; Delecluse, Henri-Jacques

    2015-01-01

    ABSTRACT Elevated secretion of inflammatory factors is associated with latent Epstein-Barr virus (EBV) infection and the pathology of EBV-associated diseases; however, knowledge of the inflammatory response and its biological significance during the lytic EBV cycle remains elusive. Here, we demonstrate that the immediate early transcriptional activator BZLF1 suppresses the proinflammatory factor tumor necrosis factor alpha (TNF-α) by binding to the promoter of TNF-α and preventing NF-κB activation. A BZLF1Δ207-210 mutant with a deletion of 4 amino acids (aa) in the protein-protein binding domain was not able to inhibit the proinflammatory factors TNF-α and gamma interferon (IFN-γ) and reduced viral DNA replication with complete transcriptional activity during EBV lytic gene expression. TNF-α depletion restored the viral replication mediated by BZLF1Δ207-210. Furthermore, a combination of TNF-α- and IFN-γ-neutralizing antibodies recovered BZLF1Δ207-210-mediated viral replication, indicating that BZLF1 attenuates the antiviral response to aid optimal lytic replication primarily through the inhibition of TNF-α and IFN-γ secretion during the lytic cycle. These results suggest that EBV BZLF1 attenuates the proinflammatory responses to facilitate viral replication. IMPORTANCE The proinflammatory response is an antiviral and anticancer strategy following the complex inflammatory phenotype. Latent Epstein-Barr virus (EBV) infection strongly correlates with an elevated secretion of inflammatory factors in a variety of severe diseases, while the inflammatory responses during the lytic EBV cycle have not been established. Here, we demonstrate that BZLF1 acts as a transcriptional suppressor of the inflammatory factors TNF-α and IFN-γ and confirm that BZLF1-facilitated escape from the TNF-α and IFN-γ response during the EBV lytic life cycle is required for optimal viral replication. This finding implies that the EBV lytic cycle employs a distinct strategy to

  1. G3BP1 restricts HIV-1 replication in macrophages and T-cells by sequestering viral RNA.

    PubMed

    Cobos Jiménez, Viviana; Martinez, Fernando O; Booiman, Thijs; van Dort, Karel A; van de Klundert, Maarten A A; Gordon, Siamon; Geijtenbeek, Teunis B H; Kootstra, Neeltje A

    2015-12-01

    HIV-1 exploits the cellular machinery for replication and therefore several interactions with cellular factors take place, some of which are yet unknown. We identified GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) as a cellular factor that restricts HIV-1, by analyzing transcriptome profiles of in vitro-cytokine-activated macrophages that are non-permissive to HIV-1 replication. Silencing of G3BP1 by RNA interference resulted in increased HIV-1 replication in primary T-cells and macrophages, but did not affect replication of other retroviruses. G3BP1 specifically interacted with HIV-1 RNA in the cytoplasm, suggesting that it sequesters viral transcripts, thus preventing translation or packaging. G3BP1 was highly expressed in resting naïve or memory T-cells from healthy donors and HIV-1 infected patients, but significantly lower in IL-2-activated T-cells. These results strongly suggest that G3BP1 captures HIV-1 RNA transcripts and thereby restricts mRNA translation, viral protein production and virus particle formation.

  2. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication

    PubMed Central

    Jiang, Zhiwu; Gu, Liming; Chen, Yanxia

    2016-01-01

    Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse cortical neurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i.) but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary cortical neurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary cortical neurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy. PMID:27525278

  3. Influenza Virus Induces Inflammatory Response in Mouse Primary Cortical Neurons with Limited Viral Replication.

    PubMed

    Wang, Gefei; Li, Rui; Jiang, Zhiwu; Gu, Liming; Chen, Yanxia; Dai, Jianping; Li, Kangsheng

    2016-01-01

    Unlike stereotypical neurotropic viruses, influenza A viruses have been detected in the brain tissues of human and animal models. To investigate the interaction between neurons and influenza A viruses, mouse cortical neurons were isolated, infected with human H1N1 influenza virus, and then examined for the production of various inflammatory molecules involved in immune response. We found that replication of the influenza virus in neurons was limited, although early viral transcription was not affected. Virus-induced neuron viability decreased at 6 h postinfection (p.i.) but increased at 24 h p.i. depending upon the viral strain. Virus-induced apoptosis and cytopathy in primary cortical neurons were not apparent at 24 h p.i. The mRNA levels of inflammatory cytokines, chemokines, and type I interferons were upregulated at 6 h and 24 h p.i. These results indicate that the influenza virus induces inflammatory response in mouse primary cortical neurons with limited viral replication. The cytokines released in viral infection-induced neuroinflammation might play critical roles in influenza encephalopathy, rather than in viral replication-induced cytopathy. PMID:27525278

  4. IL-9 Inhibits Viral Replication in Coxsackievirus B3-Induced Myocarditis

    PubMed Central

    Yu, Miao; Long, Qi; Li, Huan-Huan; Liang, Wei; Liao, Yu-Hua; Yuan, Jing; Cheng, Xiang

    2016-01-01

    Myocardial injuries in viral myocarditis (VMC) are caused by viral infection and related autoimmune disorders. Recent studies suggest that IL-9 mediated both antimicrobial immune and autoimmune responses in addition to allergic diseases. However, the role of IL-9 in viral infection and VMC remains controversial and uncertain. In this study, we infected Balb/c mice with Coxsackievirus B3 (CVB3), and found that IL-9 was enriched in the blood and hearts of VMC mice on days 5 and 7 after virus infection. Most of IL-9 was secreted by CD8+ T cells on day 5 and CD4+ T cells on day 7 in the myocardium. Further, IL-9 knockout exacerbated cardiac damage following CVB3 infection, along with a sharp increase in viral replication and IL-17a expression, as well as a decrease in TGF-β. In contrast, the repletion of IL-9 in Balb/c mice with CVB infection induced the opposite effect. Studies in vitro further revealed that IL-9 directly inhibited viral replication in cardiomyocytes by reducing coxsackie and adenovirus receptor expression, which might be associated with upregulation of TGF-β autocrine effect in these cells. However, IL-9 had no direct effect on apoptosis in cardiomyocytes. Our data indicated that IL-9 played a protective role in disease progression by inhibiting CVB3 replication in the early stages of VMC. PMID:27766098

  5. Role of pentraxin 3 in shaping arthritogenic alphaviral disease: from enhanced viral replication to immunomodulation.

    PubMed

    Foo, Suan-Sin; Chen, Weiqiang; Taylor, Adam; Sheng, Kuo-Ching; Yu, Xing; Teng, Terk-Shin; Reading, Patrick C; Blanchard, Helen; Garlanda, Cecilia; Mantovani, Alberto; Ng, Lisa F P; Herrero, Lara J; Mahalingam, Suresh

    2015-02-01

    The rising prevalence of arthritogenic alphavirus infections, including chikungunya virus (CHIKV) and Ross River virus (RRV), and the lack of antiviral treatments highlight the potential threat of a global alphavirus pandemic. The immune responses underlying alphavirus virulence remain enigmatic. We found that pentraxin 3 (PTX3) was highly expressed in CHIKV and RRV patients during acute disease. Overt expression of PTX3 in CHIKV patients was associated with increased viral load and disease severity. PTX3-deficient (PTX3(-/-)) mice acutely infected with RRV exhibited delayed disease progression and rapid recovery through diminished inflammatory responses and viral replication. Furthermore, binding of the N-terminal domain of PTX3 to RRV facilitated viral entry and replication. Thus, our study demonstrates the pivotal role of PTX3 in shaping alphavirus-triggered immunity and disease and provides new insights into alphavirus pathogenesis. PMID:25695775

  6. Analysis of the viral replication cycle of adenovirus serotype 2 after inactivation by free chlorine.

    PubMed

    Gall, Aimee M; Shisler, Joanna L; Mariñas, Benito J

    2015-04-01

    Free chlorine is effective at inactivating a wide range of waterborne viral pathogens including human adenovirus (HAdV), but the mechanisms by which free chlorine inactivates HAdV and other human viruses remain to be elucidated. Such advances in fundamental knowledge are key for development of new disinfection technologies and novel sensors to detect infectious viruses in drinking water. We developed and tested a quantitative assay to analyze several steps in the HAdV replication cycle upon increasing free chlorine exposure. We used quantitative polymerase chain reaction (qPCR) to detect HAdV genomic DNA as a means to quantify attachment and genome replication of untreated and treated virions. Also, we used quantitative reverse-transcription PCR (RT-qPCR) to quantify the transcription of E1A (first early protein) and hexon mRNA. We compared these replication cycle events to virus inactivation kinetics to determine what stage of the virus replication cycle was inhibited as a function of free chlorine exposure. We observed that adenovirus inactivated at levels up to 99.99% by free chlorine still attached to host cells; however, viral DNA synthesis and early E1A and late hexon gene transcription were inhibited. We conclude that free chlorine exposure interferes with a replication cycle event occurring postbinding but prior to early viral protein synthesis.

  7. Human miR-3145 inhibits influenza A viruses replication by targeting and silencing viral PB1 gene

    PubMed Central

    Khongnomnan, Kritsada; Makkoch, Jarika; Poomipak, Witthaya; Poovorawan, Yong

    2015-01-01

    MicroRNAs (miRNAs) play an important role in the regulation of gene expression and are involved in many cellular processes including inhibition of viral replication in infected cells. In this study, three subtypes of influenza A viruses (pH1N1, H5N1 and H3N2) were analyzed to identify candidate human miRNAs targeting and silencing viral genes expression. Candidate human miRNAs were predicted by miRBase and RNAhybrid based on minimum free energy (MFE) and hybridization patterns between human miRNAs and viral target genes. In silico analysis presented 76 miRNAs targeting influenza A viruses, including 70 miRNAs that targeted specific subtypes (21 for pH1N1, 27 for H5N1 and 22 for H3N2) and 6 miRNAs (miR-216b, miR-3145, miR-3682, miR-4513, miR-4753 and miR-5693) that targeted multiple subtypes of influenza A viruses. Interestingly, miR-3145 is the only candidate miRNA targeting all three subtypes of influenza A viruses. The miR-3145 targets to PB1 encoding polymerase basic protein 1, which is the main component of the viral polymerase complex. The silencing effect of miR-3145 was validated by 3′-UTR reporter assay and inhibition of influenza viral replication in A549 cells. In 3′-UTR reporter assay, results revealed that miR-3145 triggered significant reduction of the luciferase activity. Moreover, expression of viral PB1 genes was also inhibited considerably (P value < 0.05) in viral infected cells expressing mimic miR-3145. In conclusion, this study demonstrated that human miR-3145 triggered silencing of viral PB1 genes and lead to inhibition of multiple subtypes of influenza viral replication. Therefore, hsa-miR-3145 might be useful for alternative treatment of influenza A viruses in the future. PMID:26080461

  8. Hepatitis B viral replication influences the expression of natural killer cell ligands

    PubMed Central

    Koumbi, Lemonica; Pollicino, Teresa; Raimondo, Giovanni; Kumar, Naveenta; Karayiannis, Peter; Khakoo, Salim I.

    2016-01-01

    Background Hepatitis B virus (HBV) is accounting for over one million deaths annually due to immune-mediated chronic liver damage. Natural killer (NK) cells are abundant in the liver and contribute in HBV persistence. NK cytotoxic effects are controlled by signals from activating and inhibitory receptors. HBV may circumvent host antiviral immunity via the regulation of NK receptors and their ligands. We investigated the effect of viral replication and HBeAg mutations on NK mediators expression in the livers of chronic HBV (CHB) patients and in cell cultures. Methods HBV monomers bearing hotspot mutations in the basal core promoter and precore region were transfected into HepG2 cells using a plasmid-free assay. Serum viremia and liver HBV RNA were measured in 19 CHB patients. The expression of HBV RNA and of NKG2D ligands, B7H6, DNAX accessory molecule-1, lectin-like transcript 1 (LLT1), LFA-1 and TRAIL was measured in the livers of CHB patients and transfected cells. Results In general, high HBV replication in CHB patients and cell lines upregulated the mRNA of all NK cell ligands and particularly the inhibitory NK cell ligand, LLT1. The exception was the NKG2D ligand, MICA, that was significantly decreased in patients with high serum viremia and intrahepatic HBV RNA levels. Conclusions HBV replication has differential effects on NK cell ligands suggesting a potential escape mechanisms through up-regulation of LLT1 and down-regulation of MICA. A general trend towards upregulating NK cell ligands can be counteracted by decreasing MICA and hence weakening NK surveillance. PMID:27366037

  9. The function of temporally ordered viral gene expression in the intracellular replication of herpes simplex virus type 1 (HSV-1).

    PubMed

    Nakabayashi, Jun; Sasaki, Akira

    2009-11-01

    In the reproduction of HSV-1, the temporal profile of the viral gene expressions and the molecular mechanisms regulating the expressions are extensively studied. Functional roles of the temporally ordered gene expressions has not yet been clarified. We construct a simple mathematical model for the intracellular replication of HSV-1 to investigate the function of the ordered gene expressions. We obtain the condition for the 'explosion' of the virus from our model. The expression ratio of the early gene to the late gene must be higher than the ratio of the reaction rate of the encapsidation to that of the viral DNA replication for viruses to reproduce successfully. The preceded accumulation of the early gene product prevents the growth arrest. Further, as promoter activity of the early gene becomes higher, the replication speed of virus becomes faster. The structure of early gene promoter that has many binding motif to transcription factor accelerates the replication speed of HSV-1. This structure of the early gene promoter might be selectively maintained by allowing fast growth of the virus. With amino acid limitation, there exist finite optimal ratio of early/late gene promoter activity.

  10. Capsid-Targeted Viral Inactivation: A Novel Tactic for Inhibiting Replication in Viral Infections

    PubMed Central

    Zhang, Xingcui; Jia, Renyong; Zhou, Jiakun; Wang, Mingshu; Yin, Zhongqiong; Cheng, Anchun

    2016-01-01

    Capsid-targeted viral inactivation (CTVI), a conceptually powerful new antiviral strategy, is attracting increasing attention from researchers. Specifically, this strategy is based on fusion between the capsid protein of a virus and a crucial effector molecule, such as a nuclease (e.g., staphylococcal nuclease, Barrase, RNase HI), lipase, protease, or single-chain antibody (scAb). In general, capsid proteins have a major role in viral integration and assembly, and the effector molecule used in CTVI functions to degrade viral DNA/RNA or interfere with proper folding of viral key proteins, thereby affecting the infectivity of progeny viruses. Interestingly, such a capsid–enzyme fusion protein is incorporated into virions during packaging. CTVI is more efficient compared to other antiviral methods, and this approach is promising for antiviral prophylaxis and therapy. This review summarizes the mechanism and utility of CTVI and provides some successful applications of this strategy, with the ultimate goal of widely implementing CTVI in antiviral research. PMID:27657114

  11. Autophagic machinery activated by dengue virus enhances virus replication

    SciTech Connect

    Lee, Y.-R.; Lei, H.-Y.; Liu, M.-T.; Wang, J.-R.; Chen, S.-H.; Jiang-Shieh, Y.-F.; Lin, Y.-S.; Yeh, T.-M.; Liu, C.-C.; Liu, H.-S.

    2008-05-10

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

  12. A cell-based screening system for influenza A viral RNA transcription/replication inhibitors

    PubMed Central

    Ozawa, Makoto; Shimojima, Masayuki; Goto, Hideo; Watanabe, Shinji; Hatta, Yasuko; Kiso, Maki; Furuta, Yousuke; Horimoto, Taisuke; Peters, Noel R.; Hoffmann, F. Michael; Kawaoka, Yoshihiro

    2013-01-01

    Although two classes of antivirals, NA inhibitors and M2 ion channel blockers, are licensed for influenza treatment, dual resistant mutants, including highly pathogenic H5N1 viruses, have appeared. Alternative treatment options are, therefore, needed. Influenza A viral RNA (vRNA) transcription/replication is a promising target for antiviral development, since it is essential for virus replication. Accordingly, an efficient and reliable method to identify vRNA transcription/replication inhibitors is desirable. Here, we developed a cell-based screening system by establishing a cell line that stably expresses influenza viral ribonucleoprotein complex (vRNP). Compound library screening using this cell line allowed us to identify a compound that inhibits vRNA transcription/replication by using reporter protein expression from virus-like RNA as a readout and virus replication in vitro. vRNP-expressing cells have potential as a simple and convenient high-throughput screening (HTS) system, and, thus, are promising to identify vRNA transcription/replication inhibitors for various RNA viruses, especially for primary screens. PMID:23346363

  13. Mitochondrial Bioenergetic Alterations in Mouse Neuroblastoma Cells Infected with Sindbis Virus: Implications to Viral Replication and Neuronal Death

    PubMed Central

    Silva da Costa, Leandro; Pereira da Silva, Ana Paula; Da Poian, Andrea T.; El-Bacha, Tatiana

    2012-01-01

    The metabolic resources crucial for viral replication are provided by the host. Details of the mechanisms by which viruses interact with host metabolism, altering and recruiting high free-energy molecules for their own replication, remain unknown. Sindbis virus, the prototype of and most widespread alphavirus, causes outbreaks of arthritis in humans and serves as a model for the study of the pathogenesis of neurological diseases induced by alphaviruses in mice. In this work, respirometric analysis was used to evaluate the effects of Sindbis virus infection on mitochondrial bioenergetics of a mouse neuroblastoma cell lineage, Neuro 2a. The modulation of mitochondrial functions affected cellular ATP content and this was synchronous with Sindbis virus replication cycle and cell death. At 15 h, irrespective of effects on cell viability, viral replication induced a decrease in oxygen consumption uncoupled to ATP synthesis and a 36% decrease in maximum uncoupled respiration, which led to an increase of 30% in the fraction of oxygen consumption used for ATP synthesis. Decreased proton leak associated to complex I respiration contributed to the apparent improvement of mitochondrial function. Cellular ATP content was not affected by infection. After 24 h, mitochondria dysfunction was clearly observed as maximum uncoupled respiration reduced 65%, along with a decrease in the fraction of oxygen consumption used for ATP synthesis. Suppressed respiration driven by complexes I- and II-related substrates seemed to play a role in mitochondrial dysfunction. Despite the increase in glucose uptake and glycolytic flux, these changes were followed by a 30% decrease in ATP content and neuronal death. Taken together, mitochondrial bioenergetics is modulated during Sindbis virus infection in such a way as to favor ATP synthesis required to support active viral replication. These early changes in metabolism of Neuro 2a cells may form the molecular basis of neuronal dysfunction and Sindbis

  14. RNA polymerase II is aberrantly phosphorylated and localized to viral replication compartments following herpes simplex virus infection.

    PubMed Central

    Rice, S A; Long, M C; Lam, V; Spencer, C A

    1994-01-01

    During lytic infection, herpes simplex virus subverts the host cell RNA polymerase II transcription machinery to efficiently express its own genome while repressing the expression of most cellular genes. The mechanism by which RNA polymerase II is directed to the viral delayed-early and late genes is still unresolved. We report here that RNA polymerase II is preferentially localized to viral replication compartments early after infection with herpes simplex virus type 1. Concurrent with recruitment of RNA polymerase II into viral compartments is a rapid and aberrant phosphorylation of the large subunit carboxy-terminal domain (CTD). Aberrant phosphorylation of the CTD requires early viral gene expression but is not dependent on viral DNA replication or on the formation of viral replication compartments. Localization of RNA polymerase II and modifications to the CTD may be instrumental in favoring transcription of viral genes and repressing specific transcription of cellular genes. Images PMID:8289400

  15. Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62

    PubMed Central

    Metz, Philippe; Chiramel, Abhilash; Chatel-Chaix, Laurent; Alvisi, Gualtiero; Bankhead, Peter; Mora-Rodríguez, Rodrigo; Long, Gang; Hamacher-Brady, Anne

    2015-01-01

    ABSTRACT Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or the mTOR inhibitor Torin1. We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased. We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV. IMPORTANCE Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the

  16. Heme Oxygenase-1 Suppresses Bovine Viral Diarrhoea Virus Replication in vitro.

    PubMed

    Zhang, Chong; Pu, Fengxing; Zhang, Angke; Xu, Lele; Li, Na; Yan, Yunhuan; Gao, Jiming; Liu, Hongliang; Zhang, Gaiping; Goodfellow, Ian G; Zhou, En-Min; Xiao, Shuqi

    2015-10-29

    Viral cycle progression depends upon host-cell processes in infected cells, and this is true for bovine viral diarrhoea virus (BVDV), the causative agent of BVD that is a worldwide threat to the bovine industry. Heme oxygenase-1 (HO-1) is a ubiquitously expressed inducible isoform of the first and rate-limiting enzyme for heme degradation. Recent studies have demonstrated that HO-1 has significant antiviral properties, inhibiting the replication of viruses such as ebola virus, human immunodeficiency virus, hepatitis C virus, and porcine reproductive and respiratory syndrome virus. However, the function of HO-1 in BVDV infection is unclear. In the present study, the relationship between HO-1 and BVDV was investigated. In vitro analysis of HO-1 expression in BVDV-infected MDBK cells demonstrated that a decrease in HO-1 as BVDV replication increased. Increasing HO-1 expression through adenoviral-mediated overexpression or induction with cobalt protoporphyrin (CoPP, a potent HO-1 inducer), pre- and postinfection, effectively inhibited BVDV replication. In contrast, HO-1 siRNA knockdown in BVDV-infected cells increased BVDV replication. Therefore, the data were consistent with HO-1 acting as an anti-viral factor and these findings suggested that induction of HO-1 may be a useful prevention and treatment strategy against BVDV infection.

  17. Heme Oxygenase-1 Suppresses Bovine Viral Diarrhoea Virus Replication in vitro

    PubMed Central

    Zhang, Chong; Pu, Fengxing; Zhang, Angke; Xu, Lele; Li, Na; Yan, Yunhuan; Gao, Jiming; Liu, Hongliang; Zhang, Gaiping; Goodfellow, Ian G.; Zhou, En-Min; Xiao, Shuqi

    2015-01-01

    Viral cycle progression depends upon host-cell processes in infected cells, and this is true for bovine viral diarrhoea virus (BVDV), the causative agent of BVD that is a worldwide threat to the bovine industry. Heme oxygenase-1 (HO-1) is a ubiquitously expressed inducible isoform of the first and rate-limiting enzyme for heme degradation. Recent studies have demonstrated that HO-1 has significant antiviral properties, inhibiting the replication of viruses such as ebola virus, human immunodeficiency virus, hepatitis C virus, and porcine reproductive and respiratory syndrome virus. However, the function of HO-1 in BVDV infection is unclear. In the present study, the relationship between HO-1 and BVDV was investigated. In vitro analysis of HO-1 expression in BVDV-infected MDBK cells demonstrated that a decrease in HO-1 as BVDV replication increased. Increasing HO-1 expression through adenoviral-mediated overexpression or induction with cobalt protoporphyrin (CoPP, a potent HO-1 inducer), pre- and postinfection, effectively inhibited BVDV replication. In contrast, HO-1 siRNA knockdown in BVDV-infected cells increased BVDV replication. Therefore, the data were consistent with HO-1 acting as an anti-viral factor and these findings suggested that induction of HO-1 may be a useful prevention and treatment strategy against BVDV infection. PMID:26510767

  18. 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.

  19. The extent of early viral replication is a critical determinant of the natural history of simian immunodeficiency virus infection.

    PubMed Central

    Lifson, J D; Nowak, M A; Goldstein, S; Rossio, J L; Kinter, A; Vasquez, G; Wiltrout, T A; Brown, C; Schneider, D; Wahl, L; Lloyd, A L; Williams, J; Elkins, W R; Fauci, A S; Hirsch, V M

    1997-01-01

    Different patterns of viral replication correlate with the natural history of disease progression in humans and macaques infected with human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV), respectively. However, the viral and host factors influencing these patterns of viral replication in vivo are poorly understood. We intensively studied viral replication in macaques receiving identical inocula of SIV. Marked differences in viral replication patterns were apparent within the first week following inoculation, a time prior to the development of measurable specific immune effector responses to viral antigens. Plasma viral RNA levels measured on day 7 postinoculation correlated with levels measured in the postacute phase of infection. Differences in the susceptibility of host cells from different animals to in vitro SIV infection correlated with the permissiveness of the animals for early in vivo viral replication and hence with the postacute set point level of plasma viremia. These results suggest that host factors that exert their effects prior to full development of specific immune responses are critical in establishing the in vivo viral replication pattern and associated clinical course in subjects infected with SIV and, by extension, with HIV-1. PMID:9371613

  20. MDA7/IL-24 is an anti-viral factor that inhibits influenza virus replication.

    PubMed

    Seong, Rak-Kyun; Choi, Young-Ki; Shin, Ok Sarah

    2016-10-01

    Melanoma differentiation associated gene-7 (mda-7)/interleukin- 24 (IL-24) is a secreted cytokine, which plays an essential role in tumor suppression. Although its role as a multifunctional protein affecting broad types of cancers is well described, functions of IL-24 in host defense against virus infection are yet to be determined. In this study, we explored the anti-viral effect of recombinant IL-24 treatment during influenza infection. Infection of human lung adenocarcinoma cells (A549) with the influenza A virus up-regulated IL-24 mRNA and protein expression in a time-dependent manner. Pre-treatment of A549 cells with recombinant IL-24 protein effectively suppressed viral plaque formation. Furthermore, IL-24 treatment of A549 cells reduced viral non-structural protein 1 (NS1) synthesis, whereas IL-24 knockdown resulted in increased viral replication. Interestingly, IL-24 treatment following influenza A virus infection led to up-regulation of interferon (IFN)-induced antiviral signaling. Taken together, our results suggest that IL-24 exerts a potent suppressive effect on influenza viral replication and can be used in the treatment of influenza infection. PMID:27687232

  1. MDA7/IL-24 is an anti-viral factor that inhibits influenza virus replication.

    PubMed

    Seong, Rak-Kyun; Choi, Young-Ki; Shin, Ok Sarah

    2016-10-01

    Melanoma differentiation associated gene-7 (mda-7)/interleukin- 24 (IL-24) is a secreted cytokine, which plays an essential role in tumor suppression. Although its role as a multifunctional protein affecting broad types of cancers is well described, functions of IL-24 in host defense against virus infection are yet to be determined. In this study, we explored the anti-viral effect of recombinant IL-24 treatment during influenza infection. Infection of human lung adenocarcinoma cells (A549) with the influenza A virus up-regulated IL-24 mRNA and protein expression in a time-dependent manner. Pre-treatment of A549 cells with recombinant IL-24 protein effectively suppressed viral plaque formation. Furthermore, IL-24 treatment of A549 cells reduced viral non-structural protein 1 (NS1) synthesis, whereas IL-24 knockdown resulted in increased viral replication. Interestingly, IL-24 treatment following influenza A virus infection led to up-regulation of interferon (IFN)-induced antiviral signaling. Taken together, our results suggest that IL-24 exerts a potent suppressive effect on influenza viral replication and can be used in the treatment of influenza infection.

  2. The polymerase-like core of brome mosaic virus 2a protein, lacking a region interacting with viral 1a protein in vitro, maintains activity and 1a selectivity in RNA replication.

    PubMed Central

    Smirnyagina, E; Lin, N S; Ahlquist, P

    1996-01-01

    Brome mosaic virus (BMV), a member of the alphavirus-like super-family of positive-strand RNA viruses, encodes two proteins required for viral RNA replication: 1a and 2a. 1a contains m7G methyltransferase- and helicase-like domains, while 2a contains a polymerase (pol)-like core flanked by N- and C-terminal extensions. Genetic studies show that BMV RNA replication requires 1a-2a compatibility implying direct or indirect 1a-2a interaction in vivo. In vitro, la interacts with the N-terminal 125-amino-acid segment of 2a preceding the pol-like core, and prior deletion studies suggested that this 2a segment was essential for RNA replication. We have now used protein fusions and deletions to explore possible parallels between noncovalent 1a-2a interaction and covalent fusion of similar protein domains in tobacco mosaic virus and to see whether the N-terminal 2a-1a interaction was the primary basis for 1a-2a compatibility in vivo. We found that 2a can function as part of a tobacco mosaic virus-like 1a-2a fusion and that a 2a segment (amino acids 162 to 697) comprising the pol-like core was sufficient to provide 2a functions in such a fusion. Unexpectedly, the unfused 2a core segment also supported RNA replication when it and wild-type la were expressed as separate proteins. Moreover, in gene reassortant experiments with the related cowpea chlorotic mottle virus, the unfused 2a core segment showed the same 1a compatibility requirements as did wild-type BMV 2a. Thus, the pol-like core of 2a must interact with la in a way that is selective and essential for RNA synthesis, and 1a-2a interactions are more complex than the single, previously mapped interaction of the N-terminal 2a segment with 1a. PMID:8676500

  3. Intrinsic host restriction factors of human cytomegalovirus replication and mechanisms of viral escape.

    PubMed

    Landolfo, Santo; De Andrea, Marco; Dell'Oste, Valentina; Gugliesi, Francesca

    2016-08-12

    Before a pathogen even enters a cell, intrinsic immune defenses are active. This first-line defense is mediated by a variety of constitutively expressed cell proteins collectively termed "restriction factors" (RFs), and they form a vital element of the immune response to virus infections. Over time, however, viruses have evolved in a variety ways so that they are able to overcome these RF defenses via mechanisms that are specific for each virus. This review provides a summary of the universal characteristics of RFs, and goes on to focus on the strategies employed by some of the most important RFs in their attempt to control human cytomegalovirus (HCMV) infection. This is followed by a discussion of the counter-restriction mechanisms evolved by viruses to circumvent the host cell's intrinsic immune defenses. RFs include nuclear proteins IFN-γ inducible protein 16 (IFI16) (a Pyrin/HIN domain protein), Sp100, promyelocytic leukemia, and hDaxx; the latter three being the keys elements of nuclear domain 10 (ND10). IFI16 inhibits the synthesis of virus DNA by down-regulating UL54 transcription - a gene encoding a CMV DNA polymerase; in response, the virus antagonizes IFI16 via a process involving viral proteins UL97 and pp65 (pUL83), which results in the mislocalizing of IFI16 into the cytoplasm. In contrast, viral regulatory proteins, including pp71 and IE1, seek to modify or disrupt the ND10 proteins and thus block or reverse their inhibitory effects upon virus replication. All in all, detailed knowledge of these HCMV counter-restriction mechanisms will be fundamental for the future development of new strategies for combating HCMV infection and for identifying novel therapeutic agents. PMID:27563536

  4. Intrinsic host restriction factors of human cytomegalovirus replication and mechanisms of viral escape

    PubMed Central

    Landolfo, Santo; De Andrea, Marco; Dell’Oste, Valentina; Gugliesi, Francesca

    2016-01-01

    Before a pathogen even enters a cell, intrinsic immune defenses are active. This first-line defense is mediated by a variety of constitutively expressed cell proteins collectively termed “restriction factors” (RFs), and they form a vital element of the immune response to virus infections. Over time, however, viruses have evolved in a variety ways so that they are able to overcome these RF defenses via mechanisms that are specific for each virus. This review provides a summary of the universal characteristics of RFs, and goes on to focus on the strategies employed by some of the most important RFs in their attempt to control human cytomegalovirus (HCMV) infection. This is followed by a discussion of the counter-restriction mechanisms evolved by viruses to circumvent the host cell’s intrinsic immune defenses. RFs include nuclear proteins IFN-γ inducible protein 16 (IFI16) (a Pyrin/HIN domain protein), Sp100, promyelocytic leukemia, and hDaxx; the latter three being the keys elements of nuclear domain 10 (ND10). IFI16 inhibits the synthesis of virus DNA by down-regulating UL54 transcription - a gene encoding a CMV DNA polymerase; in response, the virus antagonizes IFI16 via a process involving viral proteins UL97 and pp65 (pUL83), which results in the mislocalizing of IFI16 into the cytoplasm. In contrast, viral regulatory proteins, including pp71 and IE1, seek to modify or disrupt the ND10 proteins and thus block or reverse their inhibitory effects upon virus replication. All in all, detailed knowledge of these HCMV counter-restriction mechanisms will be fundamental for the future development of new strategies for combating HCMV infection and for identifying novel therapeutic agents. PMID:27563536

  5. Biological roles and functional mechanisms of arenavirus Z protein in viral replication.

    PubMed

    Wang, Jialong; Danzy, Shamika; Kumar, Naveen; Ly, Hinh; Liang, Yuying

    2012-09-01

    Arenaviruses can cause severe hemorrhagic fever diseases in humans, with limited prophylactic or therapeutic measures. A small RING-domain viral protein Z has been shown to mediate the formation of virus-like particles and to inhibit viral RNA synthesis, although its biological roles in an infectious viral life cycle have not been directly addressed. By taking advantage of the available reverse genetics system for a model arenavirus, Pichinde virus (PICV), we provide the direct evidence for the essential biological roles of the Z protein's conserved residues, including the G2 myristylation site, the conserved C and H residues of RING domain, and the poorly characterized C-terminal L79 and P80 residues. Dicodon substitutions within the late (L) domain (PSAPPYEP) of the PICV Z protein, although producing viable mutant viruses, have significantly reduced virus growth, a finding suggestive of an important role for the intact L domain in viral replication. Further structure-function analyses of both PICV and Lassa fever virus Z proteins suggest that arenavirus Z proteins have similar molecular mechanisms in mediating their multiple functions, with some interesting variations, such as the role of the G2 residue in blocking viral RNA synthesis. In summary, our studies have characterized the biological roles of the Z protein in an infectious arenavirus system and have shed important light on the distinct functions of its domains in virus budding and viral RNA regulation, the knowledge of which may lead to the development of novel antiviral drugs.

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

    PubMed

    Ou, Horng D; May, Andrew P; O'Shea, Clodagh C

    2011-01-01

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

  7. Ginseng protects against respiratory syncytial virus by modulating multiple immune cells and inhibiting viral replication.

    PubMed

    Lee, Jong Seok; Lee, Yu-Na; Lee, Young-Tae; Hwang, Hye Suk; Kim, Ki-Hye; Ko, Eun-Ju; Kim, Min-Chul; Kang, Sang-Moo

    2015-01-01

    Ginseng has been used in humans for thousands of years but its effects on viral infection have not been well understood. We investigated the effects of red ginseng extract (RGE) on respiratory syncytial virus (RSV) infection using in vitro cell culture and in vivo mouse models. RGE partially protected human epithelial (HEp2) cells from RSV-induced cell death and viral replication. In addition, RGE significantly inhibited the production of RSV-induced pro-inflammatory cytokine (TNF-α) in murine dendritic and macrophage-like cells. More importantly, RGE intranasal pre-treatment prevented loss of mouse body weight after RSV infection. RGE treatment improved lung viral clearance and enhanced the production of interferon (IFN-γ) in bronchoalveolar lavage cells upon RSV infection of mice. Analysis of cellular phenotypes in bronchoalveolar lavage fluids showed that RGE treatment increased the populations of CD8+ T cells and CD11c+ dendritic cells upon RSV infection of mice. Taken together, these results provide evidence that ginseng has protective effects against RSV infection through multiple mechanisms, which include improving cell survival, partial inhibition of viral replication and modulation of cytokine production and types of immune cells migrating into the lung. PMID:25658239

  8. MAIT cells are activated during human viral infections.

    PubMed

    van Wilgenburg, Bonnie; Scherwitzl, Iris; Hutchinson, Edward C; Leng, Tianqi; Kurioka, Ayako; Kulicke, Corinna; de Lara, Catherine; Cole, Suzanne; Vasanawathana, Sirijitt; Limpitikul, Wannee; Malasit, Prida; Young, Duncan; Denney, Laura; Moore, Michael D; Fabris, Paolo; Giordani, Maria Teresa; Oo, Ye Htun; Laidlaw, Stephen M; Dustin, Lynn B; Ho, Ling-Pei; Thompson, Fiona M; Ramamurthy, Narayan; Mongkolsapaya, Juthathip; Willberg, Christian B; Screaton, Gavin R; Klenerman, Paul

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology. PMID:27337592

  9. A virally encoded small peptide regulates RTA stability and facilitates Kaposi's sarcoma-associated herpesvirus lytic replication.

    PubMed

    Jaber, Tareq; Yuan, Yan

    2013-03-01

    In both mammalian and viral genomes, a large proportion of sequences are transcribed and annotated as noncoding RNAs. A polyadenylated RNA of 3.0 kb (T3.0) is transcribed from the opposite strand of the open reading frame 50 (ORF50) DNA template in the genome of Kaposi's sarcoma-associated herpesvirus (KSHV) and has been annotated previously as a noncoding RNA. ORF50 encodes the replication and transcription activator (RTA), which controls the switch of the virus between the latent and lytic phases of the life cycle. Here we show that T3.0 encodes a small peptide of 48 amino acids (designated viral small peptide 1 [vSP-1]). vSP-1 interacts with RTA at the protein abundance regulatory signal (PARS) motifs, and the association prevents RTA from being subjected to degradation through the ubiquitin-proteasome pathway. As a consequence, vSP-1 facilitates KSHV gene expression and lytic replication. This finding reveals a novel mechanism of gene regulation in the viral life cycle. PMID:23302891

  10. hCLE/C14orf166, a cellular protein required for viral replication, is incorporated into influenza virus particles

    PubMed Central

    Rodriguez-Frandsen, Ariel; de Lucas, Susana; Pérez-González, Alicia; Pérez-Cidoncha, Maite; Roldan-Gomendio, Alejandro; Pazo, Alejandra; Marcos-Villar, Laura; Landeras-Bueno, Sara; Ortín, Juan; Nieto, Amelia

    2016-01-01

    The influenza A virus polymerase associates with a number of cellular transcription-related factors, including the RNA polymerase II (RNAP II). We previously described that the cellular protein hCLE/C14orf166 interacts with and stimulates influenza virus polymerase as well as RNAP II activities. Here we show that, despite the considerable cellular shut-off observed in infected cells, which includes RNAP II degradation, hCLE protein levels increase throughout infection in a virus replication-dependent manner. Human and avian influenza viruses of various subtypes increase hCLE levels, but other RNA or DNA viruses do not. hCLE colocalises and interacts with viral ribonucleoproteins (vRNP) in the nucleus, as well as in the cytoplasm late in infection. Furthermore, biochemical analysis of purified virus particles and immunoelectron microscopy of infected cells show hCLE in virions, in close association with viral vRNP. These findings indicate that hCLE, a cellular protein important for viral replication, is one of the very few examples of transcription factors that are incorporated into particles of an RNA-containing virus. PMID:26864902

  11. Molecular Biology of Human Herpesvirus 8: Novel Functions and Virus–Host Interactions Implicated in Viral Pathogenesis and Replication

    PubMed Central

    Cousins, Emily; Nicholas, John

    2014-01-01

    Human herpesvirus 8 (HHV-8), also known as Kaposi’s sarcoma-associated herpesvirus (KSHV), is the second identified human gammaherpesvirus. Like its relative Epstein-Barr virus, HHV-8 is linked to B-cell tumors, specifically primary effusion lymphoma and multicentric Castleman’s disease, in addition to endothelial-derived KS. HHV-8 is unusual in its possession of a plethora of “accessory” genes and encoded proteins in addition to the core, conserved herpesvirus and gammaherpesvirus genes that are necessary for basic biological functions of these viruses. The HHV-8 accessory proteins specify not only activities deducible from their cellular protein homologies but also novel, unsuspected activities that have revealed new mechanisms of virus–host interaction that serve virus replication or latency and may contribute to the development and progression of virus-associated neoplasia. These proteins include viral interleukin-6 (vIL-6), viral chemokines (vCCLs), viral G protein–coupled receptor (vGPCR), viral interferon regulatory factors (vIRFs), and viral antiapoptotic proteins homologous to FLICE (FADD-like IL-1β converting enzyme)-inhibitory protein (FLIP) and survivin. Other HHV-8 proteins, such as signaling membrane receptors encoded by open reading frames K1 and K15, also interact with host mechanisms in unique ways and have been implicated in viral pathogenesis. Additionally, a set of micro-RNAs encoded by HHV-8 appear to modulate expression of multiple host proteins to provide conditions conducive to virus persistence within the host and could also contribute to HHV-8-induced neoplasia. Here, we review the molecular biology underlying these novel virus–host interactions and their potential roles in both virus biology and virus-associated disease. PMID:24008302

  12. MAIT cells are activated during human viral infections

    PubMed Central

    van Wilgenburg, Bonnie; Scherwitzl, Iris; Hutchinson, Edward C.; Leng, Tianqi; Kurioka, Ayako; Kulicke, Corinna; de Lara, Catherine; Cole, Suzanne; Vasanawathana, Sirijitt; Limpitikul, Wannee; Malasit, Prida; Young, Duncan; Denney, Laura; Barnes, Eleanor; Ball, Jonathan; Burgess, Gary; Cooke, Graham; Dillon, John; Gore, Charles; Foster, Graham; Guha, Neil; Halford, Rachel; Herath, Cham; Holmes, Chris; Howe, Anita; Hudson, Emma; Irving, William; Khakoo, Salim; Koletzki, Diana; Martin, Natasha; Mbisa, Tamyo; McKeating, Jane; McLauchlan, John; Miners, Alec; Murray, Andrea; Shaw, Peter; Simmonds, Peter; Spencer, Chris; Targett-Adams, Paul; Thomson, Emma; Vickerman, Peter; Zitzmann, Nicole; Moore, Michael D.; Fabris, Paolo; Giordani, Maria Teresa; Oo, Ye Htun; Laidlaw, Stephen M.; Dustin, Lynn B.; Ho, Ling-Pei; Thompson, Fiona M.; Ramamurthy, Narayan; Mongkolsapaya, Juthathip; Willberg, Christian B.; Screaton, Gavin R.; Klenerman, Paul

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation—driving cytokine release and Granzyme B upregulation—is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology. PMID:27337592

  13. Combination therapy including CpG oligodeoxynucleotides and entecavir induces early viral response and enhanced inhibition of viral replication in a woodchuck model of chronic hepadnaviral infection.

    PubMed

    Meng, Zhongji; Zhang, Xiaoyong; Pei, Rongjuan; Zhang, Ejuan; Kemper, Thekla; Vollmer, Jörg; Davis, Heather L; Glebe, Dieter; Gerlich, Wolfram; Roggendorf, Michael; Lu, Mengji

    2016-01-01

    CpG oligodeoxynucleotides (ODNs) stimulate immune cells via TLR9 and are potentially useful immunomodulators for the treatment of chronic viral infections. In the present study, different classes of CpGs were tested for their capacities for innate immune activation and antiviral activities in the woodchuck model. A class P CpG ODN was found to stimulate interferon (IFN) production in woodchuck peripheral blood mononuclear cells (PBMCs) in vitro, and following subcutaneous administration in vivo, it was observed to induce IFN and MxA expression in woodchuck PBMCs. Combination treatment with CpG ODN and entecavir (ETV) led to effective suppression of the woodchuck hepatitis virus (WHV) load in the woodchucks, with early viral responses and inhibition of replication. The woodchuck hepatitis surface antigen (WHsAg) serum concentrations were strongly decreased by CpG and ETV together but not by either agent alone, indicating synergistic effects. However, viral control post-treatment was still transient, similar to that observed with ETV alone. Significantly elevated levels of serum aspartate aminotransferase (AST) but not of alanine aminotransferase (ALT) in some of the woodchucks receiving CpG ODN were noted, but these increases were resolved before the completion of treatment and were not associated with an elevated serum bilirubin level or coagulation disorders, suggesting the absence of a significant safety concern. PMID:26585244

  14. Proficient Replication of the Yeast Genome by a Viral DNA Polymerase.

    PubMed

    Stodola, Joseph L; Stith, Carrie M; Burgers, Peter M

    2016-05-27

    DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol α, Pol δ, and Pol ϵ. Pol δ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol δ is a three-subunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both iron-sulfur cluster and zinc-binding motifs, which mediate interactions with Pol31, and processive replication with the replication clamp proliferating cell nuclear antigen (PCNA), respectively. We show that the entire N-terminal domain of Pol3, containing polymerase and proofreading activities, could be effectively replaced by those from bacteriophage RB69, and could carry out chromosomal DNA replication in yeast with remarkable high fidelity, provided that adaptive mutations in the replication clamp PCNA were introduced. This result is consistent with the model that all essential interactions for DNA replication in yeast are mediated through the small C-terminal domain of Pol3. The chimeric polymerase carries out processive replication with PCNA in vitro; however, in yeast, it requires an increased involvement of the mutagenic translesion DNA polymerase ζ during DNA replication. PMID:27072134

  15. Coat Protein Activation of Alfalfa Mosaic Virus Replication Is Concentration Dependent

    PubMed Central

    Guogas, Laura M.; Laforest, Siana M.; Gehrke, Lee

    2005-01-01

    Alfalfa mosaic virus (AMV) and ilarvirus RNAs are infectious only in the presence of the viral coat protein; therefore, an understanding of coat protein's function is important for defining viral replication mechanisms. Based on in vitro replication experiments, the conformational switch model states that AMV coat protein blocks minus-strand RNA synthesis (R. C. Olsthoorn, S. Mertens, F. T. Brederode, and J. F. Bol, EMBO J. 18:4856-4864, 1999), while another report states that coat protein present in an inoculum is required to permit minus-strand synthesis (L. Neeleman and J. F. Bol, Virology 254:324-333, 1999). Here, we report on experiments that address these contrasting results with a goal of defining coat protein′s function in the earliest stages of AMV replication. To detect coat-protein-activated AMV RNA replication, we designed and characterized a subgenomic luciferase reporter construct. We demonstrate that activation of viral RNA replication by coat protein is concentration dependent; that is, replication was strongly stimulated at low coat protein concentrations but decreased progressively at higher concentrations. Genomic RNA3 mutations preventing coat protein mRNA translation or disrupting coat protein's RNA binding domain diminished replication. The data indicate that RNA binding and an ongoing supply of coat protein are required to initiate replication on progeny genomic RNA transcripts. The data do not support the conformational switch model's claim that coat protein inhibits the initial stages of viral RNA replication. Replication activation may correlate with low local coat protein concentrations and low coat protein occupancy on the multiple binding sites present in the 3′ untranslated regions of the viral RNAs. PMID:15827190

  16. Characterization of West Nile viral replication and maturation in peripheral neurons in culture.

    PubMed

    Hunsperger, Elizabeth; Roehrig, John T

    2005-02-01

    The North American West Nile virus (WNV), New York 1999 strain, appears to be highly neurotropic, and its neuroinvasiveness is an important aspect of human disease. The authors have developed an in vitro model to study WNV replication and protein processing in neurons. They compared WNV infection of the dorsal root ganglion (DRG) neurons (sensory neurons) and PC-12 cells (sympathetic neurons) to WNV infection of the mosquito cell line, C6/36, and Vero cells. WNV infection of both neuronal cell types and C6/36 cells was not cytopathic up to 30 days post infection, and continual viral shedding was observed during this period. However, WNV infection of Vero cells was lytic. Interestingly, WNV infection of neurons was not efficient, requiring a high multiplicity of infection of > or = 10. Indirect immunofluorescence assays using normal and confocal microscopy with flavivirus-reactive antibodies and WNV-infected neurons demonstrated viral antigen mostly associated with the plasma membrane and in the neurite processes. Treatment of WNV-infected C6/36, PC-12, or DRG cells with brefeldin A (BFA; a trans-Golgi inhibitor) or nocadazole (a beta-tubulin inhibitor) had little effect on viral maturation and secretion. Treatment of WNV-infected Vero cells with BFA resulted in a 1000-fold decrease in viral titer, but nocodazole had no effect. Our studies suggest that even though PC-12 and DRG neurons are mammalian cells, viral protein processing and maturation in these cells more closely resembles replication in C6/36 insect cells than in mammalian Vero cells. PMID:15804955

  17. 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. PMID:26800776

  18. 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.

  19. Viral Evasion of Natural Killer Cell Activation

    PubMed Central

    Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

    2016-01-01

    Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections. PMID:27077876

  20. Viral Evasion of Natural Killer Cell Activation.

    PubMed

    Ma, Yi; Li, Xiaojuan; Kuang, Ersheng

    2016-04-12

    Natural killer (NK) cells play a key role in antiviral innate defenses because of their abilities to kill infected cells and secrete regulatory cytokines. Additionally, NK cells exhibit adaptive memory-like antigen-specific responses, which represent a novel antiviral NK cell defense mechanism. Viruses have evolved various strategies to evade the recognition and destruction by NK cells through the downregulation of the NK cell activating receptors. Here, we review the recent findings on viral evasion of NK cells via the impairment of NK cell-activating receptors and ligands, which provide new insights on the relationship between NK cells and viral actions during persistent viral infections.

  1. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    PubMed

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin.

  2. A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

    PubMed

    Donovan, Catriona; Heyer, Antonia; Pfeifer, Eugen; Polen, Tino; Wittmann, Anja; Krämer, Reinhard; Frunzke, Julia; Bramkamp, Marc

    2015-05-26

    In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin. PMID:25916847

  3. The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA.

    PubMed Central

    Perales, B; Ortín, J

    1997-01-01

    The transcription and replication of influenza virus RNA (vRNA) were reconstituted in vivo. The experimental approach involved the transfection of plasmids encoding the viral subunits of the polymerase and the nucleoprotein into cells infected with a vaccinia virus recombinant virus expressing the T7 RNA polymerase. As templates, one of two model RNAs was transfected: vNSZ or cNSZ RNA. The RNAs were 240 nucleotides in length, contained the terminal sequences of the NS viral segment, and were of negative or positive polarity, respectively. The accumulation of cRNA and mRNA in cells transfected with vNSZ RNA and the accumulation of vRNA and mRNA in cells transfected with cNSZ RNA were determined by RNase protection assays with labeled vNSZ-L or cNSZ-L probes. The patterns of protected bands obtained indicated that both cRNA replication intermediate and mRNA accumulated when the system was reconstituted with vNSZ RNA. Likewise, both vRNA and mRNA accumulated after reconstitution with cNSZ RNA. The reconstitution of incomplete systems in which any of the subunits of the polymerase or the model RNA were omitted was completely negative for the accumulation of cRNA or vRNA, indicating that the presence of the PB2 subunit in the polymerase is required for replication of vRNA. PMID:8995663

  4. Diminished viral replication and compartmentalization of hepatitis C virus in hepatocellular carcinoma tissue

    PubMed Central

    Harouaka, Djamila; Engle, Ronald E.; Wollenberg, Kurt; Diaz, Giacomo; Tice, Ashley B.; Zamboni, Fausto; Govindarajan, Sugantha; Alter, Harvey; Kleiner, David E.; Farci, Patrizia

    2016-01-01

    Analysis of hepatitis C virus (HCV) replication and quasispecies distribution within the tumor of patients with HCV-associated hepatocellular carcinoma (HCC) can provide insight into the role of HCV in hepatocarcinogenesis and, conversely, the effect of HCC on the HCV lifecycle. In a comprehensive study of serum and multiple liver specimens from patients with HCC who underwent liver transplantation, we found a sharp and significant decrease in HCV RNA in the tumor compared with surrounding nontumorous tissues, but found no differences in multiple areas of control non-HCC cirrhotic livers. Diminished HCV replication was not associated with changes in miR-122 expression. HCV genetic diversity was significantly higher in livers containing HCC compared with control non-HCC cirrhotic livers. Tracking of individual variants demonstrated changes in the viral population between tumorous and nontumorous areas, the extent of which correlated with the decline in HCV RNA, suggesting HCV compartmentalization within the tumor. In contrast, compartmentalization was not observed between nontumorous areas and serum, or in controls between different areas of the cirrhotic liver or between liver and serum. Our findings indicate that HCV replication within the tumor is restricted and compartmentalized, suggesting segregation of specific viral variants in malignant hepatocytes. PMID:26787866

  5. Hepatitis B virus (HBV) X protein-mediated regulation of hepatocyte metabolic pathways affects viral replication.

    PubMed

    Bagga, Sumedha; Rawat, Siddhartha; Ajenjo, Marcia; Bouchard, Michael J

    2016-11-01

    Chronic HBV infection is a risk factor for hepatocellular carcinoma (HCC). The HBV HBx protein stimulates HBV replication and likely influences the development of HBV-associated HCC. Whether HBx affects regulators of metabolism in normal hepatocytes has not been addressed. We used an ex vivo, cultured primary rat hepatocyte system to assess the interplay between HBV replication and mechanistic target of rapamycin complex 1 (mTORC1) signaling. HBx activated mTORC1 signaling; however, inhibition of mTORC1 enhanced HBV replication. HBx also decreased ATP levels and activated the energy-sensing factor AMP-activated protein kinase (AMPK). Inhibition of AMPK decreased HBV replication. Inhibition of AMPK activates mTORC1, and we showed that activated mTORC1 is one factor that reduces HBV replication when AMPK is inhibited. HBx activation of both AMPK and mTORC1 suggests that these activities could provide a balancing mechanism to facilitate persistent HBV replication. HBx activation of mTORC1 and AMPK could also influence HCC development.

  6. Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo.

    PubMed

    Marión, R M; Zürcher, T; de la Luna, S; Ortín, J

    1997-10-01

    The interaction of influenza virus NS1 protein with other viral products in the infected cell was analysed by co-immunoprecipitation studies. The three subunits of the polymerase and the nucleoprotein, but not M1 protein, were co-immunoprecipitated by NS1-specific serum but not when control serum was used. Such co-immunoprecipitation was not sensitive to RNase treatment of the immunoprecipitates. Co-immunoprecipitation was also obtained when the viral transcription-replication system was reconstituted in vivo by transfection of cDNAs and model vRNA template into vaccinia virus-T7-infected cells. Analysis of the RNA pulled-down in the NS1-specific precipitates indicated the presence of both vRNA and mRNA. These results are discussed in the context of the phenotype of virus temperature-sensitive mutants affected in the NS1 gene.

  7. Enhancement of autophagy during lytic replication by the Kaposi's sarcoma-associated herpesvirus replication and transcription activator.

    PubMed

    Wen, Hui-Ju; Yang, Zhilong; Zhou, You; Wood, Charles

    2010-08-01

    Autophagy is one of two major degradation systems in eukaryotic cells. The degradation mechanism of autophagy is required to maintain the balance between the biosynthetic and catabolic processes and also contributes to defense against invading pathogens. Recent studies suggest that a number of viruses can evade or subvert the host cell autophagic pathway to enhance their own replication. Here, we investigated the effect of autophagy on the KSHV (Kaposi's sarcoma-associated herpesvirus) life cycle. We found that the inhibition of autophagy reduces KSHV lytic reactivation from latency, and an enhancement of autophagy can be detected during KSHV lytic replication. In addition, RTA (replication and transcription activator), an essential viral protein for KSHV lytic reactivation, is able to enhance the autophagic process, leading to an increase in the number of autophagic vacuoles, an increase in the level of the lipidated LC3 protein, and the formation of autolysosomes. Moreover, the inhibition of autophagy affects RTA-mediated lytic gene expression and viral DNA replication. These results suggest that RTA increases autophagy activation to facilitate KSHV lytic replication. This is the first report demonstrating that autophagy is involved in the lytic reactivation of KSHV. PMID:20484505

  8. A DNA Binding Protein Is Required for Viral Replication and Transcription in Bombyx mori Nucleopolyhedrovirus.

    PubMed

    Zhao, Cui; Zhang, Chen; Chen, Bin; Shi, Yanghui; Quan, Yanping; Nie, Zuoming; Zhang, Yaozhou; Yu, Wei

    2016-01-01

    A DNA-binding protein (DBP) [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05), indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (p<0.01). The transcriptional level of dbp-ko-Bacmid early gene lef-3, ie-1, dnapol, late gene vp39 and very late gene p10 were statistically significantly lower than dbp-re-Bacmid and wtBacmid (p<0.01). The results presented are based on Western blot analysis, which indicated that the lack of dbp gene would lead to low expressions of lef3, vp39, and p10. In conclusion, dbp was not only essential for early viral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle. PMID:27414795

  9. A DNA Binding Protein Is Required for Viral Replication and Transcription in Bombyx mori Nucleopolyhedrovirus

    PubMed Central

    Chen, Bin; Shi, Yanghui; Quan, Yanping; Nie, Zuoming; Zhang, Yaozhou; Yu, Wei

    2016-01-01

    A DNA-binding protein (DBP) [GenBank accession number: M63416] of Bombyx mori nuclear polyhedrosis virus (BmNPV) has been reported to be a regulatory factor in BmNPV, but its detailed functions remain unknown. In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system. Then, the Bacmids were transfected into BmN cells. The results of this viral titer experiment revealed that the TCID50 of the dbp-ko-Bacmid was 0; however, the dbp-re-Bacmid was similar to the wtBacmid (p>0.05), indicating that the dbp-deficient would lead to failure in the assembly of virus particles. In the next step, Real-Time PCR was used to analyze the transcriptional phases of dbp gene in BmN cells, which had been infected with BmNPV. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR. The demonstrating that the replication level of genome was lower than that of wtBacmid and dbp-re-Bacmid (p<0.01). The transcriptional level of dbp-ko-Bacmid early gene lef-3, ie-1, dnapol, late gene vp39 and very late gene p10 were statistically significantly lower than dbp-re-Bacmid and wtBacmid (p<0.01). The results presented are based on Western blot analysis, which indicated that the lack of dbp gene would lead to low expressions of lef3, vp39, and p10. In conclusion, dbp was not only essential for early viral replication, but also a viral gene that has a significant impact on transcription and expression during all periods of baculovirus life cycle. PMID:27414795

  10. Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees.

    PubMed

    Di Prisco, Gennaro; Cavaliere, Valeria; Annoscia, Desiderato; Varricchio, Paola; Caprio, Emilio; Nazzi, Francesco; Gargiulo, Giuseppe; Pennacchio, Francesco

    2013-11-12

    Large-scale losses of honey bee colonies represent a poorly understood problem of global importance. Both biotic and abiotic factors are involved in this phenomenon that is often associated with high loads of parasites and pathogens. A stronger impact of pathogens in honey bees exposed to neonicotinoid insecticides has been reported, but the causal link between insecticide exposure and the possible immune alteration of honey bees remains elusive. Here, we demonstrate that the neonicotinoid insecticide clothianidin negatively modulates NF-κB immune signaling in insects and adversely affects honey bee antiviral defenses controlled by this transcription factor. We have identified in insects a negative modulator of NF-κB activation, which is a leucine-rich repeat protein. Exposure to clothianidin, by enhancing the transcription of the gene encoding this inhibitor, reduces immune defenses and promotes the replication of the deformed wing virus in honey bees bearing covert infections. This honey bee immunosuppression is similarly induced by a different neonicotinoid, imidacloprid, but not by the organophosphate chlorpyriphos, which does not affect NF-κB signaling. The occurrence at sublethal doses of this insecticide-induced viral proliferation suggests that the studied neonicotinoids might have a negative effect at the field level. Our experiments uncover a further level of regulation of the immune response in insects and set the stage for studies on neural modulation of immunity in animals. Furthermore, this study has implications for the conservation of bees, as it will contribute to the definition of more appropriate guidelines for testing chronic or sublethal effects of pesticides used in agriculture.

  11. Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees.

    PubMed

    Di Prisco, Gennaro; Cavaliere, Valeria; Annoscia, Desiderato; Varricchio, Paola; Caprio, Emilio; Nazzi, Francesco; Gargiulo, Giuseppe; Pennacchio, Francesco

    2013-11-12

    Large-scale losses of honey bee colonies represent a poorly understood problem of global importance. Both biotic and abiotic factors are involved in this phenomenon that is often associated with high loads of parasites and pathogens. A stronger impact of pathogens in honey bees exposed to neonicotinoid insecticides has been reported, but the causal link between insecticide exposure and the possible immune alteration of honey bees remains elusive. Here, we demonstrate that the neonicotinoid insecticide clothianidin negatively modulates NF-κB immune signaling in insects and adversely affects honey bee antiviral defenses controlled by this transcription factor. We have identified in insects a negative modulator of NF-κB activation, which is a leucine-rich repeat protein. Exposure to clothianidin, by enhancing the transcription of the gene encoding this inhibitor, reduces immune defenses and promotes the replication of the deformed wing virus in honey bees bearing covert infections. This honey bee immunosuppression is similarly induced by a different neonicotinoid, imidacloprid, but not by the organophosphate chlorpyriphos, which does not affect NF-κB signaling. The occurrence at sublethal doses of this insecticide-induced viral proliferation suggests that the studied neonicotinoids might have a negative effect at the field level. Our experiments uncover a further level of regulation of the immune response in insects and set the stage for studies on neural modulation of immunity in animals. Furthermore, this study has implications for the conservation of bees, as it will contribute to the definition of more appropriate guidelines for testing chronic or sublethal effects of pesticides used in agriculture. PMID:24145453

  12. Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees

    PubMed Central

    Di Prisco, Gennaro; Cavaliere, Valeria; Annoscia, Desiderato; Varricchio, Paola; Caprio, Emilio; Nazzi, Francesco; Gargiulo, Giuseppe; Pennacchio, Francesco

    2013-01-01

    Large-scale losses of honey bee colonies represent a poorly understood problem of global importance. Both biotic and abiotic factors are involved in this phenomenon that is often associated with high loads of parasites and pathogens. A stronger impact of pathogens in honey bees exposed to neonicotinoid insecticides has been reported, but the causal link between insecticide exposure and the possible immune alteration of honey bees remains elusive. Here, we demonstrate that the neonicotinoid insecticide clothianidin negatively modulates NF-κB immune signaling in insects and adversely affects honey bee antiviral defenses controlled by this transcription factor. We have identified in insects a negative modulator of NF-κB activation, which is a leucine-rich repeat protein. Exposure to clothianidin, by enhancing the transcription of the gene encoding this inhibitor, reduces immune defenses and promotes the replication of the deformed wing virus in honey bees bearing covert infections. This honey bee immunosuppression is similarly induced by a different neonicotinoid, imidacloprid, but not by the organophosphate chlorpyriphos, which does not affect NF-κB signaling. The occurrence at sublethal doses of this insecticide-induced viral proliferation suggests that the studied neonicotinoids might have a negative effect at the field level. Our experiments uncover a further level of regulation of the immune response in insects and set the stage for studies on neural modulation of immunity in animals. Furthermore, this study has implications for the conservation of bees, as it will contribute to the definition of more appropriate guidelines for testing chronic or sublethal effects of pesticides used in agriculture. PMID:24145453

  13. Autophagy during early stages contributes to bovine viral diarrhea virus replication in MDBK cells.

    PubMed

    Fu, Qiang; Shi, Huijun; Zhang, Hui; Ren, Yan; Guo, Fei; Qiao, Jun; Jia, Bin; Wang, Pengyan; Chen, Chuangfu

    2014-10-01

    Autophagy (or autophagocytosis) is an essential and precise control process by which cells degrade unnecessary or dysfunctional cellular components or organelles in the cytoplasm in response to nutrient depletion, exogenous pathogens, or other stimuli. This process results in the removal of damaged or surplus organelles and macromolecular complexes via a lysosome-dependent mechanism. Bovine viral diarrhea virus (BVDV) is a ssRNA virus of the Flaviviridae family (genus Pestivirus). BVDV infection results in major economic losses due to poor reproductive performance and poor calf performance in cattle herds. In our previous studies, we have shown that BVDV NADL infection significantly increases autophagy in MDBK cells. To further define the interactions between autophagy and BVDV infection, we investigated the effects of autophagy on the replication of BVDV NADL. The findings showed that autophagy was inhibited by treatment with 3-methyladenine (3-MA) or wortmannin and that the knockdown of LC3 and Beclin1 using lentivirus-mediated RNA interference (RNAi) suppressed BVDV NADL replication. In contrast, the findings showed the replication of BVDV NADL was significantly increased by treatment with the autophagy inducer rapamycin within 18 h post-infection (pi). However, the mRNA levels of BVDV NADL 5'UTRs showed a downward trend after 18 h pi, and this effect was reversed by chloroquine treatment. Therefore, we inferred that infection with BVDV NADL increases autophagy, which in turn favors BVDV NADL replication at early stages.

  14. Selective serotonin reuptake inhibitor fluoxetine inhibits replication of human enteroviruses B and D by targeting viral protein 2C.

    PubMed

    Ulferts, Rachel; van der Linden, Lonneke; Thibaut, Hendrik Jan; Lanke, Kjerstin H W; Leyssen, Pieter; Coutard, Bruno; De Palma, Armando M; Canard, Bruno; Neyts, Johan; van Kuppeveld, Frank J M

    2013-04-01

    Although the genus Enterovirus contains many important human pathogens, there is no licensed drug for either the treatment or the prophylaxis of enterovirus infections. We report that fluoxetine (Prozac)--a selective serotonin reuptake inhibitor--inhibits the replication of human enterovirus B (HEV-B) and HEV-D but does not affect the replication of HEV-A and HEV-C or human rhinovirus A or B. We show that fluoxetine interferes with viral RNA replication, and we identified viral protein 2C as the target of this compound. PMID:23335743

  15. Environmental risk assessment of replication competent viral vectors applied in clinical trials: potential effects of inserted sequences.

    PubMed

    van den Akker, Eric; van der Vlugt, Cecile J B; Bleijs, Diederik A; Bergmans, Hans E

    2013-12-01

    Risk assessments of clinical applications involving genetically modified viral vectors are carried out according to general principles that are implemented in many national and regional legislations, e.g., in Directive 2001/18/EC of the European Union. Recent developments in vector design have a large impact on the concepts that underpin the risk assessments of viral vectors that are used in clinical trials. The use of (conditionally) replication competent viral vectors (RCVVs) may increase the likelihood of the exposure of the environment around the patient, compared to replication defective viral vectors. Based on this assumption we have developed a methodology for the environmental risk assessment of replication competent viral vectors, which is presented in this review. Furthermore, the increased likelihood of exposure leads to a reevaluation of what would constitute a hazardous gene product in viral vector therapies, and a keen interest in new developments in the inserts used. One of the trends is the use of inserts produced by synthetic biology. In this review the implications of these developments for the environmental risk assessment of RCVVs are highlighted, with examples from current clinical trials. The conclusion is drawn that RCVVs, notwithstanding their replication competency, can be applied in an environmentally safe way, in particular if adequate built-in safeties are incorporated, like conditional replication competency, as mitigating factors to reduce adverse environmental effects that could occur.

  16. The Sendai virus V protein interacts with the NP protein to regulate viral genome RNA replication.

    PubMed

    Horikami, S M; Smallwood, S; Moyer, S A

    1996-08-15

    The interactions of Sendai virus proteins required for viral RNA synthesis have been characterized both by the yeast two-hybrid system and through the use of glutathione S-transferase (gst)-viral fusion proteins synthesized in mammalian cells. Using the two-hybrid system we have confirmed the previously identified P-L (RNA polymerase), NPo-P (encapsidation substrate), and P-P complexes and now demonstrate NP-NP and NPo-V protein interactions. Expression of gstP and P proteins and binding to glutathione-Sepharose beads as a measure of complex formation confirmed the P-P interaction. The P-gstP binding occurred only on expression of the proteins in the same cell and was mapped to amino acids 345-411. We also show that full-length and deletion gstV and gstW proteins bound NPo protein when these sets of proteins were coexpressed and have identified one required region from amino acids 78-316. Neither gstV nor gstW bound NP assembled into nucleocapsids. Furthermore, both V and W proteins lacking the N-terminal 77 amino acids inhibited DI-H genome replication in vitro, showing the biological relevance of the remaining region. We propose that the specific inhibition of genome replication by V and W proteins occurs through interference with either the formation or the use of the NPo-P encapsidation substrate.

  17. Endemic versus epidemic viral spreads display distinct patterns of HTLV-2b replication

    SciTech Connect

    Gabet, Anne-Sophie; Moules, Vincent; Sibon, David; Nass, Catharie C.; Mortreux, Franck; Mauclere, Philippe; Gessain, Antoine; Murphy, Edward L.; Wattel, Eric . E-mail: wattel@lyon.fnclcc.fr

    2006-02-05

    As the replication pattern of leukemogenic PTLVs possesses a strong pathogenic impact, we investigated HTLV-2 replication in vivo in asymptomatic carriers belonging into 2 distinct populations infected by the same HTLV-2b subtype. They include epidemically infected American blood donors, in whom HTLV-2b has been present for only 30 years, and endemically infected Bakola Pygmies from Cameroon, characterized by a long viral endemicity (at least few generations). In blood donors, both the circulating proviral loads and the degree of infected cell proliferation were largely lower than those characterizing asymptomatic carriers infected with leukemogenic PTLVs (HTLV-1, STLV-1). This might contribute to explain the lack of known link between HTLV-2b infection and the development of malignancies in this population. In contrast, endemically infected individuals displayed high proviral loads resulting from the extensive proliferation of infected cells. The route and/or the duration of infection, viral genetic drift, host immune response, genetic background, co-infections or a combination thereof might have contributed to these differences between endemically and epidemically infected subjects. As the clonality pattern observed in endemically infected individuals is very reminiscent of that of leukemogenic PTLVs at the pre-leukemic stage, our results highlight the possible oncogenic effect of HTLV-2b infection in such population.

  18. Human papillomavirus type 31 replication modes during the early phases of the viral life cycle depend on transcriptional and posttranscriptional regulation of E1 and E2 expression.

    PubMed

    Hubert, Walter G; Laimins, Laimonis A

    2002-03-01

    The E1 and E2 proteins are both required for papillomavirus DNA replication, and replication efficiency is controlled by the abundance of these factors. In human papillomaviruses (HPVs), the regulation of E1 and E2 expression and its effect on viral replication are not well understood. In particular, it is not known if E1 and E2 modulate their own expression and how posttranscriptional mechanisms may affect the levels of the replication proteins. Previous studies have implicated splicing within the E6 open reading frame (ORF) as being important for modulating replication of HPV type 31 (HPV31) through altered expression of E1 and E2. To analyze the function of the E6 intron in viral replication more specifically, we examined the effects of E6 splicing mutations in the context of entire viral genomes in transient assays. HPV31 genomes which had mutations in the splice donor site (E6SD) or the splice acceptor site (E6SA), a deletion of the intron (E6ID), or substituted heterologous intron sequences (E6IS) were constructed. Compared to wild-type (wt) HPV31, pHPV31-E6SD, -E6SA, and -E6IS replicated inefficiently while pHPV31-E6ID replicated at an intermediate level. Cotransfection of the E6 mutant genomes with an E1 expression vector strongly activated their replication levels, indicating that efficient expression of E1 requires E6 internal splicing. In contrast, replication was activated only moderately with an E2 expression vector. Replacing the wt E6 intron in HPV31 with a heterologous intron from simian virus 40 (E6SR2) resulted in replication levels similar to that of the wt in the absence of expression vectors, suggesting that mRNA splicing upstream of the E1 ORF is important for high-level replication. To examine the effects of E6 intron splicing on E1 and E2 expression directly, we constructed reporter DNAs in which the luciferase coding sequences were fused in frame to the E1 (E1Luc) or E2 (E2Luc) gene. Reporter activities were then analyzed in transient

  19. Sequences within the early and late promoters of archetype JC virus restrict viral DNA replication and infectivity.

    PubMed

    Daniel, A M; Swenson, J J; Mayreddy, R P; Khalili, K; Frisque, R J

    1996-02-01

    Two forms of JC virus (JCV) have been isolated from its human host, an archetype found in kidney tissue and urine of nonimmunocompromised individuals and a rearranged type detected in lymphocytes and brain tissue of patients with and without progressive multifocal leukoencephalopathy. To investigate the hypothesis that alterations to the archetype transcriptional control region yield rearranged forms of the virus exhibiting new tissue tropic and pathogenic potentials, attempts were made to propagate archetype JCV in human renal and glial cell cultures. Although rearranged forms of JCV multiplied in these cells, archetype JCV failed to do so. Through the use of chimeric and mutant viral genomes, and a cell line that constitutively expresses viral T protein, we demonstrated that archetype's inactivity relative to that of rearranged forms was due to differences in the promoter-enhancer and not in the protein coding regions or origin of DNA replication. Additional analyses revealed that the absence of a large tandem duplication and the presence of a 23- and a 66-base pair sequence in the archetype transcriptional control region were responsible for this restricted lytic behavior. We discuss the possibility that deletion and duplication events within the archetype promoter-enhancer might yield more active viral variants via the loss of a negative, or the creation of a positive, transcriptional control signal(s).

  20. Herpes Simplex Virus Replication: Roles of Viral Proteins and Nucleoporins in Capsid-Nucleus Attachment▿

    PubMed Central

    Copeland, Anna Maria; Newcomb, William W.; Brown, Jay C.

    2009-01-01

    Replication of herpes simplex virus type 1 (HSV-1) involves a step in which a parental capsid docks onto a host nuclear pore complex (NPC). The viral genome then translocates through the nuclear pore into the nucleoplasm, where it is transcribed and replicated to propagate infection. We investigated the roles of viral and cellular proteins in the process of capsid-nucleus attachment. Vero cells were preloaded with antibodies specific for proteins of interest and infected with HSV-1 containing a green fluorescent protein-labeled capsid, and capsids bound to the nuclear surface were quantified by fluorescence microscopy. Results showed that nuclear capsid attachment was attenuated by antibodies specific for the viral tegument protein VP1/2 (UL36 gene) but not by similar antibodies specific for UL37 (a tegument protein), the major capsid protein (VP5), or VP23 (a minor capsid protein). Similar studies with antibodies specific for nucleoporins demonstrated attenuation by antibodies specific for Nup358 but not Nup214. The role of nucleoporins was further investigated with the use of small interfering RNA (siRNA). Capsid attachment to the nucleus was attenuated in cells treated with siRNA specific for either Nup214 or Nup358 but not TPR. The results are interpreted to suggest that VP1/2 is involved in specific attachment to the NPC and/or in migration of capsids to the nuclear surface. Capsids are suggested to attach to the NPC by way of the complex of Nup358 and Nup214, with high-resolution immunofluorescence studies favoring binding to Nup358. PMID:19073727

  1. 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

    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

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

    PubMed

    Kazlauskas, Darius; Krupovic, Mart; Venclovas, Česlovas

    2016-06-01

    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

  3. Enterovirus 71 induces dsRNA/PKR-dependent cytoplasmic redistribution of GRP78/BiP to promote viral replication.

    PubMed

    Jheng, Jia-Rong; Wang, Shin-Chyang; Jheng, Chao-Rih; Horng, Jim-Tong

    2016-01-01

    GRP78/BiP is an endoplasmic reticulum (ER) chaperone protein with the important function of maintaining ER homeostasis, and the overexpression of GRP78/BiP alleviates ER stress. Our previous studies showed that infection with enterovirus 71 (EV71), a (+)RNA picornavirus, induced GRP78/BiP upregulation; however, ectopic GRP78/BiP overexpression in ER downregulates virus replication and viral particle formation. The fact that a virus infection increases GRP78/BiP expression, which is unfavorable for virus replication, is counterintuitive. In this study, we found that the GRP78/BiP protein level was elevated in the cytoplasm instead of in the ER in EV71-infected cells. Cells transfected with polyinosinic-polycytidylic acid, a synthetic analog of replicative double-stranded RNA (dsRNA), but not with viral proteins, also exhibited upregulation and elevation of GRP78/BiP in the cytosol. Our results further demonstrate that EV71 infections induce the dsRNA/protein kinase R-dependent cytosolic accumulation of GRP78/BiP. The overexpression of a GRP78/BiP mutant lacking a KDEL retention signal failed to inhibit both dithiothreitol-induced eIF2α phosphorylation and viral replication in the context of viral protein synthesis and viral titers. These data revealed that EV71 infection might cause upregulation and aberrant redistribution of GRP78/BiP to the cytosol, thereby facilitating virus replication. PMID:27004760

  4. Enterovirus 71 induces dsRNA/PKR-dependent cytoplasmic redistribution of GRP78/BiP to promote viral replication

    PubMed Central

    Jheng, Jia-Rong; Wang, Shin-Chyang; Jheng, Chao-Rih; Horng, Jim-Tong

    2016-01-01

    GRP78/BiP is an endoplasmic reticulum (ER) chaperone protein with the important function of maintaining ER homeostasis, and the overexpression of GRP78/BiP alleviates ER stress. Our previous studies showed that infection with enterovirus 71 (EV71), a (+)RNA picornavirus, induced GRP78/BiP upregulation; however, ectopic GRP78/BiP overexpression in ER downregulates virus replication and viral particle formation. The fact that a virus infection increases GRP78/BiP expression, which is unfavorable for virus replication, is counterintuitive. In this study, we found that the GRP78/BiP protein level was elevated in the cytoplasm instead of in the ER in EV71-infected cells. Cells transfected with polyinosinic–polycytidylic acid, a synthetic analog of replicative double-stranded RNA (dsRNA), but not with viral proteins, also exhibited upregulation and elevation of GRP78/BiP in the cytosol. Our results further demonstrate that EV71 infections induce the dsRNA/protein kinase R-dependent cytosolic accumulation of GRP78/BiP. The overexpression of a GRP78/BiP mutant lacking a KDEL retention signal failed to inhibit both dithiothreitol-induced eIF2α phosphorylation and viral replication in the context of viral protein synthesis and viral titers. These data revealed that EV71 infection might cause upregulation and aberrant redistribution of GRP78/BiP to the cytosol, thereby facilitating virus replication. PMID:27004760

  5. The N-terminus of classical swine fever virus (CSFV) nonstructural protein 2 modulates viral genome RNA replication.

    PubMed

    Li, Ling; Wu, Rui; Zheng, Fengwei; Zhao, Cheng; Pan, Zishu

    2015-12-01

    Pestivirus nonstructural protein 2 (NS2) is a multifunctional, hydrophobic protein with an important but poorly understood role in viral RNA replication and infectious virus production. In the present study, based on sequence analysis, we mutated several representative conserved residues within the N-terminus of NS2 of classical swine fever virus (CSFV) and investigated how these mutations affected viral RNA replication and infectious virus production. Our results demonstrated that the mutation of two aspartic acids, NS2/D60A or NS2/D60K and NS2/D78K, in the N-terminus of NS2 abolished infectious virus production and that the substitution of arginine for alanine at position 100 (NS2/R100A) resulted in significantly decreased viral titer. The serial passage of cells containing viral genomic RNA molecules generated the revertants NS2/A60D, NS2/K60D and NS2/K78D, leading to the recovery of infectious virus. In the context of the NS2/R100A mutant, the NS2/I90L mutation compensated for infectious virus production. The regulatory roles of the indicated amino acid residues were identified to occur at the viral RNA replication level. These results revealed a novel function for the NS2 N-terminus of CSFV in modulating viral RNA replication. PMID:26232654

  6. Replication-independent long-distance trafficking by viral RNAs in Nicotiana benthamiana.

    PubMed

    Gopinath, Kodetham; Kao, C Cheng

    2007-04-01

    Viruses with separately encapsidated genomes could have their genomes introduced into different leaves of a plant, thus necessitating long-distance trafficking of the viral RNAs for successful infection. To examine this possibility, individual or combinations of genome segments from the tripartite Brome mosaic virus (BMV) were transiently expressed in leaves of Nicotiana benthamiana plants using engineered Agrobacterium tumefaciens. BMV RNA3 was found to traffic from the initial site of expression to other leaves of the plant, as detected by RNA gel blot analyses and also by the expression of an endoplasmic reticulum-targeted green fluorescent protein. When RNA3 trafficked into leaves containing the BMV replication enzymes, RNA replication, transcription, and virion production were observed. RNA3 trafficking occurred even when it did not encode the movement or capsid proteins. However, coexpression of the movement protein increased the trafficking of BMV RNAs. BMV RNA1 and RNA2 could also traffic throughout the plant, but less efficiently than RNA3. All three BMV RNAs trafficked bidirectionally to sink leaves near the apical meristem as well as to the source leaves at the bottom of the stem, suggesting that trafficking used the phloem. These results demonstrate that BMV RNAs can use a replication-independent mechanism to traffic in N. benthamiana.

  7. Innate lymphotoxin receptor mediated signaling promotes HSV-1 associated neuroinflammation and viral replication

    PubMed Central

    Liang, Yong; Yang, Kaiting; Guo, Jingya; Wroblewska, Joanna; Fu, Yang-Xin; Peng, Hua

    2015-01-01

    Host anti-viral innate immunity plays important roles in the defense against HSV-1 infection. In this study, we find an unexpected role for innate LT/LIGHT signaling in promoting HSV-1 replication and virus induced inflammation in immunocompromised mice. Using a model of footpad HSV-1 infection in Rag1–/– mice, we observed that blocking LT/LIGHT signaling with LTβR-Ig could significantly delay disease progression and extend the survival of infected mice. LTβR-Ig treatment reduced late proinflammatory cytokine release in the serum and nervous tissue, and inhibited chemokine expression and inflammatory cells infiltration in the dorsal root ganglia (DRG). Intriguingly, LTβR-Ig treatment restricted HSV-1 replication in the DRG but not the footpad. These findings demonstrate a critical role for LT/LIGHT signaling in modulating innate inflammation and promoting HSV-1 replication in the nervous system, and suggest a new target for treatment of virus-induced adverse immune response and control of severe HSV-1 infection. PMID:25993659

  8. Ex Vivo and In Vivo Inhibition of Human Rhinovirus Replication by a New Pseudosubstrate of Viral 2A Protease

    PubMed Central

    Falah, Nisrine; Violot, Sébastien; Décimo, Didier; Berri, Fatma; Foucault-Grunenwald, Marie-Laure; Ohlmann, Théophile; Schuffenecker, Isabelle; Morfin, Florence; Lina, Bruno; Riteau, Béatrice

    2012-01-01

    Human rhinoviruses (HRVs) remain a significant public health problem as they are the major cause of both upper and lower respiratory tract infections. Unfortunately, to date no vaccine or antiviral against these pathogens is available. Here, using a high-throughput yeast two-hybrid screening, we identified a 6-amino-acid hit peptide, LVLQTM, which acted as a pseudosubstrate of the viral 2A cysteine protease (2Apro) and inhibited its activity. This peptide was chemically modified with a reactive electrophilic fluoromethylketone group to form a covalent linkage with the nucleophilic active-site thiol of the enzyme. Ex vivo and in vivo experiments showed that thus converted, LVLQTM was a strong inhibitor of HRV replication in both A549 cells and mice. To our knowledge, this is the first report validating a compound against HRV infection in a mouse model. PMID:22072773

  9. Early Cytokine Dysregulation and Viral Replication Are Associated with Mortality During Lethal Influenza Infection

    PubMed Central

    Vogel, Alexander J.; Harris, Seth; Marsteller, Nathan; Condon, Shirley A.

    2014-01-01

    Abstract Infection with influenza A virus (IAV) leads to acute lung injury and possibly fatal complications, especially in immunocompromised, elderly, or chronically infected individuals. Therefore, it is important to study the factors that lead to pathology and mortality in infected hosts. In this report, we analyze immune responses to infection at a sublethal (0.1 LD50) and lethal (1 LD50) dose of the highly pathogenic IAV A/Puerto Rico/8/34 (PR8). Our experiments revealed that infection with a 1 LD50 dose induced peak viral titers at day 2 compared to day 4 in the 0.1 LD50 dose. Moreover, early cytokine dysregulation was observed in the lethal dose with significantly elevated levels of IFN-α, TNF-α, CXCL9, IL-6, and MCP-1 produced at day 2. Early inflammatory responses following infection with 1 LD50 correlated with a greater influx of neutrophils into the lung. However, depletion of neutrophils enhanced morbidity following IAV infection. Though no differences in CD8+ cell function were observed, CD4+ effector responses were impaired in the lungs 8 days after infection with 1 LD50. Histological analysis revealed significant pathology in lethally infected mice at day 2 and day 6 postinfection, when viral titers remained high. Treating lethally infected mice with oseltamivir inhibited viral titers to sublethal levels, and abrogated the pathology associated with the lethal dose. Together, these results suggest that early cytokine dysregulation and viral replication play a role in pulmonary damage and high mortality in lethally infected mice. PMID:24787235

  10. 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.

  11. Coagulation, Protease Activated Receptors and Viral Myocarditis

    PubMed Central

    Antoniak, Silvio; Mackman, Nigel

    2013-01-01

    The coagulation protease cascade plays an essential role in hemostasis. In addition, a clot contributes to host defense by limiting the spread of pathogens. Coagulation proteases induce intracellular signaling by cleavage of cell surface receptors called protease-activated receptors (PARs). These receptors allow cells to sense changes in the extracellular environment, such as infection. Viruses activate the coagulation cascade by inducing tissue factor expression and by disrupting the endothelium. Virus infection of the heart can cause myocarditis, cardiac remodeling and heart failure. Recent studies using a mouse model have shown that tissue factor, thrombin and PAR-1 signaling all positively regulate the innate immune during viral myocarditis. In contrast, PAR-2 signaling was found to inhibit interferon-β expression and the innate immune response. These observations suggest that anticoagulants may impair the innate immune response to viral infection and that inhibition of PAR-2 may be a new target to reduce viral myocarditis.. PMID:24203054

  12. Binding sites for the herpes simplex virus immediate-early protein ICP4 impose an increased dependence on viral DNA replication on simple model promoters located in the viral genome.

    PubMed

    Koop, K E; Duncan, J; Smiley, J R

    1993-12-01

    We examined the ability of binding sites for the herpes simplex virus immediate-early protein ICP4 to alter the regulation of closely linked promoters by placing strong ICP4 binding sites upstream or downstream of simple TATA promoters in the intact viral genome. We found that binding sites strongly reduced the levels of expression at early times postinfection and that this effect was partially overcome after the onset of viral DNA replication. These data confirm that DNA-bound ICP4 can inhibit the activity of a closely linked promoter and raise the possibility that ICP4 binding sites contribute to temporal regulation during infection.

  13. Replication of the rotavirus genome requires an active ubiquitin-proteasome system.

    PubMed

    López, Tomás; Silva-Ayala, Daniela; López, Susana; Arias, Carlos F

    2011-11-01

    Here we show that the ubiquitin-proteasome system is required for the efficient replication of rotavirus RRV in MA104 cells. The proteasome inhibitor MG132 decreased the yield of infectious virus under conditions where it severely reduces the synthesis of not only viral but also cellular proteins. Addition of nonessential amino acids to the cell medium restored both viral protein synthesis and cellular protein synthesis, but the production of progeny viruses was still inhibited. In medium supplemented with nonessential amino acids, we showed that MG132 does not affect rotavirus entry but inhibits the replication of the viral genome. It was also shown that it prevents the efficient incorporation into viroplasms of viral polymerase VP1 and the capsid proteins VP2 and VP6, which could explain the inhibitory effect of MG132 on genome replication and infectious virus yield. We also showed that ubiquitination is relevant for rotavirus replication since the yield of rotavirus progeny in cells carrying a temperature-sensitive mutation in the E1 ubiquitin-activating enzyme was reduced at the restrictive temperature. In addition, overexpression of ubiquitin in MG132-treated MA104 cells partially reversed the effect of the inhibitor on virus yield. Altogether, these data suggest that the ubiquitin-proteasome (UP) system has a very complex interaction with the rotavirus life cycle, with both the ubiquitination and proteolytic activities of the system being relevant for virus replication.

  14. Small interfering RNAs targeting viral structural envelope protein genes and the 5ʹ-UTR inhibit replication of bovine viral diarrhea virus in MDBK cells.

    PubMed

    Mishra, N; Rajukumar, K; Kalaiyarasu, S; Behera, S P; Nema, R K; Dubey, S C

    2011-01-01

    Bovine viral diarrhea viruses (BVDVs) are important pathogens of cattle that occur worldwide, and for which no antiviral therapy is available. In the present study, the inhibitory effect of small interfering (si) RNAs on bovine viral diarrhea virus 1 (BVDV-1) replication in cultured bovine cells was explored. Four synthetic siRNAs were designed to target structural envelope region genes (Erns, E1, and E2) and one cocktail of siRNA was generated to target the 5ʹ-UTR of the BVDV-1 genome. The inhibitory effects of siRNAs were assessed by determination of infectious viral titer, viral antigen and viral RNA. The siRNA cocktail and three of the synthetic siRNAs produced moderate anti-BVDV-1 effect in vitro as shown by 25%-40% reduction in BVDV-1 antigen production, 7.9-19.9-fold reduction in viral titer and 21-48-fold reduction in BVDV-1 RNA copy number. Our findings suggest that siRNA cocktail targeted at the 5ʹ-UTR is a stronger inhibitor of BVDV-1 replication and the targets for siRNA inhibition can be extended to BVDV-1 structural envelope protein genes.

  15. A conserved amphipathic helix in the N-terminal regulatory region of the papillomavirus E1 helicase is required for efficient viral DNA replication.

    PubMed

    Morin, Geneviève; Fradet-Turcotte, Amélie; Di Lello, Paola; Bergeron-Labrecque, Fanny; Omichinski, James G; Archambault, Jacques

    2011-06-01

    The papillomavirus E1 helicase, with the help of E2, assembles at the viral origin into a double hexamer that orchestrates replication of the viral genome. The N-terminal region (NTR) of E1 is essential for DNA replication in vivo but dispensable in vitro, suggesting that it has a regulatory function. By deletion analysis, we identified a conserved region of the E1 NTR needed for efficient replication of viral DNA. This region is predicted to form an amphipathic α-helix (AH) and shows sequence similarity to portions of the p53 and herpes simplex virus (HSV) VP16 transactivation domains known as transactivation domain 2 (TAD2) and VP16C, which fold into α-helices upon binding their target proteins, including the Tfb1/p62 (Saccharomyces cerevisiae/human) subunit of general transcription factor TFIIH. By nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we found that a peptide spanning the E1 AH binds Tfb1 on the same surface as TAD2/VP16C and with a comparable affinity, suggesting that it does bind as an α-helix. Furthermore, the E1 NTRs from several human papillomavirus (HPV) types could activate transcription in yeast, and to a lesser extent in mammalian cells, when fused to a heterologous DNA-binding domain. Mutation of the three conserved hydrophobic residues in the E1 AH, analogous to those in TAD2/VP16C that directly contact their target proteins, decreased transactivation activity and, importantly, also reduced by 50% the ability of E1 to support transient replication of DNA in C33A cells, at a step following assembly of the E1-E2-ori preinitiation complex. These results demonstrate the existence of a conserved TAD2/VP16C-like AH in E1 that is required for efficient replication of viral DNA.

  16. A conserved amphipathic helix in the N-terminal regulatory region of the papillomavirus E1 helicase is required for efficient viral DNA replication.

    PubMed

    Morin, Geneviève; Fradet-Turcotte, Amélie; Di Lello, Paola; Bergeron-Labrecque, Fanny; Omichinski, James G; Archambault, Jacques

    2011-06-01

    The papillomavirus E1 helicase, with the help of E2, assembles at the viral origin into a double hexamer that orchestrates replication of the viral genome. The N-terminal region (NTR) of E1 is essential for DNA replication in vivo but dispensable in vitro, suggesting that it has a regulatory function. By deletion analysis, we identified a conserved region of the E1 NTR needed for efficient replication of viral DNA. This region is predicted to form an amphipathic α-helix (AH) and shows sequence similarity to portions of the p53 and herpes simplex virus (HSV) VP16 transactivation domains known as transactivation domain 2 (TAD2) and VP16C, which fold into α-helices upon binding their target proteins, including the Tfb1/p62 (Saccharomyces cerevisiae/human) subunit of general transcription factor TFIIH. By nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we found that a peptide spanning the E1 AH binds Tfb1 on the same surface as TAD2/VP16C and with a comparable affinity, suggesting that it does bind as an α-helix. Furthermore, the E1 NTRs from several human papillomavirus (HPV) types could activate transcription in yeast, and to a lesser extent in mammalian cells, when fused to a heterologous DNA-binding domain. Mutation of the three conserved hydrophobic residues in the E1 AH, analogous to those in TAD2/VP16C that directly contact their target proteins, decreased transactivation activity and, importantly, also reduced by 50% the ability of E1 to support transient replication of DNA in C33A cells, at a step following assembly of the E1-E2-ori preinitiation complex. These results demonstrate the existence of a conserved TAD2/VP16C-like AH in E1 that is required for efficient replication of viral DNA. PMID:21450828

  17. Production of recombinant snakehead rhabdovirus: the NV protein is not required for viral replication.

    PubMed

    Johnson, M C; Simon, B E; Kim, C H; Leong, J A

    2000-03-01

    Snakehead rhabdovirus (SHRV) affects warm water fish in Southeast Asia and belongs to the genus Novirhabdovirus by virtue of its nonvirion gene (NV). Because SHRV grows best at temperatures between 28 and 31 degrees C, we were able to use the T7 expression system to produce viable recombinant SHRV from a cloned cDNA copy of the viral genome. Expression of a positive-strand RNA copy of the 11, 550-nucleotide SHRV genome along with the viral nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins resulted in the generation of infectious SHRV in cells preinfected with a vaccinia virus vector for T7 polymerase expression. Recombinant virus production was verified by detection of a unique restriction site engineered into the SHRV genome between the NV and L genes. Since we were now able to begin examining the function of the NV gene, we constructed a recombinant virus containing a nonsense mutation located 22 codons into the coding sequence of the NV protein. The NV knockout virus was produced at a concentration as high as that of wild-type virus in cultured fish cells, and the resulting virions appeared to be identical to the wild-type virions in electron micrographs. These initial studies suggest that NV has no critical function in SHRV replication in cultured fish cells.

  18. Activation of the Antiviral Kinase PKR and Viral Countermeasures

    PubMed Central

    Dauber, Bianca; Wolff, Thorsten

    2009-01-01

    The interferon-induced double-stranded (ds)RNA-dependent protein kinase (PKR) limits viral replication by an eIF2α-mediated block of translation. Although many negative-strand RNA viruses activate PKR, the responsible RNAs have long remained elusive, as dsRNA, the canonical activator of PKR, has not been detected in cells infected with such viruses. In this review we focus on the activating RNA molecules of different virus families, in particular the negative-strand RNA viruses. We discuss the recently identified non-canonical activators 5′-triphosphate RNA and the vRNP of influenza virus and give an update on strategies of selected RNA and DNA viruses to prevent activation of PKR. PMID:21994559

  19. 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.

  20. Phosphorylation of Hepatitis C Virus RNA Polymerases Ser29 and Ser42 by Protein Kinase C-Related Kinase 2 Regulates Viral RNA Replication

    PubMed Central

    Han, Song-Hee; Kim, Seong-Jun; Kim, Eun-Jung; Kim, Tae-Eun; Moon, Jae-Su; Kim, Geon-Woo; Lee, Seung-Hoon; Cho, Kun; Yoo, Jong Shin; Son, Woo Sung; Rhee, Jin-Kyu; Han, Seung Hyun

    2014-01-01

    ABSTRACT Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the Δ1 finger loop region that interacts with the thumb subdomain of NS5B. Colony-forming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its Δ1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. IMPORTANCE While the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed

  1. Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo.

    PubMed

    Lappalainen, Suvi; Pastor, Ana Ruth; Tamminen, Kirsi; López-Guerrero, Vanessa; Esquivel-Guadarrama, Fernando; Palomares, Laura A; Vesikari, Timo; Blazevic, Vesna

    2014-01-01

    Rotavirus (RV) is a common cause of severe gastroenteritis (GE) in children worldwide. Live oral RV vaccines protect against severe RVGE, but the immune correlates of protection are not yet clearly defined. Inner capsid VP6 protein is a highly conserved, abundant, and immunogenic RV protein, and VP6-specific mucosal antibodies, especially IgA, have been implicated to protect against viral challenge in mice. In the present study systemic and mucosal IgG and IgA responses were induced by immunizing BALB/c mice intranasally with a combination of recombinant RV VP6 protein (subgroup II [SGII]) and norovirus (NoV) virus-like particles (VLPs) used in a candidate vaccine. Following immunization mice were challenged orally with murine RV strain EDIMwt (SG non-I-non-II, G3P10[16]). In order to determine neutralizing activity of fecal samples, sera, and vaginal washes (VW) against human Wa RV (SGII, G1P1A[8]) and rhesus RV (SGI, G3P5B[3]), the RV antigen production was measured with an ELISA-based antigen reduction neutralization assay. Only VWs of immunized mice inhibited replication of both RVs, indicating heterotypic protection of induced antibodies. IgA antibody depletion and blocking experiments using recombinant VP6 confirmed that neutralization was mediated by anti-VP6 IgA antibodies. Most importantly, after the RV challenge significant reduction in viral shedding was observed in feces of immunized mice. These results suggest a significant role for mucosal RV VP6-specific IgA for the inhibition of RV replication in vitro and in vivo. In addition, these results underline the importance of non-serotype-specific immunity induced by the conserved subgroup-specific RV antigen VP6 in clearance of RV infection. PMID:25424814

  2. Immune responses elicited against rotavirus middle layer protein VP6 inhibit viral replication in vitro and in vivo

    PubMed Central

    Lappalainen, Suvi; Pastor, Ana Ruth; Tamminen, Kirsi; López-Guerrero, Vanessa; Esquivel-Guadarrama, Fernando; Palomares, Laura A; Vesikari, Timo; Blazevic, Vesna

    2014-01-01

    Rotavirus (RV) is a common cause of severe gastroenteritis (GE) in children worldwide. Live oral RV vaccines protect against severe RVGE, but the immune correlates of protection are not yet clearly defined. Inner capsid VP6 protein is a highly conserved, abundant, and immunogenic RV protein, and VP6-specific mucosal antibodies, especially IgA, have been implicated to protect against viral challenge in mice. In the present study systemic and mucosal IgG and IgA responses were induced by immunizing BALB/c mice intranasally with a combination of recombinant RV VP6 protein (subgroup II [SGII]) and norovirus (NoV) virus-like particles (VLPs) used in a candidate vaccine. Following immunization mice were challenged orally with murine RV strain EDIMwt (SG non-I-non-II, G3P10[16]). In order to determine neutralizing activity of fecal samples, sera, and vaginal washes (VW) against human Wa RV (SGII, G1P1A[8]) and rhesus RV (SGI, G3P5B[3]), the RV antigen production was measured with an ELISA-based antigen reduction neutralization assay. Only VWs of immunized mice inhibited replication of both RVs, indicating heterotypic protection of induced antibodies. IgA antibody depletion and blocking experiments using recombinant VP6 confirmed that neutralization was mediated by anti-VP6 IgA antibodies. Most importantly, after the RV challenge significant reduction in viral shedding was observed in feces of immunized mice. These results suggest a significant role for mucosal RV VP6-specific IgA for the inhibition of RV replication in vitro and in vivo. In addition, these results underline the importance of non-serotype-specific immunity induced by the conserved subgroup-specific RV antigen VP6 in clearance of RV infection. PMID:25424814

  3. 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

    2010-01-01

    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. PMID:20949022

  4. Analysis of the interactions of viral and cellular factors with human cytomegalovirus lytic origin of replication, oriLyt

    PubMed Central

    Kagele, Dominique; Rossetto, Cyprian C.; Elorza, Margret; Pari, Gregory S.

    2011-01-01

    Human cytomegalovirus transient lytic DNA replication relies on the cis-acting element oriLyt, six viral-encoded core proteins, the proposed DNA replication initiator protein UL84, IE2, IRS1 and the gene products from the UL112/113 loci. In an effort to elucidate cellular and viral-encoded factors that may play a role in oriLyt-dependent replication we used DNA-affinity purification and mass spectrometry to isolate and identify several previously unknown cellular and viral factors that interact with HCMV oriLyt DNA. These proteins include the multifunctional hnRNP-K, BUB3, HMGB1, PTB-1, UL83, UL112/113, and IRS1. Chromatin immunoprecipitation (ChIP) assays confirmed an interaction of several of these factors with oriLyt. Co-immunoprecipitation experiments detected an interaction between UL84 and hnRNP-K in infected and transfected cells. Knockdown of hnRNP K expression by siRNA inhibited the amplification of oriLyt in the transient assay. Together, these data suggest a possible regulatory role in DNA replication for several previously unidentified viral and cellular factors. PMID:22236369

  5. Temporal order of evolution of DNA replication systems inferred by comparison of cellular and viral DNA polymerases

    PubMed Central

    Koonin, Eugene V

    2006-01-01

    Background The core enzymes of the DNA replication systems show striking diversity among cellular life forms and more so among viruses. In particular, and counter-intuitively, given the central role of DNA in all cells and the mechanistic uniformity of replication, the core enzymes of the replication systems of bacteria and archaea (as well as eukaryotes) are unrelated or extremely distantly related. Viruses and plasmids, in addition, possess at least two unique DNA replication systems, namely, the protein-primed and rolling circle modalities of replication. This unexpected diversity makes the origin and evolution of DNA replication systems a particularly challenging and intriguing problem in evolutionary biology. Results I propose a specific succession for the emergence of different DNA replication systems, drawing argument from the differences in their representation among viruses and other selfish replicating elements. In a striking pattern, the DNA replication systems of viruses infecting bacteria and eukaryotes are dominated by the archaeal-type B-family DNA polymerase (PolB) whereas the bacterial replicative DNA polymerase (PolC) is present only in a handful of bacteriophage genomes. There is no apparent mechanistic impediment to the involvement of the bacterial-type replication machinery in viral DNA replication. Therefore, I hypothesize that the observed, markedly unequal distribution of the replicative DNA polymerases among the known cellular and viral replication systems has a historical explanation. I propose that, among the two types of DNA replication machineries that are found in extant life forms, the archaeal-type, PolB-based system evolved first and had already given rise to a variety of diverse viruses and other selfish elements before the advent of the bacterial, PolC-based machinery. Conceivably, at that stage of evolution, the niches for DNA-viral reproduction have been already filled with viruses replicating with the help of the archaeal

  6. A herpes simplex virus scaffold peptide that binds the portal vertex inhibits early steps in viral replication.

    PubMed

    Yang, Kui; Wills, Elizabeth; Baines, Joel D

    2013-06-01

    Previous experiments identified a 12-amino-acid (aa) peptide that was sufficient to interact with the herpes simplex virus 1 (HSV-1) portal protein and was necessary to incorporate the portal into capsids. In the present study, cells were treated at various times postinfection with peptides consisting of a portion of the Drosophila antennapedia protein, previously shown to enter cells efficiently, fused to either wild-type HSV-1 scaffold peptide (YPYYPGEARGAP) or a control peptide that contained changes at positions 4 and 5. These 4-tyrosine and 5-proline residues are highly conserved in herpesvirus scaffold proteins and were previously shown to be critical for the portal interaction. Treatment early in infection with subtoxic levels of wild-type peptide reduced viral infectivity by over 1,000-fold, while the mutant peptide had little effect on viral yields. In cells infected for 3 h in the presence of wild-type peptide, capsids were observed to transit to the nuclear rim normally, as viewed by fluorescence microscopy. However, observation by electron microscopy in thin sections revealed an aberrant and significant increase of DNA-containing capsids compared to infected cells treated with the mutant peptide. Early treatment with peptide also prevented formation of viral DNA replication compartments. These data suggest that the antiviral peptide stabilizes capsids early in infection, causing retention of DNA within them, and that this activity correlates with peptide binding to the portal protein. The data are consistent with the hypothesis that the portal vertex is the conduit through which DNA is ejected to initiate infection.

  7. Geneticin Stabilizes the Open Conformation of the 5′ Region of Hepatitis C Virus RNA and Inhibits Viral Replication

    PubMed Central

    Ariza-Mateos, Ascensión; Díaz-Toledano, Rosa; Block, Timothy M.; Prieto-Vega, Samuel

    2015-01-01

    The aminoglycoside Geneticin (G418) is known to inhibit cell culture proliferation, via virus-specific mechanisms, of two different virus genera from the family Flaviviridae. Here, we tried to determine whether Geneticin can selectively alter the switching of the nucleotide 1 to 570 RNA region of hepatitis C virus (HCV) and, if so, whether this inhibits viral growth. Two structure-dependent RNases known to specifically cleave HCV RNA were tested in the presence or absence of the drug. One was the Synechocystis sp. RNase P ribozyme, which cleaves the tRNA-like domain around the AUG start codon under high-salt buffer conditions; the second was Escherichia coli RNase III, which recognizes a double-helical RNA switch element that changes the internal ribosome entry site (IRES) from a closed (C) conformation to an open (O) one. While the drug did not affect RNase P activity, it did inhibit RNase III in the micromolar range. Kinetic studies indicated that the drug favors the switch from the C to the O conformation of the IRES by stabilizing the distal double-stranded element and inhibiting further processing of the O form. We demonstrate that, because the RNA in this region is highly conserved and essential for virus survival, Geneticin inhibits HCV Jc1 NS3 expression, the release of the viral genomic RNA, and the propagation of HCV in Huh 7.5 cells. Our study highlights the crucial role of riboswitches in HCV replication and suggests the therapeutic potential of viral-RNA-targeted antivirals. PMID:26621620

  8. Sphingosine kinase 2 is a chikungunya virus host factor co-localized with the viral replication complex.

    PubMed

    Reid, St Patrick; Tritsch, Sarah R; Kota, Krishna; Chiang, Chih-Yuan; Dong, Lian; Kenny, Tara; Brueggemann, Ernest E; Ward, Michael D; Cazares, Lisa H; Bavari, Sina

    2015-10-01

    Chikungunya virus (CHIKV) is a re-emerging alphavirus which causes severe and prolonged arthralgic febrile illness. The recent global spread of the virus and lack of approved therapeutic options makes it imperative to gain greater insight into the molecular mechanisms underlying CHIKV pathogenesis, in particular host factors recruited by the virus. In the current study, we identify sphingosine kinase 2 (SK2) as a CHIKV host factor co-localized with the viral replication complex (VRC) during infection. SK2 was demonstrated to co-localize with viral RNA and nonstructural proteins. Targeted impairment of SK2 expression or function significantly inhibited CHIKV infection. Furthermore, affinity purification-mass spectrometry studies revealed that SK2 associates with a number of proteins involved in cellular gene expression specifically during viral infection, suggesting a role in replication. Collectively these results identify SK2 as a novel CHIKV host factor.

  9. The nsp2 Replicase Proteins of Murine Hepatitis Virus and Severe Acute Respiratory Syndrome Coronavirus Are Dispensable for Viral Replication

    PubMed Central

    Graham, Rachel L.; Sims, Amy C.; Brockway, Sarah M.; Baric, Ralph S.; Denison, Mark R.

    2005-01-01

    The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVΔnsp2 and SARSΔnsp2, respectively). Infectious MHVΔnsp2 and SARSΔnsp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Δnsp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVΔnsp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVΔnsp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function. PMID:16227261

  10. The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication.

    PubMed

    Graham, Rachel L; Sims, Amy C; Brockway, Sarah M; Baric, Ralph S; Denison, Mark R

    2005-11-01

    The positive-stranded RNA genome of the coronaviruses is translated from ORF1 to yield polyproteins that are proteolytically processed into intermediate and mature nonstructural proteins (nsps). Murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) polyproteins incorporate 16 protein domains (nsps), with nsp1 and nsp2 being the most variable among the coronaviruses and having no experimentally confirmed or predicted functions in replication. To determine if nsp2 is essential for viral replication, MHV and SARS-CoV genome RNA was generated with deletions of the nsp2 coding sequence (MHVDeltansp2 and SARSDeltansp2, respectively). Infectious MHVDeltansp2 and SARSDeltansp2 viruses recovered from electroporated cells had 0.5 to 1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis that was not specific for any positive-stranded RNA species. The Deltansp2 mutant viruses lacked expression of both nsp2 and an nsp2-nsp3 precursor, but cleaved the engineered chimeric nsp1-nsp3 cleavage site as efficiently as the native nsp1-nsp2 cleavage site. Replication complexes in MHVDeltansp2-infected cells lacked nsp2 but were morphologically indistinguishable from those of wild-type MHV by immunofluorescence. nsp2 expressed in cells by stable retroviral transduction was specifically recruited to viral replication complexes upon infection with MHVDeltansp2. These results demonstrate that while nsp2 of MHV and SARS-CoV is dispensable for viral replication in cell culture, deletion of the nsp2 coding sequence attenuates viral growth and RNA synthesis. These findings also provide a system for the study of determinants of nsp targeting and function. PMID:16227261

  11. Activation of DNA Damage Response Pathways during Lytic Replication of KSHV.

    PubMed

    Hollingworth, Robert; Skalka, George L; Stewart, Grant S; Hislop, Andrew D; Blackbourn, David J; Grand, Roger J

    2015-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of several human malignancies. Human tumour viruses such as KSHV are known to interact with the DNA damage response (DDR), the molecular pathways that recognise and repair lesions in cellular DNA. Here it is demonstrated that lytic reactivation of KSHV leads to activation of the ATM and DNA-PK DDR kinases resulting in phosphorylation of multiple downstream substrates. Inhibition of ATM results in the reduction of overall levels of viral replication while inhibition of DNA-PK increases activation of ATM and leads to earlier viral release. There is no activation of the ATR-CHK1 pathway following lytic replication and CHK1 phosphorylation is inhibited at later times during the lytic cycle. Despite evidence of double-strand breaks and phosphorylation of H2AX, 53BP1 foci are not consistently observed in cells containing lytic virus although RPA32 and MRE11 localise to sites of viral DNA synthesis. Activation of the DDR following KSHV lytic reactivation does not result in a G1 cell cycle block and cells are able to proceed to S-phase during the lytic cycle. KSHV appears then to selectively activate DDR pathways, modulate cell cycle progression and recruit DDR proteins to sites of viral replication during the lytic cycle.

  12. Analysis of the viral determinants underlying replication kinetics and cellular tropism of human immunodeficiency virus.

    PubMed

    Nagashunmugam, T; Velpandi, A; Otsuka, T; Cartas, M; Srinivasan, A

    1992-01-01

    Human immunodeficiency viruses (HIVs) isolated from infected individuals show genetic and biological diversity. To delineate the genetic determinants underlying specific biological characteristics such as rate of replication and cellular tropism, generation of hybrid HIV using viruses which exhibit distinct biological feature is essential. We have used three different infectious HIV proviral DNAs, designated pZ6, pHXB2 and pARV, derived from HIVZr6, HIVHTLV-IIIB and HIVSF-2 strains, respectively, to construct hybrid HIV. Proviral DNAs differed in their ability to direct the synthesis of viral particles upon transfection into cells and the viruses derived from the molecular clones exhibited different cellular tropism. Three different methods were utilized to generate hybrid HIV, including construction of hybrid proviral DNA using molecular techniques, intracellular ligation of viral DNA fragments and the homologous recombination approach. The chimeric proviral DNAs with exchanges involving only the long terminal repeat (LTR) region indicated that LTR does not exert influence on the overall level of virus production despite extensive differences in the U3 region of the LTR. Regarding the cellular tropism of HIV, the virus derived from pHXB2 productively infected CEMx174 cells. On the other hand, pARV-derived virus did not show productive infection of CEMx174 cells. The hybrid HIV containing the 3'-end of the genome from pARV and the 5'-end of the genome from pHXB2 was effective in infecting CEMx174 cells. However, the converse hybrid containing the 5'-pARV and the 3'-pHXB2 was not effective in infecting CEMx174 cells. These results suggest that differences in the genes outside of env and nef may play a role in the ability of virus to infect a certain cell type.

  13. [ANTIVIRAL ACTIVITY OF THE DIHYDROQUERCETIN DURING THE COXSACKIEVIRUS B4 REPLICATION IN VITRO].

    PubMed

    Galochkina, A V; Zarubaev, V V; Kiselev, O I; Babkin, V A; Ostroukhova, L A

    2016-01-01

    A study of the antiviral activity of antioxidants against viral infections is believed to be essential for creating complex antiviral agents. Dihydroquercetin is considered as the most active antioxidant extracted from Larix gmelinii. In this work, we present results of experiments of the antiviral properties of dihydroquercetin against a member of the family Picarnaviridae--Coxsackievirus B4 in vitro. We have estimated that dihydroquercetin reduces viral titers at 100 µg/ml concentration as compared with control of virus. We have shown using the plaque assay that CPE of virusis reduced in the presence of dihydroquercetin at 100 µg/ml. Study of the phase of viral lifecycle, in which dihydroquercetin acted, demonstrated that the highest efficacy of the antiviral therapy was reached at early stages of virus reproduction (1-3 hours post infection). These results show that dihydroquercetin has antiviralproperty against Coxsackievirus B4. This drug and other antioxidants can be tested as inhibitors of viral replication.

  14. The HIV-1 Tat Protein Has a Versatile Role in Activating Viral Transcription ▿

    PubMed Central

    Das, Atze T.; Harwig, Alex; Berkhout, Ben

    2011-01-01

    It is generally acknowledged that the Tat protein has a pivotal role in HIV-1 replication because it stimulates transcription from the viral long terminal repeat (LTR) promoter by binding to the TAR hairpin in the nascent RNA transcript. However, a multitude of additional Tat functions have been suggested. The importance of these functions is difficult to assess in replication studies with Tat-mutated HIV-1 variants because of the dominant negative effect on viral gene expression. We therefore used an HIV-1 construct that does not depend on the Tat-TAR interaction for transcription to reevaluate whether or not Tat has a second essential function in HIV-1 replication. This HIV-rtTA variant uses the incorporated Tet-On gene expression system for activation of transcription and replicates efficiently upon complete TAR deletion. Here we demonstrated that Tat inactivation does nevertheless severely inhibit replication. Upon long-term culturing, the Tat-minus HIV-rtTA variant acquired mutations in the U3 region that improved promoter activity and reestablished replication. We showed that in the absence of a functional TAR, Tat remains important for viral transcription via Sp1 sequence elements in the U3 promoter region. Substitution of these U3 sequences with nonrelated promoter elements created a virus that replicates efficiently without Tat in SupT1 T cells. These results indicate that Tat has a versatile role in transcription via TAR and U3 elements. The results also imply that Tat has no other essential function in viral replication in cultured T cells. PMID:21752913

  15. 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

  16. The lncRNA NRON modulates HIV-1 replication in a NFAT-dependent manner and is differentially regulated by early and late viral proteins.

    PubMed

    Imam, Hasan; Bano, Aalia Shahr; Patel, Paresh; Holla, Prasida; Jameel, Shahid

    2015-03-02

    A majority of the human genome is transcribed into noncoding RNAs, of which the functions of long noncoding RNAs (lncRNAs) are poorly understood. Many host proteins and RNAs have been characterized for their roles in HIV/AIDS pathogenesis, but there is only one lncRNA, NEAT1, which is shown to affect the HIV-1 life cycle. We profiled 90 disease-related lncRNAs and found NRON (noncoding repressor of Nuclear Factor of Activated T cells [NFAT]) to be one of several lncRNAs whose expression was significantly altered following HIV-1 infection. The regulation of NRON expression during the HIV-1 life cycle was complex; its levels were reduced by the early viral accessory protein Nef and increased by the late protein Vpu. Consequently, Nef and Vpu also modulated activity of the transcription factor NFAT. The knockdown of NRON enhanced HIV-1 replication through increased activity of NFAT and the viral LTR. Using siRNA-mediated NFAT knockdown, we show the effects of NRON on HIV-1 replication to be mediated by NFAT, and the viral Nef and Vpu proteins to modulate NFAT activity through their effects on NRON. These findings add the lncRNA, NRON to the vast repertoire of host factors utilized by HIV for infection and persistence.

  17. An Acute Immune Response to Middle East Respiratory Syndrome Coronavirus Replication Contributes to Viral Pathogenicity.

    PubMed

    Baseler, Laura J; Falzarano, Darryl; Scott, Dana P; Rosenke, Rebecca; Thomas, Tina; Munster, Vincent J; Feldmann, Heinz; de Wit, Emmie

    2016-03-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in a human with severe pneumonia in 2012. Since then, infections have been detected in >1500 individuals, with disease severity ranging from asymptomatic to severe, fatal pneumonia. To elucidate the pathogenesis of this virus and investigate mechanisms underlying disease severity variation in the absence of autopsy data, a rhesus macaque and common marmoset model of MERS-CoV disease were analyzed. Rhesus macaques developed mild disease, and common marmosets exhibited moderate to severe, potentially lethal, disease. Both nonhuman primate species exhibited respiratory clinical signs after inoculation, which were more severe and of longer duration in the marmosets, and developed bronchointerstitial pneumonia. In marmosets, the pneumonia was more extensive, with development of severe airway lesions. Quantitative analysis showed significantly higher levels of pulmonary neutrophil infiltration and higher amounts of pulmonary viral antigen in marmosets. Pulmonary expression of the MERS-CoV receptor, dipeptidyl peptidase 4, was similar in marmosets and macaques. These results suggest that increased virus replication and the local immune response to MERS-CoV infection likely play a role in pulmonary pathology severity. Together, the rhesus macaque and common marmoset models of MERS-CoV span the wide range of disease severity reported in MERS-CoV-infected humans, which will aid in investigating MERS-CoV disease pathogenesis. PMID:26724387

  18. The Ubiquitin-Conjugating System: Multiple Roles in Viral Replication and Infection

    PubMed Central

    Calistri, Arianna; Munegato, Denis; Carli, Ilaria; Parolin, Cristina; Palù, Giorgio

    2014-01-01

    Through the combined action of ubiquitinating and deubiquitinating enzymes, conjugation of ubiquitin to a target protein acts as a reversible post-translational modification functionally similar to phosphorylation. Indeed, ubiquitination is more and more recognized as a central process for the fine regulation of many cellular pathways. Due to their nature as obligate intracellular parasites, viruses rely on the most conserved host cell machineries for their own replication. Thus, it is not surprising that members from almost every viral family are challenged by ubiquitin mediated mechanisms in different steps of their life cycle and have evolved in order to by-pass or exploit the cellular ubiquitin conjugating system to maximize their chance to establish a successful infection. In this review we will present several examples of the complex interplay that links viruses and the ubiquitin conjugation machinery, with a special focus on the mechanisms evolved by the human immunodeficiency virus to escape from cellular restriction factors and to exit from infected cells. PMID:24805990

  19. Exercise effects on IFN-beta expression and viral replication in lung macrophages after HSV-1 infection.

    PubMed

    Kohut, M L; Davis, J M; Jackson, D A; Jani, P; Ghaffar, A; Mayer, E P; Essig, D A

    1998-12-01

    Mice exercised to fatigue and exposed to herpes simplex virus type 1 (HSV-1) exhibit greater mortality than control mice. In this study, we examined lung macrophage resistance to HSV-1 after exercise in terms of both viral replication and interferon (IFN)-beta production. We utilized the reverse transcriptase-rapid polymerase chain reaction to measure the IFN-beta mRNA content in alveolar macrophages. IFN release was measured with a bioassay, and viral replication within the macrophage was assessed by plaque titration. Exercised (Ex) mice ran on a treadmill until fatigue while control (Con) mice remained in lanes above the treadmill. After exercise, alveolar macrophages were removed and incubated with HSV-1. Alveolar macrophage IFN-beta mRNA was greater in Ex than in Con mice. Culture supernatant from infected macrophages showed a higher degree of IFN release and a higher number of infectious viral particles in Ex vs. Con mice. It is likely that the increase in IFN-beta mRNA occurs in response to a higher degree of viral replication. These results suggest that macrophages from Ex mice are less resistant to infection with HSV-1.

  20. The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms

    PubMed Central

    Jungfleisch, Jennifer; Chowdhury, Ashis; Alves-Rodrigues, Isabel; Tharun, Sundaresan; Díez, Juana

    2015-01-01

    The Lsm1-7-Pat1 complex binds to the 3′ end of cellular mRNAs and promotes 3′ end protection and 5′–3′ decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes. PMID:26092942

  1. The Lsm1-7-Pat1 complex promotes viral RNA translation and replication by differential mechanisms.

    PubMed

    Jungfleisch, Jennifer; Chowdhury, Ashis; Alves-Rodrigues, Isabel; Tharun, Sundaresan; Díez, Juana

    2015-08-01

    The Lsm1-7-Pat1 complex binds to the 3' end of cellular mRNAs and promotes 3' end protection and 5'-3' decay. Interestingly, this complex also specifically binds to cis-acting regulatory sequences of viral positive-strand RNA genomes promoting their translation and subsequent recruitment from translation to replication. Yet, how the Lsm1-7-Pat1 complex regulates these two processes remains elusive. Here, we show that Lsm1-7-Pat1 complex acts differentially in these processes. By using a collection of well-characterized lsm1 mutant alleles and a system that allows the replication of Brome mosaic virus (BMV) in yeast we show that the Lsm1-7-Pat1 complex integrity is essential for both, translation and recruitment. However, the intrinsic RNA-binding ability of the complex is only required for translation. Consistent with an RNA-binding-independent function of the Lsm1-7-Pat1 complex on BMV RNA recruitment, we show that the BMV 1a protein, the sole viral protein required for recruitment, interacts with this complex in an RNA-independent manner. Together, these results support a model wherein Lsm1-7-Pat1 complex binds consecutively to BMV RNA regulatory sequences and the 1a protein to promote viral RNA translation and later recruitment out of the host translation machinery to the viral replication complexes.

  2. DNA replication origin activation in space and time.

    PubMed

    Fragkos, Michalis; Ganier, Olivier; Coulombe, Philippe; Méchali, Marcel

    2015-06-01

    DNA replication begins with the assembly of pre-replication complexes (pre-RCs) at thousands of DNA replication origins during the G1 phase of the cell cycle. At the G1-S-phase transition, pre-RCs are converted into pre-initiation complexes, in which the replicative helicase is activated, leading to DNA unwinding and initiation of DNA synthesis. However, only a subset of origins are activated during any S phase. Recent insights into the mechanisms underlying this choice reveal how flexibility in origin usage and temporal activation are linked to chromosome structure and organization, cell growth and differentiation, and replication stress.

  3. Porcine epidemic diarrhea virus (PEDV) co-infection induced chlamydial persistence/stress does not require viral replication

    PubMed Central

    Schoborg, Robert V.; Borel, Nicole

    2014-01-01

    Chlamydiae may exist at the site of infection in an alternative replicative form, called the aberrant body (AB). ABs are produced during a viable but non-infectious developmental state termed “persistence” or “chlamydial stress.” As persistent/stressed chlamydiae: (i) may contribute to chronic inflammation observed in diseases like trachoma; and (ii) are more resistant to current anti-chlamydial drugs of choice, it is critical to better understand this developmental stage. We previously demonstrated that porcine epidemic diarrhea virus (PEDV) co-infection induced Chlamydia pecorum persistence/stress in culture. One critical characteristic of persistence/stress is that the chlamydiae remain viable and can reenter the normal developmental cycle when the stressor is removed. Thus, we hypothesized that PEDV-induced persistence would be reversible if viral replication was inhibited. Therefore, we performed time course experiments in which Vero cells were C. pecorum/PEDV infected in the presence of cycloheximide (CHX), which inhibits viral but not chlamydial protein synthesis. CHX-exposure inhibited PEDV replication, but did not inhibit induction of C. pecorum persistence at 24 h post-PEDV infection, as indicated by AB formation and reduced production of infectious EBs. Interestingly, production of infectious EBs resumed when CHX-exposed, co-infected cells were incubated 48–72 h post-PEDV co-infection. These data demonstrate that PEDV co-infection-induced chlamydial persistence/stress is reversible and suggest that this induction (i) does not require viral replication in host cells; and (ii) does not require de novo host or viral protein synthesis. These data also suggest that viral binding and/or entry may be required for this effect. Because the PEDV host cell receptor (CD13 or aminopeptidase N) stimulates cellular signaling pathways in the absence of PEDV infection, we suspect that PEDV co-infection might alter CD13 function and induce the chlamydiae to

  4. Identification of Proteins Bound to Dengue Viral RNA In Vivo Reveals New Host Proteins Important for Virus Replication

    PubMed Central

    Phillips, Stacia L.; Soderblom, Erik J.

    2016-01-01

    ABSTRACT Dengue virus is the most prevalent cause of arthropod-borne infection worldwide. Due to the limited coding capacity of the viral genome and the complexity of the viral life cycle, host cell proteins play essential roles throughout the course of viral infection. Host RNA-binding proteins mediate various aspects of virus replication through their physical interactions with viral RNA. Here we describe a technique designed to identify such interactions in the context of infected cells using UV cross-linking followed by antisense-mediated affinity purification and mass spectrometry. Using this approach, we identified interactions, several of them novel, between host proteins and dengue viral RNA in infected Huh7 cells. Most of these interactions were subsequently validated using RNA immunoprecipitation. Using small interfering RNA (siRNA)-mediated gene silencing, we showed that more than half of these host proteins are likely involved in regulating virus replication, demonstrating the utility of this method in identifying biologically relevant interactions that may not be identified using traditional in vitro approaches. PMID:26733069

  5. Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    PubMed Central

    Trabattoni, Daria; Gnudi, Federica; Ibba, Salomè V.; Saulle, Irma; Agostini, Simone; Masetti, Michela; Biasin, Mara; Rossignol, Jean-Francois; Clerici, Mario

    2016-01-01

    Nitazoxanide (Alinia®, NTZ) and tizoxanide (TIZ), its active circulating metabolite, belong to a class of agents known as thiazolides (TZD) endowed with broad anti-infective activities. TIZ and RM-4848, the active metabolite of RM-5038, were shown to stimulate innate immunity in vitro. Because natural resistance to HIV-1 infection in HIV-exposed seronegative (HESN) individuals is suggested to be associated with strong innate immune responses, we verified whether TIZ and RM-4848 could reduce the in vitro infectiousness of HIV-1. Peripheral blood mononuclear cells (PBMCs) from 20 healthy donors were infected in vitro with HIV-1BaL in the presence/absence of TIZ or RM4848. HIV-1 p24 were measured at different timepoints. The immunomodulatory abilities of TZD were evaluated by the expression of type I IFN pathway genes and the production of cytokines and chemokines. TZD drastically inhibited in vitro HIV-1 replication (>87%). This was associated with the activation of innate immune responses and with the up-regulation of several interferon-stimulated genes (ISGs), including those involved in cholesterol pathway, particularly the cholesterol-25 hydroxylase (CH25H). TZD inhibition of HIV-1 replication in vitro could be due to their ability to stimulate potent and multifaceted antiviral immune responses. These data warrant the exploration of TZD as preventive/therapeutic agent in HIV infection. PMID:27250526

  6. Involvement of the PI3K and ERK signaling pathways in largemouth bass virus-induced apoptosis and viral replication.

    PubMed

    Huang, Xiaohong; Wang, Wei; Huang, Youhua; Xu, Liwen; Qin, Qiwei

    2014-12-01

    Increased reports demonstrated that largemouth Bass, Micropterus salmoides in natural and artificial environments were always suffered from an emerging iridovirus disease, largemouth Bass virus (LMBV). However, the underlying mechanism of LMBV pathogenesis remained largely unknown. Here, we investigated the cell signaling events involved in virus induced cell death and viral replication in vitro. We found that LMBV infection in epithelioma papulosum cyprini (EPC) cells induced typical apoptosis, evidenced by the appearance of apoptotic bodies, cytochrome c release, mitochondrial membrane permeabilization (MMP) destruction and reactive oxygen species (ROS) generation. Two initiators of apoptosis, caspase-8 and caspase-9, and the executioner of apoptosis, caspase-3, were all significantly activated with the infection time, suggested that not only mitochondrion-mediated, but also death receptor-mediated apoptosis were involved in LMBV infection. Reporter gene assay showed that the promoter activity of transcription factors including p53, NF-κB, AP-1 and cAMP response element-binding protein (CREB) were decreased during LMBV infection. After treatment with different signaling pathway inhibitors, virus production were significantly suppressed by the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway and extracellular-signal-regulated kinases (ERK) signaling pathway. Furthermore, LMBV infection induced apoptosis was enhanced by PI3K inhibitor LY294002, but decreased by addition of ERK inhibitor UO126. Therefore, we speculated that apoptosis was sophisticatedly regulated by a series of cell signaling events for efficient virus propagation. Taken together, our results provided new insights into the molecular mechanism of ranavirus infection. PMID:25260912

  7. Involvement of the PI3K and ERK signaling pathways in largemouth bass virus-induced apoptosis and viral replication.

    PubMed

    Huang, Xiaohong; Wang, Wei; Huang, Youhua; Xu, Liwen; Qin, Qiwei

    2014-12-01

    Increased reports demonstrated that largemouth Bass, Micropterus salmoides in natural and artificial environments were always suffered from an emerging iridovirus disease, largemouth Bass virus (LMBV). However, the underlying mechanism of LMBV pathogenesis remained largely unknown. Here, we investigated the cell signaling events involved in virus induced cell death and viral replication in vitro. We found that LMBV infection in epithelioma papulosum cyprini (EPC) cells induced typical apoptosis, evidenced by the appearance of apoptotic bodies, cytochrome c release, mitochondrial membrane permeabilization (MMP) destruction and reactive oxygen species (ROS) generation. Two initiators of apoptosis, caspase-8 and caspase-9, and the executioner of apoptosis, caspase-3, were all significantly activated with the infection time, suggested that not only mitochondrion-mediated, but also death receptor-mediated apoptosis were involved in LMBV infection. Reporter gene assay showed that the promoter activity of transcription factors including p53, NF-κB, AP-1 and cAMP response element-binding protein (CREB) were decreased during LMBV infection. After treatment with different signaling pathway inhibitors, virus production were significantly suppressed by the inhibition of phosphatidylinositol 3-kinase (PI3K) pathway and extracellular-signal-regulated kinases (ERK) signaling pathway. Furthermore, LMBV infection induced apoptosis was enhanced by PI3K inhibitor LY294002, but decreased by addition of ERK inhibitor UO126. Therefore, we speculated that apoptosis was sophisticatedly regulated by a series of cell signaling events for efficient virus propagation. Taken together, our results provided new insights into the molecular mechanism of ranavirus infection.

  8. Viral Replication and Lung Lesions in BALB/c Mice Experimentally Inoculated with Avian Metapneumovirus Subgroup C Isolated from Chickens

    PubMed Central

    She, Ruiping; Hu, Fengjiao; Wang, Jing; Yan, Xu; Zhang, Chunyan; Liu, Shuhang; Quan, Rong; Li, Zixuan; Du, Fang; Wei, Ting; Liu, Jue

    2014-01-01

    Avian metapneumovirus (aMPV) emerged as an important respiratory pathogen causing acute respiratory tract infection in avian species. Here we used a chicken aMPV subgroup C (aMPV/C) isolate to inoculate experimentally BALB/c mice and found that the aMPV/C can efficiently replicate and persist in the lungs of mice for at least 21 days with a peak viral load at day 6 postinoculation. Lung pathological changes were characterized by increased inflammatory cells. Immunochemical assay showed the presence of viral antigens in the lungs and significant upregulation of pulmonary inflammatory cytokines and chemokines including MCP-1, MIP-1α, RANTES, IL-1β, IFN-γ, and TNF-α were detected following inoculation. These results indicate for the first time that chicken aMPV/C may replicate in the lung of mice. Whether aMPV/C has potential as zoonotic pathogen, further investigation will be required. PMID:24637582

  9. Dichloroacetate blocks aerobic glycolytic adaptation to attenuated measles virus and promotes viral replication leading to enhanced oncolysis in glioblastoma.

    PubMed

    Li, Chunyan; Meng, Gang; Su, Lei; Chen, Aiping; Xia, Mao; Xu, Chun; Yu, Decai; Jiang, Aiqin; Wei, Jiwu

    2015-01-30

    Targeting reprogrammed energy metabolism such as aerobic glycolysis is a potential strategy for cancer treatment. However, tumors exhibiting low-rate glycolysis or metabolic heterogeneity might be resistant to such treatment. We hypothesized that a therapeutic modality that drove cancer cells to high-rate glycolysis might sensitize cancer cells to interference directed against metabolic flux. In this study, we found that attenuated oncolytic measles virus Edmonston strain (MV-Edm) caused glioblastoma cells to shift to high-rate aerobic glycolysis; this adaptation was blocked by dichloroacetate (DCA), an inhibitor of glycolysis, leading to profound cell death of cancer cells but not of normal cells. DCA enhanced viral replication by mitigating mitochondrial antiviral signaling protein (MAVS)-mediated innate immune responses. In a subcutaneous glioblastoma (GBM) xenograft mouse model, low-dose MV-Edm and DCA significantly inhibited tumor growth in vivo. We found that DCA impaired glycolysis (blocking bioenergetic generation) and enhanced viral replication (increasing bioenergetic consumption), which, in combination, accelerated bioenergetic exhaustion leading to necrotic cell death. Taken together, oncolytic MV-Edm sensitized cancer cells to DCA, and in parallel, DCA promoted viral replication, thus, improving oncolysis. This novel therapeutic approach should be readily incorporated into clinical trials.

  10. Brome mosaic virus capsid protein regulates accumulation of viral replication proteins by binding to the replicase assembly RNA element.

    PubMed

    Yi, Guanghui; Letteney, Ester; Kim, Chul-Hyun; Kao, C Cheng

    2009-04-01

    Viruses provide valuable insights into the regulation of molecular processes. Brome mosaic virus (BMV) is one of the simplest entities with four viral proteins and three genomic RNAs. Here we report that the BMV capsid protein (CP), which functions in RNA encapsidation and virus trafficking, also represses viral RNA replication in a concentration-dependent manner by inhibiting the accumulation of the RNA replication proteins. Expression of the replication protein 2a in trans can partially rescue BMV RNA accumulation. A mutation in the CP can decrease the repression of translation. Translation repression by the CP requires a hairpin RNA motif named the B Box that contains seven loop nucleotides (nt) within the 5' untranslated regions (UTR) of BMV RNA1 and RNA2. Purified CP can bind directly to the B Box RNA with a K (d) of 450 nM. The secondary structure of the B Box RNA was determined to contain a highly flexible 7-nt loop using NMR spectroscopy, native gel analysis, and thermal denaturation studies. The B Box is also recognized by the BMV 1a protein to assemble the BMV replicase, suggesting that the BMV CP can act to regulate several viral infection processes.

  11. Nuclear localization of dengue virus nonstructural protein 5 does not strictly correlate with efficient viral RNA replication and inhibition of type I interferon signaling.

    PubMed

    Kumar, Anil; Bühler, Sandra; Selisko, Barbara; Davidson, Andrew; Mulder, Klaas; Canard, Bruno; Miller, Sven; Bartenschlager, Ralf

    2013-04-01

    Dengue virus (DENV) is an important human pathogen, especially in the tropical and subtropical parts of the world, causing considerable morbidity and mortality. DENV replication occurs in the cytoplasm; however, a high proportion of nonstructural protein 5 (NS5), containing methyltransferase (MTase) and RNA-dependent RNA polymerase (RdRp) activities, accumulates in the nuclei of infected cells. The present study investigates the impact of nuclear localization of NS5 on its known functions, including viral RNA replication and subversion of the type I interferon response. By using a mutation analysis approach, we identified the most critical residues within the αβ nuclear localization signal (αβNLS), which are essential for the nuclear accumulation of this protein. Although we observed an overall correlation between reduced nuclear accumulation of NS5 and impaired RNA replication, we identified one mutant with drastically reduced amounts of nuclear NS5 and virtually unaffected RNA replication, arguing that nuclear localization of NS5 does not correlate strictly with DENV replication, at least in cell culture. Because NS5 plays an important role in blocking interferon signaling via STAT-2 (signal transducer and activator of transcription 2) degradation, the abilities of the NLS mutants to block this pathway were investigated. All mutants were able to degrade STAT-2, with accordingly similar type I interferon resistance phenotypes. Since the NLS is contained within the RdRp domain, the MTase and RdRp activities of the mutants were determined by using recombinant full-length NS5. We found that the C-terminal region of the αβNLS is a critical functional element of the RdRp domain required for polymerase activity. These results indicate that efficient DENV RNA replication requires only minimal, if any, nuclear NS5, and they identify the αβNLS as a structural element required for proper RdRp activity. PMID:23408610

  12. Development of viable TAP-tagged dengue virus for investigation of host-virus interactions in viral replication.

    PubMed

    Poyomtip, Teera; Hodge, Kenneth; Matangkasombut, Ponpan; Sakuntabhai, Anavaj; Pisitkun, Trairak; Jirawatnotai, Siwanon; Chimnaronk, Sarin

    2016-03-01

    Dengue virus (DENV) is a mosquito-borne flavivirus responsible for life-threatening dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The viral replication machinery containing the core non-structural protein 5 (NS5) is implicated in severe dengue symptoms but molecular details remain obscure. To date, studies seeking to catalogue and characterize interaction networks between viral NS5 and host proteins have been limited to the yeast two-hybrid system, computational prediction and co-immunoprecipitation (IP) of ectopically expressed NS5. However, these traditional approaches do not reproduce a natural course of infection in which a number of DENV NS proteins colocalize and tightly associate during the replication process. Here, we demonstrate the development of a recombinant DENV that harbours a TAP tag in NS5 to study host-virus interactions in vivo. We show that our engineered DENV was infective in several human cell lines and that the tags were stable over multiple viral passages, suggesting negligible structural and functional disturbance of NS5. We further provide proof-of-concept for the use of rationally tagged virus by revealing a high confidence NS5 interaction network in human hepatic cells. Our analysis uncovered previously unrecognized hnRNP complexes and several low-abundance fatty acid metabolism genes, which have been implicated in the viral life cycle. This study sets a new standard for investigation of host-flavivirus interactions.

  13. The C-terminal 50 amino acid residues of dengue NS3 protein are important for NS3-NS5 interaction and viral replication.

    PubMed

    Tay, Moon Y F; Saw, Wuan Geok; Zhao, Yongqian; Chan, Kitti W K; Singh, Daljit; Chong, Yuwen; Forwood, Jade K; Ooi, Eng Eong; Grüber, Gerhard; Lescar, Julien; Luo, Dahai; Vasudevan, Subhash G

    2015-01-23

    Dengue virus multifunctional proteins NS3 protease/helicase and NS5 methyltransferase/RNA-dependent RNA polymerase form part of the viral replication complex and are involved in viral RNA genome synthesis, methylation of the 5'-cap of viral genome, and polyprotein processing among other activities. Previous studies have shown that NS5 residue Lys-330 is required for interaction between NS3 and NS5. Here, we show by competitive NS3-NS5 interaction ELISA that the NS3 peptide spanning residues 566-585 disrupts NS3-NS5 interaction but not the null-peptide bearing the N570A mutation. Small angle x-ray scattering study on NS3(172-618) helicase and covalently linked NS3(172-618)-NS5(320-341) reveals a rigid and compact formation of the latter, indicating that peptide NS5(320-341) engages in specific and discrete interaction with NS3. Significantly, NS3:Asn-570 to alanine mutation introduced into an infectious DENV2 cDNA clone did not yield detectable virus by plaque assay even though intracellular double-stranded RNA was detected by immunofluorescence. Detection of increased negative-strand RNA synthesis by real time RT-PCR for the NS3:N570A mutant suggests that NS3-NS5 interaction plays an important role in the balanced synthesis of positive- and negative-strand RNA for robust viral replication. Dengue virus infection has become a global concern, and the lack of safe vaccines or antiviral treatments urgently needs to be addressed. NS3 and NS5 are highly conserved among the four serotypes, and the protein sequence around the pinpointed amino acids from the NS3 and NS5 regions are also conserved. The identification of the functionally essential interaction between the two proteins by biochemical and reverse genetics methods paves the way for rational drug design efforts to inhibit viral RNA synthesis.

  14. Replicative homeostasis II: Influence of polymerase fidelity on RNA virus quasispecies biology: Implications for immune recognition, viral autoimmunity and other "virus receptor" diseases

    PubMed Central

    Sallie, Richard

    2005-01-01

    Much of the worlds' population is in active or imminent danger from established infectious pathogens, while sporadic and pandemic infections by these and emerging agents threaten everyone. RNA polymerases (RNApol) generate enormous genetic and consequent antigenic heterogeneity permitting both viruses and cellular pathogens to evade host defences. Thus, RNApol causes more morbidity and premature mortality than any other molecule. The extraordinary genetic heterogeneity defining viral quasispecies results from RNApol infidelity causing rapid cumulative genomic RNA mutation a process that, if uncontrolled, would cause catastrophic loss of sequence integrity and inexorable quasispecies extinction. Selective replication and replicative homeostasis, an epicyclical regulatory mechanism dynamically linking RNApol fidelity and processivity with quasispecies phenotypic diversity, modulating polymerase fidelity and, hence, controlling quasispecies behaviour, prevents this happening and also mediates immune escape. Perhaps more importantly, ineluctable generation of broad phenotypic diversity after viral RNA is translated to protein quasispecies suggests a mechanism of disease that specifically targets, and functionally disrupts, the host cell surface molecules – including hormone, lipid, cell signalling or neurotransmitter receptors – that viruses co-opt for cell entry. This mechanism – "Viral Receptor Disease (VRD)" – may explain so-called "viral autoimmunity", some classical autoimmune disorders and other diseases, including type II diabetes mellitus, and some forms of obesity. Viral receptor disease is a unifying hypothesis that may also explain some diseases with well-established, but multi-factorial and apparently unrelated aetiologies – like coronary artery and other vascular diseases – in addition to diseases like schizophrenia that are poorly understood and lack plausible, coherent, pathogenic explanations. PMID:16115320

  15. CTCF and Rad21 act as host cell restriction factors for Kaposi's sarcoma-associated herpesvirus (KSHV) lytic replication by modulating viral gene transcription.

    PubMed

    Li, Da-Jiang; Verma, Dinesh; Mosbruger, Tim; Swaminathan, Sankar

    2014-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus that causes Kaposi's sarcoma and is associated with the development of lymphoproliferative diseases. KSHV reactivation from latency and virion production is dependent on efficient transcription of over eighty lytic cycle genes and viral DNA replication. CTCF and cohesin, cellular proteins that cooperatively regulate gene expression and mediate long-range DNA interactions, have been shown to bind at specific sites in herpesvirus genomes. CTCF and cohesin regulate KSHV gene expression during latency and may also control lytic reactivation, although their role in lytic gene expression remains incompletely characterized. Here, we analyze the dynamic changes in CTCF and cohesin binding that occur during the process of KSHV viral reactivation and virion production by high resolution chromatin immunoprecipitation and deep sequencing (ChIP-Seq) and show that both proteins dissociate from viral genomes in kinetically and spatially distinct patterns. By utilizing siRNAs to specifically deplete CTCF and Rad21, a cohesin component, we demonstrate that both proteins are potent restriction factors for KSHV replication, with cohesin knockdown leading to hundred-fold increases in viral yield. High-throughput RNA sequencing was used to characterize the transcriptional effects of CTCF and cohesin depletion, and demonstrated that both proteins have complex and global effects on KSHV lytic transcription. Specifically, both proteins act as positive factors for viral transcription initially but subsequently inhibit KSHV lytic transcription, such that their net effect is to limit KSHV RNA accumulation. Cohesin is a more potent inhibitor of KSHV transcription than CTCF but both proteins are also required for efficient transcription of a subset of KSHV genes. These data reveal novel effects of CTCF and cohesin on transcription from a relatively small genome that resemble their effects on the cellular genome by acting as

  16. Molecular biology of adenovirus type 2 semipermissive infections. I. Viral growth and expression of viral replicative functions during restricted adenovirus infection.

    PubMed

    Eggerding, F A; Pierce, W C

    1986-01-15

    As an initial step toward understanding the mechanisms underlying host cell restriction of adenovirus 2 (Ad2) replication, we have studied various cell lines derived from hamster (CHO-K1), rat (CREF, NRK-49F, C-3, C-9), and mouse (3T3-Swiss) tissues to determine their degree of permissivity to Ad2 replication. For each cell line tested, the time course of Ad2 growth was determined; the yield of infectious virus, as measured by titration on HeLa cell monolayers, was reduced 3 to 5 logs. This result is independent of the multiplicity of infection at multiplicities between 4 and 100 plaque-forming units (PFU) per cell. The Western immunoblotting technique was used to quantitate the amounts of early proteins (E1A 45-54K proteins, E1B 21 and 58K proteins, E2A 72K DNA binding protein) and late structural proteins (hexon, fiber) produced during restricted infections. All cell lines expressed 72K DNA binding protein and variable levels of other early proteins. C-3, C-9, and NRK-49F cells expressed hexon as well as low, but detectable levels of fiber protein. Mouse 3T3-Swiss cells failed to synthesize any detectable levels of late structural proteins. DNA synthesis analysis indicated all rodent cell lines were capable of replicating viral DNA. A decreased rate of viral DNA synthesis was observed in CREF cells. Evidence is presented which suggests newly synthesized viral DNA is unstable in 3T3-Swiss cells.

  17. BRD4 Phosphorylation Regulates HPV E2-Mediated Viral Transcription, Origin Replication, and Cellular MMP-9 Expression.

    PubMed

    Wu, Shwu-Yuan; Nin, Dawn Sijin; Lee, A-Young; Simanski, Scott; Kodadek, Thomas; Chiang, Cheng-Ming

    2016-08-01

    Post-translational modification can modulate protein conformation and alter binding partner recruitment within gene regulatory regions. Here, we report that bromodomain-containing protein 4 (BRD4), a transcription co-factor and chromatin regulator, uses a phosphorylation-induced switch mechanism to recruit E2 protein encoded by cancer-associated human papillomavirus (HPV) to viral early gene and cellular matrix metalloproteinase-9 (MMP-9) promoters. Enhanced MMP-9 expression, induced upon keratinocyte differentiation, occurs via BRD4-dependent recruitment of active AP-1 and NF-κB to their target sequences. This is triggered by replacement of AP-1 family members JunB and JunD by c-Jun and by re-localization of NF-κB from the cytoplasm to the nucleus. In addition, BRD4 phosphorylation is critical for E2- and origin-dependent HPV DNA replication. A class of phospho-BRD4-targeting compounds, distinct from the BET bromodomain inhibitors, effectively blocks BRD4 phosphorylation-specific functions in transcription and factor recruitment. PMID:27477287

  18. Singapore grouper iridovirus-encoded semaphorin homologue (SGIV-sema) contributes to viral replication, cytoskeleton reorganization and inhibition of cellular immune responses.

    PubMed

    Yan, Yang; Cui, Huachun; Guo, Chuanyu; Wei, Jingguang; Huang, Youhua; Li, Lili; Qin, Qiwei

    2014-05-01

    Semaphorins are a large, phylogenetically conserved family of proteins that are involved in a wide range of biological processes including axonal steering, organogenesis, neoplastic transformation, as well as immune responses. In this study, a novel semaphorin homologue gene belonging to the Singapore grouper iridovirus (SGIV), ORF155R (termed SGIV-sema), was cloned and characterized. The coding region of SGIV-sema is 1728 bp in length, encoding a predicted protein with 575 aa. SGIV-sema contains a ~370 aa N-terminal Sema domain, a conserved plexin-semaphorin-integrin (PSI) domain, and an immunoglobulin (Ig)-like domain near the C terminus. SGIV-sema is an early gene product during viral infection and predominantly distributed in the cytoplasm with a speckled and clubbed pattern of appearance. Functionally, SGIV-sema could promote viral replication during SGIV infection in vitro, with no effect on the proliferation of host cells. Intriguingly, ectopically expressed SGIV-sema could alter the cytoskeletal structure of fish cells, characterized by a circumferential ring of microtubules near the nucleus and a disrupted microfilament organization. Furthermore, SGIV-sema was able to attenuate the cellular immune response, as demonstrated by decreased expression of inflammation/immune-related genes such as IL-8, IL-15, TNF-α and mediator of IRF3 activation (MITA), in SGIV-sema-expressing cells before and after SGIV infection. Ultimately, our study identified a novel, functional SGIV gene that could regulate cytoskeletal structure, immune responses and facilitate viral replication.

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

    SciTech Connect

    Sasaki, Yutaka; Kakisaka, Michinori; Chutiwitoonchai, Nopporn; Tajima, Shigeru; Hikono, Hirokazu; Saito, Takehiko; Aida, Yoko

    2014-07-18

    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 a 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.

  20. The Isomerase Active Site of Cyclophilin A Is Critical for Hepatitis C Virus Replication*

    PubMed Central

    Chatterji, Udayan; Bobardt, Michael; Selvarajah, Suganya; Yang, Feng; Tang, Hengli; Sakamoto, Noayo; Vuagniaux, Gregoire; Parkinson, Tanya; Gallay, Philippe

    2009-01-01

    Cyclosporine A and nonimmunosuppressive cyclophilin (Cyp) inhibitors such as Debio 025, NIM811, and SCY-635 block hepatitis C virus (HCV) replication in vitro. This effect was recently confirmed in HCV-infected patients where Debio 025 treatment dramatically decreased HCV viral load, suggesting that Cyps inhibitors represent a novel class of anti-HCV agents. However, it remains unclear how these compounds control HCV replication. Recent studies suggest that Cyps are important for HCV replication. However, a profound disagreement currently exists as to the respective roles of Cyp members in HCV replication. In this study, we analyzed the respective contribution of Cyp members to HCV replication by specifically knocking down their expression by both transient and stable small RNA interference. Only the CypA knockdown drastically decreased HCV replication. The re-expression of an exogenous CypA escape protein, which contains escape mutations at the small RNA interference recognition site, restored HCV replication, demonstrating the specificity for the CypA requirement. We then mutated residues that reside in the hydrophobic pocket of CypA where proline-containing peptide substrates and cyclosporine A bind and that are vital for the enzymatic or the hydrophobic pocket binding activity of CypA. Remarkably, these CypA mutants fail to restore HCV replication, suggesting for the first time that HCV exploits either the isomerase or the chaperone activity of CypA to replicate in hepatocytes and that CypA is the principal mediator of the Cyp inhibitor anti-HCV activity. Moreover, we demonstrated that the HCV NS5B polymerase associates with CypA via its enzymatic pocket. The study of the roles of Cyps in HCV replication should lead to the identification of new targets for the development of alternate anti-HCV therapies. PMID:19380579

  1. Transcription elongation regulator 1 (TCERG1) regulates competent RNA polymerase II-mediated elongation of HIV-1 transcription and facilitates efficient viral replication

    PubMed Central

    2013-01-01

    Background Control of RNA polymerase II (RNAPII) release from pausing has been proposed as a checkpoint mechanism to ensure optimal RNAPII activity, especially in large, highly regulated genes. HIV-1 gene expression is highly regulated at the level of elongation, which includes transcriptional pausing that is mediated by both viral and cellular factors. Here, we present evidence for a specific role of the elongation-related factor TCERG1 in regulating the extent of HIV-1 elongation and viral replication in vivo. Results We show that TCERG1 depletion diminishes the basal and viral Tat-activated transcription from the HIV-1 LTR. In support of a role for an elongation mechanism in the transcriptional control of HIV-1, we found that TCERG1 modifies the levels of pre-mRNAs generated at distal regions of HIV-1. Most importantly, TCERG1 directly affects the elongation rate of RNAPII transcription in vivo. Furthermore, our data demonstrate that TCERG1 regulates HIV-1 transcription by increasing the rate of RNAPII elongation through the phosphorylation of serine 2 within the carboxyl-terminal domain (CTD) of RNAPII and suggest a mechanism for the involvement of TCERG1 in relieving pausing. Finally, we show that TCERG1 is required for HIV-1 replication. Conclusions Our study reveals that TCERG1 regulates HIV-1 transcriptional elongation by increasing the elongation rate of RNAPII and phosphorylation of Ser 2 within the CTD. Based on our data, we propose a general mechanism for TCERG1 acting on genes that are regulated at the level of elongation by increasing the rate of RNAPII transcription through the phosphorylation of Ser2. In the case of HIV-1, our evidence provides the basis for further investigation of TCERG1 as a potential therapeutic target for the inhibition of HIV-1 replication PMID:24165037

  2. Influenza virus gene expression: viral RNA replication in vivo and in vitro

    SciTech Connect

    Shapiro, G.I.

    1987-01-01

    To develop an overall scheme for the control of influenza virus gene expression, single-stranded M13 DNAs specific for the various genomic segments were used to analyze the synthesis of virus-specific RNAs in infected cells. The results showed that virus infection is divided into two distinct phases. During the early phase, the syntheses of specific virion RNAs (vRNAs), viral mRNAs, and viral proteins were coupled. This phase lasted for 2.5 hours in BHK-21 cells, the time when the rate of synthesis of all the viral mRNAs was maximal. During the late phase, the synthesis of all the vRNAs remained at or near maximum, whereas the rate of synthesis of all the viral mRNAs declined dramatically. Viral mRNA and protein syntheses were also not coupled, as the synthesis of all the viral proteins continued at maximum levels, indicating that protein synthesis during this phase was directed principally by previously synthesized viral mRNAs. Pulses with (/sup 3/H)uridine and nonaqueous fractionation of cells were used to show that influenza vRNA, like viral mRNAs, are synthesized in the nucleus and efficiently transported to the cytoplasm. In contrast, the full-length transcripts of the vRNAs, the templates for new vRNA synthesis, were synthesized only at early times, and remained sequestered in the nucleus to direct vRNA synthesis throughout infection.

  3. Viral precursor protein P3 and its processed products perform discrete and essential functions in the poliovirus RNA replication complex

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The differential use of protein precursors and their products is a key strategy used during poliovirus replication. To characterize the role of protein precursors during replication, we examined the complementation profiles of mutants that inhibited 3D polymerase or 3C-RNA binding activity. We showe...

  4. Potent and selective inhibition of human immunodeficiency virus (HIV)-1 and HIV-2 replication by a class of bicyclams interacting with a viral uncoating event.

    PubMed Central

    De Clercq, E; Yamamoto, N; Pauwels, R; Baba, M; Schols, D; Nakashima, H; Balzarini, J; Debyser, Z; Murrer, B A; Schwartz, D

    1992-01-01

    A series of bicyclams have been shown to be potent and selective inhibitors of human immunodeficiency virus (HIV). The compounds are inhibitory to the replication of various HIV-1 and HIV-2 strains in various human T-cell systems, including peripheral blood lymphocytes, at 0.14-1.4 microM, without being toxic to the host cells at 2.2 mM. The bicyclam JM2763 is active against 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant HIV-1 strains and acts additively with AZT. Mechanism of action studies revealed that the bicyclams (i.e., JM2763) interact with an early event of the retrovirus replicative cycle, which could be tentatively identified as a viral uncoating event. Images PMID:1608936

  5. 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.

  6. Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells

    PubMed Central

    Dixit, Updesh; Pandey, Ashutosh K.; Mishra, Priya; Sengupta, Amitabha; Pandey, Virendra N.

    2016-01-01

    Persistent hepatitis C virus (HCV) infection leads to chronic hepatitis C (CHC), which often progresses to liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The molecular mechanisms that establish CHC and cause its subsequent development into LC and HCC are poorly understood. We have identified a cytoplasmic double-stranded RNA binding protein, Stau1, which is crucial for HCV replication. In this study, Stau1 specifically interacted with the variable-stem-loop region in the 3′ NTR and domain IIId of the HCV-IRES in the 5′ NTR, and promoted HCV replication and translation. Stau1 coimmunoprecipitates HCV NS5B and a cell factor, protein kinase R (PKR), which is critical for interferon-induced cellular antiviral and antiproliferative responses. Like Stau1, PKR displayed binding specificity to domain IIId of HCV-IRES. Stau1 binds to PKR and strongly inhibits PKR-autophosphorylation. We demonstrated that the transport of HCV RNA on the polysomes is Stau1-dependent, being mainly localized in the monosome fractions when Stau1 is downregulated and exclusively localized in the polysomes when Stau1 is overexpressed. Our findings suggest that HCV may appropriate Stau1 to its advantage to prevent PKR-mediated inhibition of eIF2α, which is required for the synthesis of HCV proteins for translocation of viral RNA genome to the polysomes for efficient translation and replication. PMID:27106056

  7. Secondary mutations in viruses resistant to HIV-1 integrase inhibitors that restore viral infectivity and replication kinetics.

    PubMed

    Nakahara, Koichiro; Wakasa-Morimoto, Chiaki; Kobayashi, Masanori; Miki, Shigeru; Noshi, Takeshi; Seki, Takahiro; Kanamori-Koyama, Mikiko; Kawauchi, Shinobu; Suyama, Akemi; Fujishita, Toshio; Yoshinaga, Tomokazu; Garvey, Edward P; Johns, Brian A; Foster, Scott A; Underwood, Mark R; Sato, Akihiko; Fujiwara, Tamio

    2009-02-01

    Passage of HIV-1 in the presence of integrase inhibitors (INIs) generates resistant viruses that have mutations in the integrase region. Integrase-resistant mutations Q148K and Q148R were identified as primary mutations with the passage of HIV-1 IIIB in the presence of INIs S-1360 or S/GSK-364735, respectively. Secondary amino acid substitutions E138K or G140S were observed when passage with INI was continued. The role of these mutations was investigated with molecular clones. Relative to Q148K alone, Q148K/E138K had 2- and >6-fold increases in resistance to S-1360 and S/GSK-364735, respectively, and the double mutant had slightly better infectivity and replication kinetics. In contrast, Q148K/G140S and Q148R/E138K had nearly equivalent or slightly reduced fold resistance to the INI compared with their respective Q148 primary mutants, and had increases in infectivity and replication kinetics. Recovery of these surrogates of viral fitness coincided with the recovery of integration efficiency of viral DNA into the host cell chromosome for these double mutants. These data show that recovery of viral integration efficiency can be an important factor for the emergence and maintenance of INI-resistant mutations.

  8. An insulin-like growth factor homologue of Singapore grouper iridovirus modulates cell proliferation, apoptosis and enhances viral replication.

    PubMed

    Yan, Yang; Cui, Huachun; Guo, Chuanyu; Li, Jun; Huang, Xiaohong; Wei, Jingguang; Qin, Qiwei

    2013-12-01

    Insulin-like growth factors (IGFs) play crucial roles in regulating cell differentiation, proliferation and apoptosis. In this study, a novel IGF homologue gene (IGF-like) encoded by Singapore grouper iridovirus (SGIV) ORF062R (termed SGIV-IGF), was cloned and characterized. The coding region of SGIV-IGF is 771 bp in length, with a variable number of tandem repeats (VNTR) locus at the 3'-end. We cloned one isoform of this novel gene, 582 bp in length, containing the predicted IGF domain and 3.6 copy numbers of the 27 bp repeat unit. SGIV-IGF was an early transcribed gene during viral infection, and SGIV-IGF was distributed predominantly in the cytoplasm with a diffused granular appearance. Intriguingly, overexpression of SGIV-IGF was able to promote the growth of grouper embryonic cells (GP cells) by promoting G1/S phase transition, which was at least partially dependent on its 3'-end VNTR locus. Furthermore, viral titre assay and real-time quantitative PCR (RT-qPCR) analysis proved that SGIV-IGF could promote SGIV replication in grouper cells. In addition, overexpression of SGIV-IGF mildly facilitated apoptosis in SGIV-infected non-host fathead minnow (FHM) cells. Together, our study demonstrated a novel functional gene of SGIV which may regulate viral replication and cellular processes through multiple mechanisms that appear to be cell type-dependent.

  9. Pur-Alpha Induces JCV Gene Expression and Viral Replication by Suppressing SRSF1 in Glial Cells

    PubMed Central

    Sariyer, Ilker Kudret; Sariyer, Rahsan; Otte, Jessica; Gordon, Jennifer

    2016-01-01

    Objective PML is a rare and fatal demyelinating disease of the CNS caused by the human polyomavirus, JC virus (JCV), which occurs in AIDS patients and those on immunosuppressive monoclonal antibody therapies (mAbs). We sought to identify mechanisms that could stimulate reactivation of JCV in a cell culture model system and targeted pathways which could affect early gene transcription and JCV T-antigen production, which are key steps of the viral life cycle for blocking reactivation of JCV. Two important regulatory partners we have previously identified for T-antigen include Pur-alpha and SRSF1 (SF2/ASF). SRSF1, an alternative splicing factor, is a potential regulator of JCV whose overexpression in glial cells strongly suppresses viral gene expression and replication. Pur-alpha has been most extensively characterized as a sequence-specific DNA- and RNA-binding protein which directs both viral gene transcription and mRNA translation, and is a potent inducer of the JCV early promoter through binding to T-antigen. Methods and Results Pur-alpha and SRSF1 both act directly as transcriptional regulators of the JCV promoter and here we have observed that Pur-alpha is capable of ameliorating SRSF1-mediated suppression of JCV gene expression and viral replication. Interestingly, Pur-alpha exerted its effect by suppressing SRSF1 at both the protein and mRNA levels in glial cells suggesting this effect can occur independent of T-antigen. Pur-alpha and SRSF1 were both localized to oligodendrocyte inclusion bodies by immunohistochemistry in brain sections from patients with HIV-1 associated PML. Interestingly, inclusion bodies were typically positive for either Pur-alpha or SRSF1, though some cells appeared to be positive for both proteins. Conclusions Taken together, these results indicate the presence of an antagonistic interaction between these two proteins in regulating of JCV gene expression and viral replication and suggests that they play an important role during viral

  10. Viral replication in excised fin tissues (VREFT) corresponds with prior exposure of Pacific herring, Clupea pallasii (Valenciennes), to viral haemorrhagic septicaemia virus (VHSV)

    USGS Publications Warehouse

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

    2011-01-01

    Procedures for a viral replication in excised fin tissue (VREFT) assay were adapted to Pacific herring, Clupea pallasii, and optimized both to reduce processing time and to provide the greatest resolution between na??ve herring and those previously exposed to viral haemorrhagic septicaemia virus (VHSV), Genogroup IVa. The optimized procedures included removal of the left pectoral fin from a euthanized fish, inoculation of the fin with >105 plaque-forming units (PFU) mL-1 VHSV for 1 h, rinsing the fin in fresh medium six times to remove unadsorbed virions, incubation of the fin in fresh medium for 4 days and enumeration of the viral titre in a sample of the incubation medium by plaque assay. The optimized VREFT assay was effective at identifying the prior exposure history of laboratory-reared Pacific herring to VHSV. The geometric mean VREFT value was significantly greater (P < 0.01) among na??ve herring (1.2 ?? 103 PFU mL-1) than among groups that survived exposure to VHSV (1.0-2.9 ?? 102 PFU mL-1); additionally, the proportion of cultures with no detectable virus was significantly greater (P = 0.0002) among fish that survived exposure to VHSV (39-47%) than among na??ve fish (3.3%). The optimized VREFT assay demonstrates promise for identifying VHSV exposure history and forecasting disease potential in populations of wild Pacific herring. ?? 2010 Blackwell Publishing Ltd.

  11. Human cytomegalovirus transcriptome activity differs during replication in human fibroblast, epithelial and astrocyte cell lines

    PubMed Central

    Towler, James C.; Ebrahimi, Bahram; Lane, Brian; Davison, Andrew J.

    2012-01-01

    Broad cell tropism contributes to the pathogenesis of human cytomegalovirus (HCMV), but the extent to which cell type influences HCMV gene expression is unclear. A bespoke HCMV DNA microarray was used to monitor the transcriptome activity of the low passage Merlin strain of HCMV at 12, 24, 48 and 72 h post-infection, during a single round of replication in human fetal foreskin fibroblast cells (HFFF-2s), human retinal pigmented epithelial cells (RPE-1s) and human astrocytoma cells (U373MGs). In order to correlate transcriptome activity with concurrent biological responses, viral cytopathic effect, growth kinetics and genomic loads were examined in the three cell types. The temporal expression pattern of viral genes was broadly similar in HFFF-2s and RPE-1s, but dramatically different in U373MGs. Of the 165 known HCMV protein-coding genes, 41 and 48 were differentially regulated in RPE-1s and U373MGs, respectively, compared with HFFF-2s, and 22 of these were differentially regulated in both RPE-1s and U373MGs. In RPE-1s, all differentially regulated genes were downregulated, but, in U373MGs, some were down- and others upregulated. Differentially regulated genes were identified among the immediate-early, early, early late and true-late viral gene classes. Grouping of downregulated genes according to function at landmark stages of the replication cycle led to the identification of potential bottleneck stages (genome replication, virion assembly, and virion maturation and release) that may account for cell type-dependent viral growth kinetics. The possibility that cell type-specific differences in expressed cellular factors are responsible for modulation of viral gene expression is discussed. PMID:22258857

  12. Ebselen Inhibits Hepatitis C Virus NS3 Helicase Binding to Nucleic Acid and Prevents Viral Replication

    PubMed Central

    2015-01-01

    The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is both a protease, which cleaves viral and host proteins, and a helicase that separates nucleic acid strands, using ATP hydrolysis to fuel the reaction. Many antiviral drugs, and compounds in clinical trials, target the NS3 protease, but few helicase inhibitors that function as antivirals have been reported. This study focuses on the analysis of the mechanism by which ebselen (2-phenyl-1,2-benzisoselenazol-3-one), a compound previously shown to be a HCV antiviral agent, inhibits the NS3 helicase. Ebselen inhibited the abilities of NS3 to unwind nucleic acids, to bind nucleic acids, and to hydrolyze ATP, and about 1 μM ebselen was sufficient to inhibit each of these activities by 50%. However, ebselen had no effect on the activity of the NS3 protease, even at 100 times higher ebselen concentrations. At concentrations below 10 μM, the ability of ebselen to inhibit HCV helicase was reversible, but prolonged incubation of HCV helicase with higher ebselen concentrations led to irreversible inhibition and the formation of covalent adducts between ebselen and all 14 cysteines present in HCV helicase. Ebselen analogues with sulfur replacing the selenium were just as potent HCV helicase inhibitors as ebselen, but the length of the linker between the phenyl and benzisoselenazol rings was critical. Modifications of the phenyl ring also affected compound potency over 30-fold, and ebselen was a far more potent helicase inhibitor than other, structurally unrelated, thiol-modifying agents. Ebselen analogues were also more effective antiviral agents, and they were less toxic to hepatocytes than ebselen. Although the above structure–activity relationship studies suggest that ebselen targets a specific site on NS3, we were unable to confirm binding to either the NS3 ATP binding site or nucleic acid binding cleft by examining the effects of ebselen on NS3 proteins lacking key cysteines. PMID:25126694

  13. Vesicular stomatitis virus in Drosophila melanogaster cells: regulation of viral transcription and replication.

    PubMed Central

    Blondel, D; Petitjean, A M; Dezélée, S; Wyers, F

    1988-01-01

    Vesicular stomatitis virus RNA synthesis was investigated during the establishment of persistent infection in Drosophila melanogaster cells. The transcription rate declined as early as 5 h after infection and was strongly inhibited after 7 h, leading to a decrease in viral mRNA levels and in viral protein synthesis rates. Full-length plus-strand antigenomes and minus-strand genomes were detected after a 3-h lag time and accumulated until 15 h after infection. Short encapsidated plus-strand molecules were also generated corresponding to the 5' end of viral defective antigenomes. Assembly and release of virions were not restricted, but their infectivity was extremely reduced. In persistently infected cells, an equilibrium was reached where the level of intracellular genomes maintained was constant and maximal even after the rate of all viral syntheses had decreased. These results are discussed with regard to the establishment of persistent infection. Images PMID:2824851

  14. Foot-and-mouth disease virus virulence in cattle is co-determined by viral replication dynamics and route of infection.

    PubMed

    Arzt, Jonathan; Pacheco, Juan M; Smoliga, George R; Tucker, Meghan T; Bishop, Elizabeth; Pauszek, Steven J; Hartwig, Ethan J; de los Santos, Teresa; Rodriguez, Luis L

    2014-03-01

    Early events in the pathogenesis of foot-and-mouth disease virus (FMDV) infection in cattle were investigated through aerosol and intraepithelial lingual (IEL) inoculations of a cDNA-derived FMDV-A24 wild type virus (FMDV-WT) or a mutant derived from the same clone (FMDV-Mut). After aerosolization of FMDV-WT, primary infection sites had significantly greater quantities of FMDV, viral RNA, and type I/III interferon (IFN) activity compared to corresponding tissues from cattle infected with FMDV-Mut. Additionally, FMDV-WT-infected cattle had marked induction of systemic IFN activity in serum. In contrast, FMDV-Mut aerosol-infected cattle did not manifest systemic IFN response nor had viremia. Interestingly, IEL inoculation of FMDV-Mut in cattle restored the virulent phenotype and systemic IFN response. These data indicate that the attenuated phenotype in cattle is associated with decreased replicative efficiency, reflected by decreased innate response. However, attenuation is abrogated by bypassing the common primary infection sites, inducing accelerated viral replication at the inoculation site.

  15. Autophagy Activated by Bluetongue Virus Infection Plays a Positive Role in Its Replication.

    PubMed

    Lv, Shuang; Xu, Qingyuan; Sun, Encheng; Yang, Tao; Li, Junping; Feng, Yufei; Zhang, Qin; Wang, Haixiu; Zhang, Jikai; Wu, Donglai

    2015-08-01

    Bluetongue virus (BTV) is an important pathogen of wild and domestic ruminants. Despite extensive study in recent decades, the interplay between BTV and host cells is not clearly understood. Autophagy as a cellular adaptive response plays a part in many viral infections. In our study, we found that BTV1 infection triggers the complete autophagic process in host cells, as demonstrated by the appearance of obvious double-membrane autophagosome-like vesicles, GFP-LC3 dots accumulation, the conversion of LC3-I to LC3-II and increased levels of autophagic flux in BSR cells (baby hamster kidney cell clones) and primary lamb lingual epithelial cells upon BTV1 infection. Moreover, the results of a UV-inactivated BTV1 infection assay suggested that the induction of autophagy was dependent on BTV1 replication. Therefore, we investigated the role of autophagy in BTV1 replication. The inhibition of autophagy by pharmacological inhibitors (3-MA, CQ) and RNA interference (siBeclin1) significantly decreased viral protein synthesis and virus yields. In contrast, treating BSR cells with rapamycin, an inducer of autophagy, promoted viral protein expression and the production of infectious BTV1. These findings lead us to conclude that autophagy is activated by BTV1 and contributes to its replication, and provide novel insights into BTV-host interactions.

  16. Simian virus 40 large T-antigen point mutants that are defective in viral DNA replication but competent in oncogenic transformation.

    PubMed Central

    Manos, M M; Gluzman, Y

    1984-01-01

    The large T antigen of simian virus 40 (SV40) is a multifunctional protein that is essential in both the virus lytic cycle and the oncogenic transformation of cells by SV40. To investigate the role of the numerous biochemical and physiological activities of T antigen in the lytic and transformation processes, we have studied DNA replication-deficient, transformation-competent large T-antigen mutants. Here we describe the genetic and biochemical analyses of two such mutants, C2/SV40 and C11/SV40. The mutants were isolated by rescuing the integrated SV40 DNA from C2 and C11 cells (CV-1 cell lines transformed with UV-irradiated SV40). The mutant viral early regions were cloned into the plasmid vector pK1 to generate pC2 and pC11. The mutations that are responsible for the deficiency in viral DNA replication were localized by marker rescue. Subsequent DNA sequencing revealed point mutations that predict amino acid substitutions in the carboxyl third of the protein in both mutants. The pC2 mutation predicts the change of Lys----Arg at amino acid 516. pC11 has two mutations, one predicting a change of Pro----Ser at residue 522, and another predicting a Pro----Arg change at amino acid 549. The two C11 mutations were separated from each other to form two distinct viral genomes in pC11A and pC11B. pC2, pC11, pC11A, and pC11B are able to transform both primary and established rodent cell cultures. The C11 and C11A T antigens are defective in ATPase activity, suggesting that wild-type levels of ATPase activity are not necessary for the oncogenic transformation of cells by T antigen. Images PMID:6330530

  17. Inhibition of respiratory syncytial virus replication and virus-induced p38 kinase activity by berberine.

    PubMed

    Shin, Han-Bo; Choi, Myung-Soo; Yi, Chae-Min; Lee, Jun; Kim, Nam-Jung; Inn, Kyung-Soo

    2015-07-01

    Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection and poses a major public health threat worldwide. No effective vaccines or therapeutics are currently available; berberine, an isoquinoline alkaloid from various medicinal plants, has been shown to exert antiviral and several other biological effects. Recent studies have shown that p38 mitogen-activated protein kinase (MAPK) activity is implicated in infection by and replication of viruses such as RSV and the influenza virus. Because berberine has previously been implicated in modulating the activity of p38 MAPK, its effects on RSV infection and RSV-mediated p38 MAPK activation were examined. Replication of RSV in epithelial cells was significantly reduced by treatment with berberine. Berberine treatment caused decrease in viral protein and mRNA syntheses. Similar to previously reported findings, RSV infection caused phosphorylation of p38 MAPK at a very early time point of infection, and phosphorylation was dramatically reduced by berberine treatment. In addition, production of interleukin-6 mRNA upon RSV infection was significantly suppressed by treatment with berberine, suggesting the anti-inflammatory role of berberine during RSV infection. Taken together, we showed that berberine, a natural compound already proven to be safe for human consumption, suppresses the replication of RSV. In addition, the current study suggests that inhibition of RSV-mediated early p38 MAPK activation, which has been implicated as an early step in viral infection, as a potential molecular mechanism.

  18. NCOA4 transcriptional coactivator inhibits activation of DNA replication origins.

    PubMed

    Bellelli, Roberto; Castellone, Maria Domenica; Guida, Teresa; Limongello, Roberto; Dathan, Nina Alayne; Merolla, Francesco; Cirafici, Anna Maria; Affuso, Andrea; Masai, Hisao; Costanzo, Vincenzo; Grieco, Domenico; Fusco, Alfredo; Santoro, Massimo; Carlomagno, Francesca

    2014-07-01

    NCOA4 is a transcriptional coactivator of nuclear hormone receptors that undergoes gene rearrangement in human cancer. By combining studies in Xenopus laevis egg extracts and mouse embryonic fibroblasts (MEFs), we show here that NCOA4 is a minichromosome maintenance 7 (MCM7)-interacting protein that is able to control DNA replication. Depletion-reconstitution experiments in Xenopus laevis egg extracts indicate that NCOA4 acts as an inhibitor of DNA replication origin activation by regulating CMG (CDC45/MCM2-7/GINS) helicase. NCOA4(-/-) MEFs display unscheduled origin activation and reduced interorigin distance; this results in replication stress, as shown by the presence of fork stalling, reduction of fork speed, and premature senescence. Together, our findings indicate that NCOA4 acts as a regulator of DNA replication origins that helps prevent inappropriate DNA synthesis and replication stress.

  19. Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity

    PubMed Central

    Jittavisutthikul, Surasak; Seesuay, Watee; Thanongsaksrikul, Jeeraphong; Thueng-in, Kanyarat; Srimanote, Potjanee; Werner, Rolf G.; Chaicumpa, Wanpen

    2016-01-01

    A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV-RNA-transfected Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV-RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies-treated transfected cells also had up-expression of the genes coding for the host innate immune response, including TRIF, TRAF3, IRF3, IL-28B, and IFN-β. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease, including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc-binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities, leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins. PMID:27617013

  20. Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity

    PubMed Central

    Jittavisutthikul, Surasak; Seesuay, Watee; Thanongsaksrikul, Jeeraphong; Thueng-in, Kanyarat; Srimanote, Potjanee; Werner, Rolf G.; Chaicumpa, Wanpen

    2016-01-01

    A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV-RNA-transfected Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV-RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies-treated transfected cells also had up-expression of the genes coding for the host innate immune response, including TRIF, TRAF3, IRF3, IL-28B, and IFN-β. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease, including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc-binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities, leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins.

  1. Human Transbodies to HCV NS3/4A Protease Inhibit Viral Replication and Restore Host Innate Immunity.

    PubMed

    Jittavisutthikul, Surasak; Seesuay, Watee; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Srimanote, Potjanee; Werner, Rolf G; Chaicumpa, Wanpen

    2016-01-01

    A safe and effective direct acting anti-hepatitis C virus (HCV) agent is still needed. In this study, human single chain variable fragments of antibody (scFvs) that bound to HCV NS3/4A protein were produced by phage display technology. The engineered scFvs were linked to nonaarginines (R9) for making them cell penetrable. HCV-RNA-transfected Huh7 cells treated with the transbodies produced from four different transformed E. coli clones had reduced HCV-RNA inside the cells and in the cell spent media, as well as fewer HCV foci in the cell monolayer compared to the transfected cells in culture medium alone. The transbodies-treated transfected cells also had up-expression of the genes coding for the host innate immune response, including TRIF, TRAF3, IRF3, IL-28B, and IFN-β. Computerized homology modeling and intermolecular docking predicted that the effective transbodies interacted with several critical residues of the NS3/4A protease, including those that form catalytic triads, oxyanion loop, and S1 and S6 pockets, as well as a zinc-binding site. Although insight into molecular mechanisms of the transbodies need further laboratory investigation, it can be deduced from the current data that the transbodies blocked the HCV NS3/4A protease activities, leading to the HCV replication inhibition and restoration of the virally suppressed host innate immunity. The engineered antibodies should be tested further for treatment of HCV infection either alone, in combination with current therapeutics, or in a mixture with their cognates specific to other HCV proteins. PMID:27617013

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

    PubMed

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

    2014-03-01

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

  3. Profiling Kinase Activity during Hepatitis C Virus Replication Using a Wortmannin Probe.

    PubMed

    Desrochers, Geneviève F; Sherratt, Allison R; Blais, David R; Nasheri, Neda; Ning, Zhibin; Figeys, Daniel; Goto, Natalie K; Pezacki, John Paul

    2015-09-11

    To complete its life cycle, the hepatitis C virus (HCV) induces changes to numerous aspects of its host cell. As kinases act as regulators of many pathways utilized by HCV, they are likely enzyme targets for virally induced inhibition or activation. Herein, we used activity-based protein profiling (ABPP), which allows for the identification of active enzymes in complex protein samples and the quantification of their activity, to identify kinases that displayed differential activity in HCV-expressing cells. We utilized an ABPP probe, wortmannin-yne, based on the kinase inhibitor wortmannin, which contains a pendant alkyne group for bioconjugation using bioorthogonal chemistry. We observed changes in the activity of kinases involved in the mitogen-activated protein kinase pathway, apoptosis pathways, and cell cycle control. These results establish changes to the active kinome, as reported by wortmannin-yne, in the proteome of human hepatoma cells actively replicating HCV. The observed changes include kinase activity that affect viral entry, replication, assembly, and secretion, implying that HCV is regulating the pathways that it uses for its life cycle through modulation of the active kinome. PMID:27617927

  4. The absence of IDO upregulates type I IFN production, resulting in suppression of viral replication in the retrovirus-infected mouse.

    PubMed

    Hoshi, Masato; Saito, Kuniaki; Hara, Akira; Taguchi, Ayako; Ohtaki, Hirofumi; Tanaka, Ryo; Fujigaki, Hidetsugu; Osawa, Yosuke; Takemura, Masao; Matsunami, Hidetoshi; Ito, Hiroyasu; Seishima, Mitsuru

    2010-09-15

    Indoleamine 2,3-dioxygenase, the L-tryptophan-degrading enzyme, plays a key role in the powerful immunomodulatory effects on several different types of cells. Because modulation of IDO activities after viral infection may have great impact on disease progression, we investigated the role of IDO following infection with LP-BM5 murine leukemia virus. We found suppressed BM5 provirus copies and increased type I IFNs in the spleen from IDO knockout (IDO(-/-)) and 1-methyl-D-L-tryptophan-treated mice compared with those from wild-type (WT) mice. Additionally, the number of plasmacytoid dendritic cells in IDO(-/-) mice was higher in the former than in the WT mice. In addition, neutralization of type I IFNs in IDO(-/-) mice resulted in an increase in LP-BM5 viral replication. Moreover, the survival rate of IDO(-/-) mice or 1-methyl-D-L-tryptophan-treated mice infected with LP-BM5 alone or with both Toxoplasma gondii and LP-BM5 was clearly greater than the survival rate of WT mice. To our knowledge, the present study is the first report to observe suppressed virus replication with upregulated type I IFN in IDO(-/-) mice, suggesting that modulation of the IDO pathway may be an effective strategy for treatment of virus infection.

  5. Influenza A Virus-Induced Degradation of Eukaryotic Translation Initiation Factor 4B Contributes to Viral Replication by Suppressing IFITM3 Protein Expression

    PubMed Central

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

    2014-01-01

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

  6. Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

    SciTech Connect

    Kohio, Hinissan P.; Adamson, Amy L.

    2013-09-15

    As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.

  7. Activation of mammalian Chk1 during DNA replication arrest

    PubMed Central

    Feijoo, Carmen; Hall-Jackson, Clare; Wu, Rong; Jenkins, David; Leitch, Jane; Gilbert, David M.; Smythe, Carl

    2001-01-01

    Checkpoints maintain order and fidelity in the cell cycle by blocking late-occurring events when earlier events are improperly executed. Here we describe evidence for the participation of Chk1 in an intra-S phase checkpoint in mammalian cells. We show that both Chk1 and Chk2 are phosphorylated and activated in a caffeine-sensitive signaling pathway during S phase, but only in response to replication blocks, not during normal S phase progression. Replication block–induced activation of Chk1 and Chk2 occurs normally in ataxia telangiectasia (AT) cells, which are deficient in the S phase response to ionizing radiation (IR). Resumption of synthesis after removal of replication blocks correlates with the inactivation of Chk1 but not Chk2. Using a selective small molecule inhibitor, cells lacking Chk1 function show a progressive change in the global pattern of replication origin firing in the absence of any DNA replication. Thus, Chk1 is apparently necessary for an intra-S phase checkpoint, ensuring that activation of late replication origins is blocked and arrested replication fork integrity is maintained when DNA synthesis is inhibited. PMID:11535615

  8. Viral activation and recruitment of metacaspases in the unicellular coccolithophore, Emiliania huxleyi.

    PubMed

    Bidle, Kay D; Haramaty, Liti; Barcelos E Ramos, Joana; Falkowski, Paul

    2007-04-01

    Lytic viral infection and programmed cell death (PCD) are thought to represent two distinct death mechanisms in phytoplankton, unicellular photoautotrophs that drift with ocean currents. Here, we demonstrate an interaction between autocatalytic PCD and lytic viral infection in the cosmopolitan coccolithophorid, Emiliania huxleyi. Successful infection of E. huxleyi strain 374 with a lytic virus, EhV1, resulted in rapid internal degradation of cellular components, a dramatic reduction in the photosynthetic efficiency (F(v)/F(m)), and an up-regulation of metacaspase protein expression, concomitant with induction of caspase-like activity. Caspase activation was confirmed through in vitro cleavage in cell extracts of the fluorogenic peptide substrate, IETD-AFC, and direct, in vivo staining of cells with the fluorescently labeled irreversible caspase inhibitor, FITC-VAD-FMK. Direct addition of z-VAD-FMK to infected cultures abolished cellular caspase activity and protein expression and severely impaired viral production. The absence of metacaspase protein expression in resistant E. huxleyi strain 373 during EhV1 infection further demonstrated the critical role of these proteases in facilitating viral lysis. Together with the presence of caspase cleavage recognition sequences within virally encoded proteins, we provide experimental evidence that coccolithoviruses induce and actively recruit host metacaspases as part of their replication strategy. These findings reveal a critical role for metacaspases in the turnover of phytoplankton biomass upon infection with viruses and point to coevolution of host-virus interactions in the activation and maintenance of these enzymes in planktonic, unicellular protists.

  9. At the crossroads of autophagy and infection: Noncanonical roles for ATG proteins in viral replication.

    PubMed

    Solvik, Tina; Debnath, Jayanta

    2016-08-29

    Autophagy-related (ATG) proteins have increasingly demonstrated functions other than cellular self-eating. In this issue, Mauthe et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201602046) conduct an unbiased RNA interference screen of the ATG proteome to reveal numerous noncanonical roles for ATG proteins during viral infection. PMID:27573461

  10. 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

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

    PubMed

    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

  12. Three-dimensional analysis of a viral RNA replication complex reveals a virus-induced mini-organelle.

    PubMed

    Kopek, Benjamin G; Perkins, Guy; Miller, David J; Ellisman, Mark H; Ahlquist, Paul

    2007-09-01

    Positive-strand RNA viruses are the largest genetic class of viruses and include many serious human pathogens. All positive-strand RNA viruses replicate their genomes in association with intracellular membrane rearrangements such as single- or double-membrane vesicles. However, the exact sites of RNA synthesis and crucial topological relationships between relevant membranes, vesicle interiors, surrounding lumens, and cytoplasm generally are poorly defined. We applied electron microscope tomography and complementary approaches to flock house virus (FHV)-infected Drosophila cells to provide the first 3-D analysis of such replication complexes. The sole FHV RNA replication factor, protein A, and FHV-specific 5-bromouridine 5'-triphosphate incorporation localized between inner and outer mitochondrial membranes inside approximately 50-nm vesicles (spherules), which thus are FHV-induced compartments for viral RNA synthesis. All such FHV spherules were outer mitochondrial membrane invaginations with interiors connected to the cytoplasm by a necked channel of approximately 10-nm diameter, which is sufficient for ribonucleotide import and product RNA export. Tomographic, biochemical, and other results imply that FHV spherules contain, on average, three RNA replication intermediates and an interior shell of approximately 100 membrane-spanning, self-interacting protein As. The results identify spherules as the site of protein A and nascent RNA accumulation and define spherule topology, dimensions, and stoichiometry to reveal the nature and many details of the organization and function of the FHV RNA replication complex. The resulting insights appear relevant to many other positive-strand RNA viruses and support recently proposed structural and likely evolutionary parallels with retrovirus and double-stranded RNA virus virions.

  13. Replication of Murine Cytomegalovirus in Differentiated Macrophages as a Determinant of Viral Pathogenesis

    PubMed Central

    Hanson, Laura K.; Slater, Jacquelyn S.; Karabekian, Zaruhi; Virgin, Herbert W.; Biron, Christine A.; Ruzek, Melanie C.; van Rooijen, Nico; Ciavarra, Richard P.; Stenberg, Richard M.; Campbell, Ann E.

    1999-01-01

    Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as “filters” in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection. PMID:10364349

  14. Coinfection of tick cell lines has variable effects on replication of intracellular bacterial and viral pathogens

    PubMed Central

    Moniuszko, Anna; Rückert, Claudia; Alberdi, M. Pilar; Barry, Gerald; Stevenson, Brian; Fazakerley, John K.; Kohl, Alain; Bell-Sakyi, Lesley

    2014-01-01

    Ticks transmit various human and animal microbial pathogens and may harbour more than one pathogen simultaneously. Both viruses and bacteria can trigger, and may subsequently suppress, vertebrate host and arthropod vector anti-microbial responses. Microbial coinfection of ticks could lead to an advantage or disadvantage for one or more of the microorganisms. In this preliminary study, cell lines derived from the ticks Ixodes scapularis and Ixodes ricinus were infected sequentially with 2 arthropod-borne pathogens, Borrelia burgdorferi s.s., Ehrlichia ruminantium, or Semliki Forest virus (SFV), and the effect of coinfection on the replication of these pathogens was measured. Prior infection of tick cell cultures with the spirochaete B. burgdorferi enhanced subsequent replication of the rickettsial pathogen E. ruminantium whereas addition of spirochaetes to cells infected with E. ruminantium had no effect on growth of the latter. Both prior and subsequent presence of B. burgdorferi also had a positive effect on SFV replication. Presence of E. ruminantium or SFV had no measurable effect on B. burgdorferi growth. In tick cells infected first with E. ruminantium and then with SFV, virus replication was significantly higher across all time points measured (24, 48, 72 h post infection), while presence of the virus had no detectable effect on bacterial growth. When cells were infected first with SFV and then with E. ruminantium, there was no effect on replication of either pathogen. The results of this preliminary study indicate that interplay does occur between different pathogens during infection of tick cells. Further study is needed to determine if this results from direct pathogen–pathogen interaction or from effects on host cell defences, and to determine if these observations also apply in vivo in ticks. If presence of one pathogen in the tick vector results in increased replication of another, this could have implications for disease transmission and incidence

  15. Coinfection of tick cell lines has variable effects on replication of intracellular bacterial and viral pathogens.

    PubMed

    Moniuszko, Anna; Rückert, Claudia; Alberdi, M Pilar; Barry, Gerald; Stevenson, Brian; Fazakerley, John K; Kohl, Alain; Bell-Sakyi, Lesley

    2014-06-01

    Ticks transmit various human and animal microbial pathogens and may harbour more than one pathogen simultaneously. Both viruses and bacteria can trigger, and may subsequently suppress, vertebrate host and arthropod vector anti-microbial responses. Microbial coinfection of ticks could lead to an advantage or disadvantage for one or more of the microorganisms. In this preliminary study, cell lines derived from the ticks Ixodes scapularis and Ixodes ricinus were infected sequentially with 2 arthropod-borne pathogens, Borrelia burgdorferi s.s., Ehrlichia ruminantium, or Semliki Forest virus (SFV), and the effect of coinfection on the replication of these pathogens was measured. Prior infection of tick cell cultures with the spirochaete B. burgdorferi enhanced subsequent replication of the rickettsial pathogen E. ruminantium whereas addition of spirochaetes to cells infected with E. ruminantium had no effect on growth of the latter. Both prior and subsequent presence of B. burgdorferi also had a positive effect on SFV replication. Presence of E. ruminantium or SFV had no measurable effect on B. burgdorferi growth. In tick cells infected first with E. ruminantium and then with SFV, virus replication was significantly higher across all time points measured (24, 48, 72h post infection), while presence of the virus had no detectable effect on bacterial growth. When cells were infected first with SFV and then with E. ruminantium, there was no effect on replication of either pathogen. The results of this preliminary study indicate that interplay does occur between different pathogens during infection of tick cells. Further study is needed to determine if this results from direct pathogen-pathogen interaction or from effects on host cell defences, and to determine if these observations also apply in vivo in ticks. If presence of one pathogen in the tick vector results in increased replication of another, this could have implications for disease transmission and incidence.

  16. PTB Binds to the 3’ Untranslated Region of the Human Astrovirus Type 8: A Possible Role in Viral Replication

    PubMed Central

    Espinosa-Hernández, Wendy; Velez-Uriza, Dora; Valdés, Jesús; Vélez-Del Valle, Cristina; Salas-Benito, Juan; Martínez-Contreras, Rebeca; García-Espítia, Matilde; Salas-Benito, Mariana; Vega-Almeida, Tania; De Nova-Ocampo, Mónica

    2014-01-01

    The 3′ untranslated region (3′UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3′UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3′UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3′UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3′UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3′UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3′UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3′UTR HAstV-8 and is required or participates in viral replication. PMID:25406089

  17. PTB binds to the 3' untranslated region of the human astrovirus type 8: a possible role in viral replication.

    PubMed

    Espinosa-Hernández, Wendy; Velez-Uriza, Dora; Valdés, Jesús; Vélez-Del Valle, Cristina; Salas-Benito, Juan; Martínez-Contreras, Rebeca; García-Espítia, Matilde; Salas-Benito, Mariana; Vega-Almeida, Tania; De Nova-Ocampo, Mónica

    2014-01-01

    The 3' untranslated region (3'UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3'UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3'UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3'UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3'UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3'UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3'UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3'UTR HAstV-8 and is required or participates in viral replication.

  18. Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag.

    PubMed

    Schneider, William M; Brzezinski, Jonathon D; Aiyer, Sriram; Malani, Nirav; Gyuricza, Mercedes; Bushman, Frederic D; Roth, Monica J

    2013-06-01

    The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA)(1-23), human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA(1-32), required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration. PMID:23661057

  19. Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag

    PubMed Central

    Schneider, William M.; Brzezinski, Jonathon D.; Aiyer, Sriram; Malani, Nirav; Gyuricza, Mercedes; Bushman, Frederic D.; Roth, Monica J.

    2013-01-01

    The p12 protein of murine leukemia virus (MuLV) group-specific antigen (Gag) is associated with the preintegration complex, and mutants of p12 (PM14) show defects in nuclear entry or retention. Here we show that p12 proteins engineered to encode peptide sequences derived from known viral tethering proteins can direct chromatin binding during the early phase of viral replication and rescue a lethal p12-PM14 mutant. Peptides studied included segments of Kaposi sarcoma herpesvirus latency-associated nuclear antigen (LANA)1–23, human papillomavirus 8 E2, and prototype foamy virus chromatin-binding sequences. Amino acid substitutions in Kaposi sarcoma herpesvirus LANA and prototype foamy virus chromatin-binding sequences that blocked nucleosome association failed to rescue MuLV p12-PM14. Rescue by a larger LANA peptide, LANA1–32, required second-site mutations that are predicted to reduce peptide binding affinity to chromosomes, suggesting that excessively high binding affinity interfered with Gag/p12 function. This is supported by confocal microscopy of chimeric p12-GFP fusion constructs showing the reverted proteins had weaker association to condensed mitotic chromosomes. Analysis of the integration-site selection of these chimeric viruses showed no significant change in integration profile compared with wild-type MuLV, suggesting release of the tethered p12 post mitosis, before viral integration. PMID:23661057

  20. Targeted disruption of influenza A virus hemagglutinin in genetically modified mice reduces viral replication and improves disease outcome.

    PubMed

    Wang, Song; Chen, Chao; Yang, Zhou; Chi, Xiaojuan; Zhang, Jing; Chen, Ji-Long

    2016-01-01

    Influenza A virus can cause acute respiratory infection in animals and humans around the globe, and is still a major threat to animal husbandry and public health. Due to antigenic drift and antigenic shift of the virus, development of novel anti-influenza strategies has become an urgent task. Here we generated transgenic (TG) mice stably expressing a short-hairpin RNA specifically targeting hemagglutinin (HA) of influenza A virus, and investigated the susceptibility of the mice to influenza virus infection. We found that HA expression was dramatically disrupted in TG mice infected with WSN or PR8 virus. Importantly, the animals showed reduced virus production in lungs, slower weight loss, attenuated acute organ injury and consequently increased survival rates as compared to wild type (WT) mice after the viral infection. Moreover, TG mice exhibited a normal level of white blood cells following the virus infection, whereas the number of these cells was significantly decreased in WT mice with same challenge. Together, these experiments demonstrate that the TG mice are less permissive for influenza virus replication, and suggest that shRNA-based efficient disruption of viral gene expression in animals may be a useful strategy for prevention and control of a viral zoonosis. PMID:27033724

  1. Local production of tumor necrosis factor encoded by recombinant vaccinia virus is effective in controlling viral replication in vivo.

    PubMed Central

    Sambhi, S K; Kohonen-Corish, M R; Ramshaw, I A

    1991-01-01

    Tumor necrosis factor (TNF) has pleiotropic effects on a wide variety of cell types. In vitro studies have demonstrated that TNF has antiviral properties and is induced in response to viral infections. However, a role for TNF in the antiviral immune response of the host has yet to be demonstrated. Here we describe the construction of and studies using a recombinant vaccinia virus that encodes the gene for murine TNF-alpha. By comparing the replication of and immune responses elicited by the TNF-encoding virus to a similarly constructed control virus, we hoped to observe immunobiological effects of TNF in the host. The in vivo experiments with this recombinant virus demonstrate that the localized production of TNF-alpha during a viral infection leads to the rapid and efficient clearance of the virus in normal mice and attenuates the otherwise lethal pathogenicity of the virus in immunodeficient animals. This attenuation occurs early in the infection (by postinfection hour 24) and is not due to the enhancement of cellular or antibody responses by the vaccinia virus-encoded TNF. This evidence suggests that attenuation of the recombinant virus is due to a direct antiviral effect of TNF on cells at the site of infection. Therefore, these results support the suggestion that TNF produced by immune cells may be an important effector mechanism of viral clearance in vivo. Images PMID:2023951

  2. Local Production of Tumor Necrosis Factor Encoded by Recombinant Vaccinia Virus is Effective in Controlling Viral Replication in vivo

    NASA Astrophysics Data System (ADS)

    Sambhi, Sharan K.; Kohonen-Corish, Maija R. J.; Ramshaw, Ian A.

    1991-05-01

    Tumor necrosis factor (TNF) has pleiotropic effects on a wide variety of cell types. In vitro studies have demonstrated that TNF has antiviral properties and is induced in response to viral infections. However, a role for TNF in the antiviral immune response of the host has yet to be demonstrated. Here we describe the construction of and studies using a recombinant vaccinia virus that encodes the gene for murine TNF-α. By comparing the replication of and immune responses elicited by the TNF-encoding virus to a similarly constructed control virus, we hoped to observe immunobiological effects of TNF in the host. The in vivo experiments with this recombinant virus demonstrate that the localized production of TNF-α during a viral infection leads to the rapid and efficient clearance of the virus in normal mice and attenuates the otherwise lethal pathogenicity of the virus in immunodeficient animals. This attenuation occurs early in the infection (by postinfection hour 24) and is not due to the enhancement of cellular or antibody responses by the vaccinia virus-encoded TNF. This evidence suggests that attenuation of the recombinant virus is due to a direct antiviral effect of TNF on cells at the site of infection. Therefore, these results support the suggestion that TNF produced by immune cells may be an important effector mechanism of viral clearance in vivo.

  3. Multiple consecutive initiation of replication producing novel brush-like intermediates at the termini of linear viral dsDNA genomes with hairpin ends

    PubMed Central

    Martínez-Alvarez, Laura; Bell, Stephen D.; Peng, Xu

    2016-01-01

    Linear dsDNA replicons with hairpin ends are found in the three domains of life, mainly associated with plasmids and viruses including the poxviruses, some phages and archaeal rudiviruses. However, their replication mechanism is not clearly understood. In this study, we find that the rudivirus SIRV2 undergoes multiple consecutive replication reinitiation events at the genomic termini. Using a strand-displacement replication strategy, the multiple reinitiation events from one parental template yield highly branched intermediates corresponding to about 30 genome units which generate exceptional ‘brush-like’ structures. Moreover, our data support the occurrence of an additional strand-coupled bidirectional replication from a circular dimeric intermediate. The multiple reinitiation process ensures rapid copying of the parental viral genome and will enable protein factors involved in viral genome replication to be specifically localised intracellularly, thereby helping the virus to avoid host defence mechanisms. PMID:27407114

  4. Antimycobacterial activity of pyrazinoate prodrugs in replicating and non-replicating Mycobacterium tuberculosis.

    PubMed

    Segretti, Natanael Dante; Simões, Cristina Kortstee; Corrêa, Michelle Fidelis; Felli, Veni Maria Andres; Miyata, Marcelo; Cho, Sang Hyun; Franzblau, Scott Gary; Fernandes, João Paulo Dos Santos

    2016-07-01

    Tuberculosis (TB) is an important infectious disease caused by Mycobacterium tuberculosis (Mtb) and responsible for thousands of deaths every year. Although there are antimycobacterial drugs available in therapeutics, just few new chemical entities have reached clinical trials, and in fact, since introduction of rifampin only two important drugs had reached the market. Pyrazinoic acid (POA), the active agent of pyrazinamide, has been explored through prodrug approach to achieve novel molecules with anti-Mtb activity, however, there is no activity evaluation of these molecules against non-replicating Mtb until the present. Additionally, pharmacokinetic must be preliminary evaluated to avoid future problems during clinical trials. In this paper, we have presented six POA esters as prodrugs in order to evaluate their anti-Mtb activity in replicating and non-replicating Mtb, and these showed activity highly influenced by medium composition (especially by albumin). Lipophilicity seems to play the main role in the activity, possibly due to controlling membrane passage. Novel duplicated prodrugs of POA were also described, presenting interesting activity. Cytotoxicity of these prodrugs set was also evaluated, and these showed no important cytotoxic profile. PMID:27449999

  5. Replication of a recombinant hepatitis E virus genome tagged with reporter genes and generation of a short-term cell line producing viral RNA and proteins.

    PubMed

    Thakral, Deepshi; Nayak, Baibaswata; Rehman, Shagufta; Durgapal, Hemlata; Panda, Subrat Kumar

    2005-04-01

    Hepatitis E virus (HEV) replication has been demonstrated in HepG2 cells transfected with full-length in vitro transcripts of an infectious cDNA clone. This cDNA clone was modified to generate several subgenomic HEV replicons with fused reporter genes. In vitro-transcribed capped RNAs generated from these were transfected into HepG2 cells. Negative-strand RNA was detected, indicating the occurrence of replication. The replicon containing an in-frame fusion of HEV ORF2 with enhanced green fluorescent protein (EGFP) was positive for fluorescence, whereas no signal was observed when the replicase domain was deleted. An HEV ORF3-EGFP in-frame fusion did not yield fluorescence. Deletions introduced into ORF2 did not affect the replication competency of the viral RNA. To explore the possibility of using a reporter-gene assay to monitor the synthesis of plus- and minus-strand RNA, the EGFP gene fused to the encephalomyocarditis virus internal ribosome entry site (IRES) was inserted into partially deleted ORF2 of HEV, in both the sense [HEV-IRES-EGFP(+)] and antisense [HEV-IRES-EGFP(-)] orientations. HepG2 cells transfected with HEV-IRES-EGFP(+) and HEV-IRES-EGFP(-) vectors were positive for EGFP fluorescence. To quantify HEV replication, EGFP was replaced with Renilla luciferase (RLuc). HEV-IRES-RLuc(+) showed approximately 10-fold higher luminescence than HEV-IRES-RLuc(-). There was complete loss of activity when the helicase-replicase domain in HEV-IRES-RLuc(-) was deleted. A short-term HepG2 cell line containing the full-length viral genome in the pcDNA3 vector was established. Viral RNA and proteins (RdRp, pORF2 and pORF3) could be detected in the geneticin-resistant cells, even after the seventh passage. In the absence of a reliable cell-culture system to study HEV biology, these reporter replicons, as well as the cell line, bestow immense utility.

  6. Kaposi's sarcoma-associated herpesvirus ORF6 gene is essential in viral lytic replication.

    PubMed

    Peng, Can; Chen, Jungang; Tang, Wei; Liu, Chunlan; Chen, Xulin

    2014-01-01

    Kaposi's sarcoma associated herpesvirus (KSHV) is associated with Kaposis's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV encodes at least 8 open reading frames (ORFs) that play important roles in its lytic DNA replication. Among which, ORF6 of KSHV encodes an ssDNA binding protein that has been proved to participate in origin-dependent DNA replication in transient assays. To define further the function of ORF6 in the virus life cycle, we constructed a recombinant virus genome with a large deletion within the ORF6 locus by using a bacterial artificial chromosome (BAC) system. Stable 293T cells carrying the BAC36 (wild type) and BACΔ6 genomes were generated. When monolayers of 293T-BAC36 and 293T-BACΔ6 cells were induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate, infectious virus was detected from the 293T-BAC36 cell supernatants only and not from the 293T- BACΔ6 cell supernatants. DNA synthesis was defective in 293T-BACΔ6 cells. Expression of ORF6 in trans in BACΔ6-containing cells was able to rescue both defects. Our results provide genetic evidence that ORF6 is essential for KSHV lytic replication. The stable 293T cells carrying the BAC36 and BACΔ6 genomes could be used as tools to investigate the detailed functions of ORF6 in the lytic replication of KSHV. PMID:24911362

  7. Effects of the W153L Substitution in HIV Reverse Transcriptase on Viral Replication and Drug Resistance to Multiple Categories of Reverse Transcriptase Inhibitors

    PubMed Central

    Xu, Hong-Tao; Colby-Germinario, Susan P.; Oliveira, Maureen; Rajotte, Daniel; Bethell, Richard

    2014-01-01

    A W153L substitution in HIV-1 reverse transcriptase (RT) was recently identified by selection with a novel nucleotide-competing RT inhibitor (NcRTI) termed compound A that is a member of the benzo[4,5]furo[3,2,d]pyrimidin-2-one NcRTI family of drugs. To investigate the impact of W153L, alone or in combination with the clinically relevant RT resistance substitutions K65R (change of Lys to Arg at position 65), M184I, K101E, K103N, E138K, and Y181C, on HIV-1 phenotypic susceptibility, viral replication, and RT enzymatic function, we generated recombinant RT enzymes and viruses containing each of these substitutions or various combinations of them. We found that W153L-containing viruses were impaired in viral replicative capacity and were hypersusceptible to tenofovir (TFV) while retaining susceptibility to most nonnucleoside RT inhibitors. The nucleoside 3TC retained potency against W153L-containing viruses but not when the M184I substitution was also present. W153L was also able to reverse the effects of the K65R substitution on resistance to TFV, and K65R conferred hypersusceptibility to compound A. Biochemical assays demonstrated that W153L alone or in combination with K65R, M184I, K101E, K103N, E138K, and Y181C impaired enzyme processivity and polymerization efficiency but did not diminish RNase H activity, providing mechanistic insights into the low replicative fitness associated with these substitutions. We show that the mechanism of the TFV hypersusceptibility conferred by W153L is mainly due to increased efficiency of TFV-diphosphate incorporation. These results demonstrate that compound A and/or derivatives thereof have the potential to be important antiretroviral agents that may be combined with tenofovir to achieve synergistic results. PMID:24867966

  8. Roles of HIV-1 capsid in viral replication and immune evasion.

    PubMed

    Le Sage, Valerie; Mouland, Andrew J; Valiente-Echeverría, Fernando

    2014-11-26

    The primary roles of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein are to encapsidate and protect the viral RNA genome. It is becoming increasing apparent that HIV-1 CA is a multifunctional protein that acts early during infection to coordinate uncoating, reverse transcription, nuclear import of the pre-integration complex and integration of double stranded viral DNA into the host genome. Additionally, numerous recent studies indicate that CA is playing a crucial function in HIV-1 immune evasion. Here we summarize the current knowledge on HIV-1 CA and its interactions with the host cell to promote infection. The fact that CA engages in a number of different protein-protein interactions with the host makes it an interesting target for the development of new potent antiviral agents.

  9. Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA.

    PubMed

    Wang, H-M; Xia, X-Z; Hu, G-X; Yu, L; He, H-B

    2016-03-01

    Innate immunity, especially the anti-viral genes, exerts an important barrier function in preventing viral infections. Myxovirus-resistant (Mx) gene take an anti-viral role, whereas its effects on foot-and-mouth disease virus (FMDV) in naturally susceptible cells are still unclear. The bovine primary fetal tracheal epithelial cell line BPTE-siMx1, in which bovine Mx1 gene was silenced, was established and treated with IFN alpha for 6 hr before FMDV infection. The copy numbers of the negative and positive strand viral RNA were determined by strand-specific real-time fluorescence quantitative RT-PCR. The TCID50 of BPTE-siMx1 cells increased at least 17-fold as compared to control cells BPTE-LacZ at 8 hr post infection, thus silencing of bovine Mx1 could promote the replication of FMDV. The amount of both the negative and positive strand viral RNA in BPTE-siMx1 cells significantly increased as compared to BPTE-LacZ cells, indicating that the replication levels of viral RNA were promoted by silencing bovine Mx1. The bovine Mx1 gene could provide resistance against FMDV in the bovine primary fetal tracheal epithelial cells via suppressing the replication of viral RNA. PMID:26982472

  10. Bovine Mx1 enables resistance against foot-and-mouth disease virus in naturally susceptible cells by inhibiting the replication of viral RNA.

    PubMed

    Wang, H-M; Xia, X-Z; Hu, G-X; Yu, L; He, H-B

    2016-03-01

    Innate immunity, especially the anti-viral genes, exerts an important barrier function in preventing viral infections. Myxovirus-resistant (Mx) gene take an anti-viral role, whereas its effects on foot-and-mouth disease virus (FMDV) in naturally susceptible cells are still unclear. The bovine primary fetal tracheal epithelial cell line BPTE-siMx1, in which bovine Mx1 gene was silenced, was established and treated with IFN alpha for 6 hr before FMDV infection. The copy numbers of the negative and positive strand viral RNA were determined by strand-specific real-time fluorescence quantitative RT-PCR. The TCID50 of BPTE-siMx1 cells increased at least 17-fold as compared to control cells BPTE-LacZ at 8 hr post infection, thus silencing of bovine Mx1 could promote the replication of FMDV. The amount of both the negative and positive strand viral RNA in BPTE-siMx1 cells significantly increased as compared to BPTE-LacZ cells, indicating that the replication levels of viral RNA were promoted by silencing bovine Mx1. The bovine Mx1 gene could provide resistance against FMDV in the bovine primary fetal tracheal epithelial cells via suppressing the replication of viral RNA.

  11. An Essential Viral Transcription Activator Modulates Chromatin Dynamics

    PubMed Central

    Gibeault, Rebecca L.; Bildersheim, Michael D.

    2016-01-01

    Although ICP4 is the only essential transcription activator of herpes simplex virus 1 (HSV-1), its mechanisms of action are still only partially understood. We and others propose a model in which HSV-1 genomes are chromatinized as a cellular defense to inhibit HSV-1 transcription. To counteract silencing, HSV-1 would have evolved proteins that prevent or destabilize chromatinization to activate transcription. These proteins should act as HSV-1 transcription activators. We have shown that HSV-1 genomes are organized in highly dynamic nucleosomes and that histone dynamics increase in cells infected with wild type HSV-1. We now show that whereas HSV-1 mutants encoding no functional ICP0 or VP16 partially enhanced histone dynamics, mutants encoding no functional ICP4 did so only minimally. Transient expression of ICP4 was sufficient to enhance histone dynamics in the absence of other HSV-1 proteins or HSV-1 DNA. The dynamics of H3.1 were increased in cells expressing ICP4 to a greater extent than those of H3.3. The dynamics of H2B were increased in cells expressing ICP4, whereas those of canonical H2A were not. ICP4 preferentially targets silencing H3.1 and may also target the silencing H2A variants. In infected cells, histone dynamics were increased in the viral replication compartments, where ICP4 localizes. These results suggest a mechanism whereby ICP4 activates transcription by disrupting, or preventing the formation of, stable silencing nucleosomes on HSV-1 genomes. PMID:27575707

  12. Ebola virus VP40 late domains are not essential for viral replication in cell culture.

    PubMed

    Neumann, Gabriele; Ebihara, Hideki; Takada, Ayato; Noda, Takeshi; Kobasa, Darwyn; Jasenosky, Luke D; Watanabe, Shinji; Kim, Jin H; Feldmann, Heinz; Kawaoka, Yoshihiro

    2005-08-01

    Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture. PMID:16051823

  13. Activity of trifluoperazine against replicating, non-replicating and drug resistant M. tuberculosis.

    PubMed

    Advani, Meeta J; Siddiqui, Imran; Sharma, Pawan; Reddy, Hemalatha

    2012-01-01

    Trifluoperazine, a known calmodulin antagonist, belongs to a class of phenothiazine compounds that have multiple sites of action in mycobacteria including lipid synthesis, DNA processes, protein synthesis and respiration. The objective of this study is to evaluate the potential of TFP to be used as a lead molecule for development of novel TB drugs by showing its efficacy on multiple drug resistant (MDR) Mycobacterium tuberculosis (M.tb) and non-replicating dormant M.tb. Wild type and MDR M.tb were treated with TFP under different growth conditions of stress like low pH, starvation, presence of nitric oxide and in THP-1 infection model. Perturbation in growth kinetics of bacilli at different concentrations of TFP was checked to determine the MIC of TFP for active as well as dormant bacilli. Results show that TFP is able to significantly reduce the actively replicating as well as non-replicating bacillary load. It has also shown inhibitory effect on the growth of MDR M.tb. TFP has shown enhanced activity against intracellular bacilli, presumably because phenothiazines are known to get accumulated in macrophages. This concentration was, otherwise, found to be non-toxic to macrophage in vitro. Our results show that TFP has the potential to be an effective killer of both actively growing and non-replicating bacilli including MDR TB. Further evaluation and in vivo studies with Trifluoperazine can finally help us know the feasibility of this compound to be used as either a lead compound for development of new TB drugs or as an adjunct in the current TB chemotherapy.

  14. Loss of CARD9-mediated innate activation attenuates severe influenza pneumonia without compromising host viral immunity.

    PubMed

    Uematsu, Takayuki; Iizasa, Ei'ichi; Kobayashi, Noritada; Yoshida, Hiroki; Hara, Hiromitsu

    2015-01-01

    Influenza virus (IFV) infection is a common cause of severe viral pneumonia associated with acute respiratory distress syndrome (ARDS), which is difficult to control with general immunosuppressive therapy including corticosteroids due to the unfavorable effect on viral replication. Studies have suggested that the excessive activation of the innate immunity by IFV is responsible for severe pathologies. In this study, we focused on CARD9, a signaling adaptor known to regulate innate immune activation through multiple innate sensor proteins, and investigated its role in anti-IFV defense and lung pathogenesis in a mouse model recapitulating severe influenza pneumonia with ARDS. We found that influenza pneumonia was dramatically attenuated in Card9-deficient mice, which showed improved mortality with reduced inflammatory cytokines and chemokines in the infected lungs. However, viral clearance, type-I interferon production, and the development of anti-viral B and T cell immunity were not compromised by CARD9 deficiency. Syk or CARD9-deficient DCs but not macrophages showed impaired cytokine but not type-I interferon production in response to IFV in vitro, indicating a possible role for the Syk-CARD9 pathway in DCs in excessive inflammation of IFV-infected lungs. Therefore, inhibition of this pathway is an ideal therapeutic target for severe influenza pneumonia without affecting viral clearance. PMID:26627732

  15. Loss of CARD9-mediated innate activation attenuates severe influenza pneumonia without compromising host viral immunity

    PubMed Central

    Uematsu, Takayuki; Iizasa, Ei’ichi; Kobayashi, Noritada; Yoshida, Hiroki; Hara, Hiromitsu

    2015-01-01

    Influenza virus (IFV) infection is a common cause of severe viral pneumonia associated with acute respiratory distress syndrome (ARDS), which is difficult to control with general immunosuppressive therapy including corticosteroids due to the unfavorable effect on viral replication. Studies have suggested that the excessive activation of the innate immunity by IFV is responsible for severe pathologies. In this study, we focused on CARD9, a signaling adaptor known to regulate innate immune activation through multiple innate sensor proteins, and investigated its role in anti-IFV defense and lung pathogenesis in a mouse model recapitulating severe influenza pneumonia with ARDS. We found that influenza pneumonia was dramatically attenuated in Card9-deficient mice, which showed improved mortality with reduced inflammatory cytokines and chemokines in the infected lungs. However, viral clearance, type-I interferon production, and the development of anti-viral B and T cell immunity were not compromised by CARD9 deficiency. Syk or CARD9-deficient DCs but not macrophages showed impaired cytokine but not type-I interferon production in response to IFV in vitro, indicating a possible role for the Syk-CARD9 pathway in DCs in excessive inflammation of IFV-infected lungs. Therefore, inhibition of this pathway is an ideal therapeutic target for severe influenza pneumonia without affecting viral clearance. PMID:26627732

  16. Interferon-beta treatment and active replication of the JC virus in relapsing-remitting multiple sclerosis patients.

    PubMed

    Alvarez-Lafuente, R; García-Montojo, M; De Las Heras, V; Bartolomé, M; Arroyo, R

    2007-02-01

    We analyzed the effect of beta-interferon (beta-IFN) treatment over the active replication of JC virus (JCV) through the evaluation of JCV DNA prevalence and viral load in peripheral blood mononuclear cells (PBMCs) and serum samples, and mRNA prevalence and viral load, in relapsing-remitting multiple sclerosis (RRMS) patients. DNA extracted from PBMCs and serum, and mRNA extracted from PBMCs were analyzed in 146 RRMS patients (73 treated with beta-IFN, and 73 untreated patients), and 73 matched healthy blood donors for the presence of JCV genomes by quantitative real-time PCR assay. We found the same DNA prevalence in PBMC samples in RRMS patients treated with beta-IFN and in untreated ones: 6.8% (5/73). When we analyzed the viral active replication in both groups through the analysis of DNA prevalence in serum samples and the mRNA extracted from PBMCs, we did not find any positive sample. Regarding the viral load of those positive samples, we did not find any statistical significant difference between treated and untreated RRMS patients: 28.6 +/- 7.2 and 32.3 +/- 8.4 copies/microg of DNA, respectively. These results lead us to conclude that beta-IFN treatment in monotherapy has not any effect on JCV active replication.

  17. 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 Central

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

    2016-01-01

    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. PMID:26950140

  18. 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

    2016-01-01

    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

  19. 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

  20. OX40 and 4-1BB downregulate Kaposi’s sarcoma-associated herpesvirus replication in lymphatic endothelial cells, but 4-1BB and not OX40 inhibits viral replication in B-cells.

    PubMed

    Cho, Min; Myoung, Jinjong

    2015-12-01

    Kaposi’s sarcoma-associated herpesvirus (KSHV) belongs to the human gammaherpesvirus subfamily and is associated with malignancies of endothelial origin (Kaposi’s sarcoma, KS) and B-cell origin [primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD)]. Viral lytic replication is known to be required for KS and MCD. As KSHV-related tumours mostly develop in human subjects when the immune system is compromised by immunosuppressive regimen, human immunodeficiency virus infection or some genetic deficiencies, KSHV-specific immune responses are believed to be important in the control of KSHV replication. However, analysis of the roles of immune cells in viral pathogenesis has been difficult due to the lack of an adequate animal model. Recently, congenital OX40 deficiency, as determined by genome-wide exome sequencing, was shown to be associated with aggressive childhood KS in a patient, suggesting that disrupted OX40–OX40L interactions might be implicated in disease development. Here, we report that interaction of recombinant OX40 protein with OX40L expressed on endothelial cells severely impaired KSHV lytic replication. Furthermore, 4-1BB–4-1BBL interactions were also capable of efficiently inhibiting viral replication in B-cells and endothelial cells. To the best of our knowledge, this is the first direct evidence that ligation of tumour necrosis factor superfamily members and their cognate receptors is important for the control of viral lytic replication. These data are likely to pave the way for the development of KSHV-specific therapies for KS and MCD, in which viral lytic replication is a disease-determining factor. PMID:26467721

  1. Replication stress activates DNA repair synthesis in mitosis.

    PubMed

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A; Bursomanno, Sara; Aleliunaite, Aiste; Wu, Wei; Mankouri, Hocine W; Shen, Huahao; Liu, Ying; Hickson, Ian D

    2015-12-10

    Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress. The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach.

  2. Replication stress activates DNA repair synthesis in mitosis.

    PubMed

    Minocherhomji, Sheroy; Ying, Songmin; Bjerregaard, Victoria A; Bursomanno, Sara; Aleliunaite, Aiste; Wu, Wei; Mankouri, Hocine W; Shen, Huahao; Liu, Ying; Hickson, Ian D

    2015-12-10

    Oncogene-induced DNA replication stress has been implicated as a driver of tumorigenesis. Many chromosomal rearrangements characteristic of human cancers originate from specific regions of the genome called common fragile sites (CFSs). CFSs are difficult-to-replicate loci that manifest as gaps or breaks on metaphase chromosomes (termed CFS 'expression'), particularly when cells have been exposed to replicative stress. The MUS81-EME1 structure-specific endonuclease promotes the appearance of chromosome gaps or breaks at CFSs following replicative stress. Here we show that entry of cells into mitotic prophase triggers the recruitment of MUS81 to CFSs. The nuclease activity of MUS81 then promotes POLD3-dependent DNA synthesis at CFSs, which serves to minimize chromosome mis-segregation and non-disjunction. We propose that the attempted condensation of incompletely duplicated loci in early mitosis serves as the trigger for completion of DNA replication at CFS loci in human cells. Given that this POLD3-dependent mitotic DNA synthesis is enhanced in aneuploid cancer cells that exhibit intrinsically high levels of chromosomal instability (CIN(+)) and replicative stress, we suggest that targeting this pathway could represent a new therapeutic approach. PMID:26633632

  3. CD200R1 supports HSV-1 viral replication and licenses pro-inflammatory signaling functions of TLR2.

    PubMed

    Soberman, Roy J; MacKay, Christopher R; Vaine, Christine A; Ryan, Glennice Bowen; Cerny, Anna M; Thompson, Mikayla R; Nikolic, Boris; Primo, Valeria; Christmas, Peter; Sheiffele, Paul; Aronov, Lisa; Knipe, David M; Kurt-Jones, Evelyn A

    2012-01-01

    The CD200R1:CD200 axis is traditionally considered to limit tissue inflammation by down-regulating pro-inflammatory signaling in myeloid cells bearing the receptor. We generated CD200R1(-/-) mice and employed them to explore both the role of CD200R1 in regulating macrophage signaling via TLR2 as well as the host response to an in vivo, TLR2-dependent model, herpes simplex virus 1 (HSV-1) infection. CD200R1(-/-) peritoneal macrophages demonstrated a 70-75% decrease in the generation of IL-6 and CCL5 (Rantes) in response to the TLR2 agonist Pam(2)CSK(4) and to HSV-1. CD200R1(-/-) macrophages could neither up-regulate the expression of TLR2, nor assemble a functional inflammasome in response to HSV-1. CD200R1(-/-) mice were protected from HSV-1 infection and exhibited dysfunctional TLR2 signaling. Finally, both CD200R1(-/-) mice and CD200R1(-/-) fibroblasts and macrophages showed a markedly reduced ability to support HSV-1 replication. In summary, our data demonstrate an unanticipated and novel requirement for CD200R1 in "licensing" pro-inflammatory functions of TLR2 and in limiting viral replication that are supported by ex vivo and in vivo evidence.

  4. A Temporal Gate for Viral Enhancers to Co-opt Toll-Like-Receptor Transcriptional Activation Pathways upon Acute Infection

    PubMed Central

    Kropp, Kai A.; Hsieh, Wei Yuan; Isern, Elena; Forster, Thorsten; Krause, Eva; Brune, Wolfram; Angulo, Ana; Ghazal, Peter

    2015-01-01

    Viral engagement with macrophages activates Toll-Like-Receptors (TLRs) and viruses must contend with the ensuing inflammatory responses to successfully complete their replication cycle. To date, known counter-strategies involve the use of viral-encoded proteins that often employ mimicry mechanisms to block or redirect the host response to benefit the virus. Whether viral regulatory DNA sequences provide an opportunistic strategy by which viral enhancer elements functionally mimic innate immune enhancers is unknown. Here we find that host innate immune genes and the prototypical viral enhancer of cytomegalovirus (CMV) have comparable expression kinetics, and positively respond to common TLR agonists. In macrophages but not fibroblasts we show that activation of NFκB at immediate-early times of infection is independent of virion-associated protein, M45. We find upon virus infection or transfection of viral genomic DNA the TLR-agonist treatment results in significant enhancement of the virus transcription-replication cycle. In macrophage time-course infection experiments we demonstrate that TLR-agonist stimulation of the viral enhancer and replication cycle is strictly delimited by a temporal gate with a determined half-maximal time for enhancer-activation of 6 h; after which TLR-activation blocks the viral transcription-replication cycle. By performing a systematic siRNA screen of 149 innate immune regulatory factors we identify not only anticipated anti-viral and pro-viral contributions but also new factors involved in the CMV transcription-replication cycle. We identify a central convergent NFκB-SP1-RXR-IRF axis downstream of TLR-signalling. Activation of the RXR component potentiated direct and indirect TLR-induced activation of CMV transcription-replication cycle; whereas chromatin binding experiments using wild-type and enhancer-deletion virus revealed IRF3 and 5 as new pro-viral host transcription factor interactions with the CMV enhancer in macrophages. In a

  5. Temperature-dependent innate defense against the common cold virus limits viral replication at warm temperature in mouse airway cells.

    PubMed

    Foxman, Ellen F; Storer, James A; Fitzgerald, Megan E; Wasik, Bethany R; Hou, Lin; Zhao, Hongyu; Turner, Paul E; Pyle, Anna Marie; Iwasaki, Akiko

    2015-01-20

    Most isolates of human rhinovirus, the common cold virus, replicate more robustly at the cool temperatures found in the nasal cavity (33-35 °C) than at core body temperature (37 °C). To gain insight into the mechanism of temperature-dependent growth, we compared the transcriptional response of primary mouse airway epithelial cells infected with rhinovirus at 33 °C vs. 37 °C. Mouse airway cells infected with mouse-adapted rhinovirus 1B exhibited a striking enrichment in expression of antiviral defense response genes at 37 °C relative to 33 °C, which correlated with significantly higher expression levels of type I and type III IFN genes and IFN-stimulated genes (ISGs) at 37 °C. Temperature-dependent IFN induction in response to rhinovirus was dependent on the MAVS protein, a key signaling adaptor of the RIG-I-like receptors (RLRs). Stimulation of primary airway cells with the synthetic RLR ligand poly I:C led to greater IFN induction at 37 °C relative to 33 °C at early time points poststimulation and to a sustained increase in the induction of ISGs at 37 °C relative to 33 °C. Recombinant type I IFN also stimulated more robust induction of ISGs at 37 °C than at 33 °C. Genetic deficiency of MAVS or the type I IFN receptor in infected airway cells permitted higher levels of viral replication, particularly at 37 °C, and partially rescued the temperature-dependent growth phenotype. These findings demonstrate that in mouse airway cells, rhinovirus replicates preferentially at nasal cavity temperature due, in part, to a less efficient antiviral defense response of infected cells at cool temperature.

  6. Comparative inhibitory activity of the stilbenes resveratrol and oxyresveratrol on African swine fever virus replication.

    PubMed

    Galindo, I; Hernáez, B; Berná, J; Fenoll, J; Cenis, J L; Escribano, J M; Alonso, C

    2011-07-01

    Stilbenols are polyphenolic phytoalexins produced by plants in response to biotic or abiotic stress. These compounds have received much attention because of their significant biological effects. One of these is their antiviral action, which has previously been documented for two members of this class, namely resveratrol and oxyresveratrol. Here we tested the antiviral effect of these two compounds on African swine fever virus, the only member of the newly created family Asfarviridae and a serious limitation to porcine production worldwide. Our results show a potent, dose-dependent antiviral effect of resveratrol and oxyresveratrol in vitro. Interestingly, this antiviral activity was found for these synthetic compounds and also for oxyresveratrol extracted from new natural sources (mulberry twigs). The antiviral effect of these two drugs was demonstrated at concentrations that do not induce cytotoxicity in cultured cells. Moreover, these antivirals achieved a 98-100% reduction in viral titers. Both compounds allowed early protein synthesis but inhibited viral DNA replication, late viral protein synthesis and viral factory formation.

  7. The Envelope Cytoplasmic Tail of HIV-1 Subtype C Contributes to Poor Replication Capacity through Low Viral Infectivity and Cell-to-Cell Transmission

    PubMed Central

    Lemaire, Morgane; Masquelier, Cécile; Beraud, Cyprien; Rybicki, Arkadiusz; Servais, Jean-Yves; Iserentant, Gilles; Schmit, Jean-Claude; Seguin-Devaux, Carole; Perez Bercoff, Danielle

    2016-01-01

    The cytoplasmic tail (gp41CT) of the HIV-1 envelope (Env) mediates Env incorporation into virions and regulates Env intracellular trafficking. Little is known about the functional impact of variability in this domain. To address this issue, we compared the replication of recombinant virus pairs carrying the full Env (Env viruses) or the Env ectodomain fused to the gp41CT of NL4.3 (EnvEC viruses) (12 subtype C and 10 subtype B pairs) in primary CD4+ T-cells and monocyte-derived-macrophages (MDMs). In CD4+ T-cells, replication was as follows: B-EnvEC = B-Env>C-EnvEC>C-Env, indicating that the gp41CT of subtype C contributes to the low replicative capacity of this subtype. In MDMs, in contrast, replication capacity was comparable for all viruses regardless of subtype and of gp41CT. In CD4+ T-cells, viral entry, viral release and viral gene expression were similar. However, infectivity of free virions and cell-to-cell transmission of C-Env viruses released by CD4+ T-cells was lower, suggestive of lower Env incorporation into virions. Subtype C matrix only minimally rescued viral replication and failed to restore infectivity of free viruses and cell-to-cell transmission. Taken together, these results show that polymorphisms in the gp41CT contribute to viral replication capacity and suggest that the number of Env spikes per virion may vary across subtypes. These findings should be taken into consideration in the design of vaccines. PMID:27598717

  8. The Envelope Cytoplasmic Tail of HIV-1 Subtype C Contributes to Poor Replication Capacity through Low Viral Infectivity and Cell-to-Cell Transmission.

    PubMed

    Santos da Silva, Eveline; Mulinge, Martin; Lemaire, Morgane; Masquelier, Cécile; Beraud, Cyprien; Rybicki, Arkadiusz; Servais, Jean-Yves; Iserentant, Gilles; Schmit, Jean-Claude; Seguin-Devaux, Carole; Perez Bercoff, Danielle

    2016-01-01

    The cytoplasmic tail (gp41CT) of the HIV-1 envelope (Env) mediates Env incorporation into virions and regulates Env intracellular trafficking. Little is known about the functional impact of variability in this domain. To address this issue, we compared the replication of recombinant virus pairs carrying the full Env (Env viruses) or the Env ectodomain fused to the gp41CT of NL4.3 (EnvEC viruses) (12 subtype C and 10 subtype B pairs) in primary CD4+ T-cells and monocyte-derived-macrophages (MDMs). In CD4+ T-cells, replication was as follows: B-EnvEC = B-Env>C-EnvEC>C-Env, indicating that the gp41CT of subtype C contributes to the low replicative capacity of this subtype. In MDMs, in contrast, replication capacity was comparable for all viruses regardless of subtype and of gp41CT. In CD4+ T-cells, viral entry, viral release and viral gene expression were similar. However, infectivity of free virions and cell-to-cell transmission of C-Env viruses released by CD4+ T-cells was lower, suggestive of lower Env incorporation into virions. Subtype C matrix only minimally rescued viral replication and failed to restore infectivity of free viruses and cell-to-cell transmission. Taken together, these results show that polymorphisms in the gp41CT contribute to viral replication capacity and suggest that the number of Env spikes per virion may vary across subtypes. These findings should be taken into consideration in the design of vaccines. PMID:27598717

  9. Viral Factors Induce Hedgehog Pathway Activation in Humans with Viral Hepatitis, Cirrhosis, and Hepatocellular Carcinoma

    PubMed Central

    Pereira, Thiago de Almeida; Witek, Rafal P.; Syn, Wing-Kin; Choi, Steve S.; Bradrick, Shelton; Karaca, Gamze F; Agboola, Kolade M.; Jung, Youngmi; Omenetti, Alessia; Moylan, Cynthia A.; Yang, Liu; Fernandez-Zapico, Martin E.; Jhaveri, Ravi; Shah, Vijay H.; Pereira, Fausto E.; Diehl, Anna Mae

    2010-01-01

    Hh pathway activation promotes many processes that occur during fibrogenic liver repair. Whether the Hh pathway modulates the outcomes of virally-mediated liver injury has never been examined. Gene-profiling studies of human hepatocellular carcinomas (HCC) demonstrate Hh pathway activation in HCCs related to chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV). Because most HCC develop in cirrhotic livers, we hypothesized that Hh pathway activation occurs during fibrogenic repair of liver damage due to chronic viral hepatitis, and that Hh-responsive cells mediate disease progression and hepatocarciongenesis in chronic viral hepatitis. Immunohistochemistry and qRTPCR analysis were used to analyze Hh pathway activation and identify Hh-responsive cell types in liver biopsies from 45 patients with chronic HBV or HCV. Hh signaling was then manipulated in cultured liver cells to directly assess the impact of Hh activity in relevant cell types. We found increased hepatic expression of Hh ligands in all patients with chronic viral hepatitis, and demonstrated that infection with HCV stimulated cultured hepatocytes to produce Hh ligands. The major cell populations that expanded during cirrhosis and HCC (i.e., liver myofibroblasts, activated endothelial cells, and progenitors expressing markers of tumor stem/initiating cells) were Hh-responsive, and higher levels of Hh pathway activity associated with cirrhosis and HCC. Inhibiting pathway activity in Hh-responsive target cells reduced fibrogenesis, angiogenesis, and growth. Conclusions HBV/HCV infection increases hepatocyte production of Hh ligands and expands types of Hh-responsive cells that promote liver fibrosis and cancer. PMID:20697376

  10. A novel viral thymidylate kinase with dual kinase activity.

    PubMed

    Guevara-Hernandez, Eduardo; Arvizu-Flores, Aldo A; Lugo-Sanchez, Maria E; Velazquez-Contreras, Enrique F; Castillo-Yañez, Francisco J; Brieba, Luis G; Sotelo-Mundo, Rogerio R

    2015-10-01

    Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 μM, 3.6 s(-1)), dTDP (251 μM, 0.9 s(-1)) and ATP (92 μM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 μM) and dTDP (10 μM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 μM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

  11. New World and Old World Alphaviruses Have Evolved to Exploit Different Components of Stress Granules, FXR and G3BP Proteins, for Assembly of Viral Replication Complexes

    PubMed Central

    Kim, Dal Young; Reynaud, Josephine M.; Rasalouskaya, Aliaksandra; Akhrymuk, Ivan; Mobley, James A.; Frolov, Ilya; Frolova, Elena I.

    2016-01-01

    The positive-strand RNA viruses initiate their amplification in the cell from a single genome delivered by virion. This single RNA molecule needs to become involved in replication process before it is recognized and degraded by cellular machinery. In this study, we show that distantly related New World and Old World alphaviruses have independently evolved to utilize different cellular stress granule-related proteins for assembly of complexes, which recruit viral genomic RNA and facilitate formation of viral replication complexes (vRCs). Venezuelan equine encephalitis virus (VEEV) utilizes all members of the Fragile X syndrome (FXR) family, while chikungunya and Sindbis viruses exploit both members of the G3BP family. Despite being in different families, these proteins share common characteristics, which determine their role in alphavirus replication, namely, the abilities for RNA-binding and for self-assembly into large structures. Both FXR and G3BP proteins interact with virus-specific, repeating amino acid sequences located in the C-termini of hypervariable, intrinsically disordered domains (HVDs) of viral nonstructural protein nsP3. We demonstrate that these host factors orchestrate assembly of vRCs and play key roles in RNA and virus replication. Only knockout of all of the homologs results in either pronounced or complete inhibition of replication of different alphaviruses. The use of multiple homologous proteins with redundant functions mediates highly efficient recruitment of viral RNA into the replication process. This independently evolved acquisition of different families of cellular proteins by the disordered protein fragment to support alphavirus replication suggests that other RNA viruses may utilize a similar mechanism of host factor recruitment for vRC assembly. The use of different host factors by alphavirus species may be one of the important determinants of their pathogenesis. PMID:27509095

  12. Lychee flower extract inhibits proliferation and viral replication of HSV-1-infected corneal epithelial cells

    PubMed Central

    Hsu, Chang-Min; Chiang, Samuel Tung-Hsing; Chang, Yuan-Yen; Chen, Yi-Chen; Yang, Deng-Jye; Chen, Ya-Yu; Lin, Hui-Wen

    2016-01-01

    Purpose Herpes simplex virus type I (HSV-1) is capable of causing a wide array of human ocular diseases. Herpes simplex virus keratitis (HSK)-induced cytopathogenicity together with the chronic immune-inflammatory reaction can trigger stromal scarring, thinning, and neovascularization which may lead to permanent vision impairment. Lychee flower extract (LFE) is known for its antioxidant and anti-inflammatory effects. Therefore, in this study, we investigated the mechanism of the Statens Seruminstitut rabbit corneal (SIRC) epithelial cells infected by HSV-1 and examined the antiviral capabilities of LFE. Methods SIRC cells were pretreated with different concentrations of LFE (0.2, 0.1, and 0.05 μg/ml) and then infected with 1 MOI of HSV-1 for 24 h. The cell viability or morphology was evaluated in this study. In addition, the supernatants and cell extracts were collected for Cell Counting Kit-8 (CCK), plaque assay, and western blotting. Results We found that HSV-1-induced cell proliferation is regulated through inhibition of the mammalian target of rapamycin (mTOR) and p70s6k phosphorylation in response to the LFE. In addition, the LFE enhanced the autophagy protein expression (Beclin-1 and light chain 3, LC3) and decreased the viral titers. Conclusions These results showed the antiviral capabilities and the protective effects of LFE. Taken together, our data indicate that LFE has potential as an anti-HSK (herpes simplex keratitis) for HSV-1 infection. PMID:26937165

  13. An Epigenetic Compound Library Screen Identifies BET Inhibitors That Promote HSV-1 and -2 Replication by Bridging P-TEFb to Viral Gene Promoters through BRD4

    PubMed Central

    Chen, Xiaoqing; Ma, Yingyu; Dai, Yue; Fan, Yimei; Hou, Yayi; Tan, Ren Xiang

    2016-01-01

    The human HSV-1 and -2 are common pathogens of human diseases. Both host and viral factors are involved in HSV lytic infection, although detailed mechanisms remain elusive. By screening a chemical library of epigenetic regulation, we identified bromodomain-containing protein 4 (BRD4) as a critical player in HSV infection. We show that treatment with pan BD domain inhibitor enhanced both HSV infection. Using JQ1 as a probe, we found that JQ1, a defined BD1 inhibitor, acts through BRD4 protein since knockdown of BRD4 expression ablated JQ1 effect on HSV infection. BRD4 regulates HSV replication through complex formation involving CDK9 and RNAP II; whereas, JQ1 promotes HSV-1 infection by allocating the complex to HSV gene promoters. Therefore, suppression of BRD4 expression or inhibition of CDK9 activity impeded HSV infection. Our data support a model that JQ1 enhances HSV infection by switching BRD4 to transcription regulation of viral gene expression from chromatin targeting since transient expression of BRD4 BD1 or BD1/2 domain had similar effect to that by JQ1 treatment. In addition to the identification that BRD4 is a modulator for JQ1 action on HSV infection, this study demonstrates BRD4 has an essential role in HSV infection. PMID:27764245

  14. Coronavirus Nsp10, a Critical Co-factor for Activation of Multiple Replicative Enzymes*

    PubMed Central

    Bouvet, Mickaël; Lugari, Adrien; Posthuma, Clara C.; Zevenhoven, Jessika C.; Bernard, Stéphanie; Betzi, Stéphane; Imbert, Isabelle; Canard, Bruno; Guillemot, Jean-Claude; Lécine, Patrick; Pfefferle, Susanne; Drosten, Christian; Snijder, Eric J.; Decroly, Etienne; Morelli, Xavier

    2014-01-01

    The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1–16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3′-5′ exoribonuclease and 2′-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2′-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses. PMID:25074927

  15. Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes.

    PubMed

    Bouvet, Mickaël; Lugari, Adrien; Posthuma, Clara C; Zevenhoven, Jessika C; Bernard, Stéphanie; Betzi, Stéphane; Imbert, Isabelle; Canard, Bruno; Guillemot, Jean-Claude; Lécine, Patrick; Pfefferle, Susanne; Drosten, Christian; Snijder, Eric J; Decroly, Etienne; Morelli, Xavier

    2014-09-12

    The RNA-synthesizing machinery of the severe acute respiratory syndrome Coronavirus (SARS-CoV) is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a/1b. The 148-amino acid nsp10 subunit contains two zinc fingers and is known to interact with both nsp14 and nsp16, stimulating their respective 3'-5' exoribonuclease and 2'-O-methyltransferase activities. Using alanine-scanning mutagenesis, in cellulo bioluminescence resonance energy transfer experiments, and in vitro pulldown assays, we have now identified the key residues on the nsp10 surface that interact with nsp14. The functional consequences of mutations introduced at these positions were first evaluated biochemically by monitoring nsp14 exoribonuclease activity. Disruption of the nsp10-nsp14 interaction abrogated the nsp10-driven activation of the nsp14 exoribonuclease. We further showed that the nsp10 surface interacting with nsp14 overlaps with the surface involved in the nsp10-mediated activation of nsp16 2'-O-methyltransferase activity, suggesting that nsp10 is a major regulator of SARS-CoV replicase function. In line with this notion, reverse genetics experiments supported an essential role of the nsp10 surface that interacts with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro yielded a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disturbed proved to be crippled but viable. These experiments imply that the nsp10 surface that interacts with nsp14 and nsp16 and possibly other subunits of the viral replication complex may be a target for the development of antiviral compounds against pathogenic coronaviruses. PMID:25074927

  16. A tumour necrosis factor receptor-like protein encoded by Singapore grouper iridovirus modulates cell proliferation, apoptosis and viral replication.

    PubMed

    Yu, Yepin; Huang, Youhua; Wei, Shina; Li, Pengfei; Zhou, Lingli; Ni, Songwei; Huang, Xiaohong; Qin, Qiwei

    2016-03-01

    It has been demonstrated that tumour necrosis factor receptor (TNFR) homologues encoded by viruses are usually involved in virus immune evasion by regulating the host immune response or mediating apoptotic cell death. Here, a novel TNFR-like protein encoded by Singapore grouper iridovirus (SGIV VP51) was cloned and characterized. Amino acid analysis showed that VP51 contained three cysteine-rich domains (CRDs) and a transmembrane domain at its C terminus. The expression of VP51 in vitro enhanced cell proliferation, and affected cell cycle progression via altering the G1/S transition. Furthermore, VP51 overexpression improved cell viability during SGIV infection via inhibiting virus-induced apoptosis, evidenced by the reduction of apoptotic bodies and the decrease of caspase-3 activation. In addition, overexpression of VP51 increased viral titre and the expression of viral structural protein gene MCP and cell proliferation promoting gene ICP-18. In contrast, the expression of the viral apoptosis inducing gene, LITAF, was significantly decreased. Although all three CRDs were essential for the action of VP51, CRD2 and CRD3 exerted more crucial roles on virus-induced apoptosis, viral gene transcription and virus production, while CRD1 was more crucial for cell proliferation. Together, SGIV TNFR-like products not only affected cell cycle progression and enhanced cell growth by increasing the expression of the virus encoded cell proliferation gene, but also inhibited virus-induced apoptotic cell death by decreasing the expression of the viral apoptosis inducing gene. Our results provided new insights into understanding the underlying mechanism by which iridovirus regulated the apoptotic pathway to complete its life cycle.

  17. Japanese encephalitis virus activates autophagy as a viral immune evasion strategy.

    PubMed

    Jin, Rui; Zhu, Wandi; Cao, Shengbo; Chen, Rui; Jin, Hui; Liu, Yang; Wang, Shaobo; Wang, Wei; Xiao, Gengfu

    2013-01-01

    In addition to manipulating cellular homeostasis and survivability, autophagy also plays a crucial role in numerous viral infections. In this study, we discover that Japanese encephalitis virus (JEV) infection results in the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II) protein and GFP-LC3 puncta in vitro and an increase in autophagosomes/autolysosomes in vivo. The fusion between autophagosomes and lysosomes is essential for virus replication. Knockdown of autophagy-related genes reduced JEV replication in vitro, as indicated by viral RNA and protein levels. We also note that JEV infection in autophagy-impaired cells displayed active caspases cleavage and cell death. Moreover, we find that JEV induces higher type I interferon (IFN) activation in cells deficient in autophagy-related genes as the cells exhibited increased phosphorylation and dimerization of interferon regulatory factor 3 (IRF3) and mitochondrial antiviral signaling protein (MAVS) aggregation. Finally, we find that autophagy is indispensable for efficient JEV replication even in an IFN-defective background. Overall, our studies provide the first description of the mechanism of the autophagic innate immune signaling pathway during JEV infection.

  18. Viral activities and life cycles in deep subseafloor sediments.

    PubMed

    Engelhardt, Tim; Orsi, William D; Jørgensen, Bo Barker

    2015-12-01

    Viruses are highly abundant in marine subsurface sediments and can even exceed the number of prokaryotes. However, their activity and quantitative impact on microbial populations are still poorly understood. Here, we use gene expression data from published continental margin subseafloor metatranscriptomes to qualitatively assess viral diversity and activity in sediments up to 159 metres below seafloor (mbsf). Mining of the metatranscriptomic data revealed 4651 representative viral homologues (RVHs), representing 2.2% of all metatranscriptome sequence reads, which have close translated homology (average 77%, range 60-97% amino acid identity) to viral proteins. Archaea-infecting RVHs are exclusively detected in the upper 30 mbsf, whereas RVHs for filamentous inoviruses predominate in the deepest sediment layers. RVHs indicative of lysogenic phage-host interactions and lytic activity, notably cell lysis, are detected at all analysed depths and suggest a dynamic virus-host association in the marine deep biosphere studied here. Ongoing lytic viral activity is further indicated by the expression of clustered, regularly interspaced, short palindromic repeat-associated cascade genes involved in cellular defence against viral attacks. The data indicate the activity of viruses in subsurface sediment of the Peruvian margin and suggest that viruses indeed cause cell mortality and may play an important role in the turnover of subseafloor microbial biomass. PMID:26109514

  19. Viral activities and life cycles in deep subseafloor sediments.

    PubMed

    Engelhardt, Tim; Orsi, William D; Jørgensen, Bo Barker

    2015-12-01

    Viruses are highly abundant in marine subsurface sediments and can even exceed the number of prokaryotes. However, their activity and quantitative impact on microbial populations are still poorly understood. Here, we use gene expression data from published continental margin subseafloor metatranscriptomes to qualitatively assess viral diversity and activity in sediments up to 159 metres below seafloor (mbsf). Mining of the metatranscriptomic data revealed 4651 representative viral homologues (RVHs), representing 2.2% of all metatranscriptome sequence reads, which have close translated homology (average 77%, range 60-97% amino acid identity) to viral proteins. Archaea-infecting RVHs are exclusively detected in the upper 30 mbsf, whereas RVHs for filamentous inoviruses predominate in the deepest sediment layers. RVHs indicative of lysogenic phage-host interactions and lytic activity, notably cell lysis, are detected at all analysed depths and suggest a dynamic virus-host association in the marine deep biosphere studied here. Ongoing lytic viral activity is further indicated by the expression of clustered, regularly interspaced, short palindromic repeat-associated cascade genes involved in cellular defence against viral attacks. The data indicate the activity of viruses in subsurface sediment of the Peruvian margin and suggest that viruses indeed cause cell mortality and may play an important role in the turnover of subseafloor microbial biomass.

  20. Immunodominant epitopes in nsp2 of porcine reproductive and respiratory syndrome virus are dispensable for replication but play an important role in viral pathogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) is the largest protein of the virus. Besides its crucial role in viral replication, recent studies indicated its involvement in modulating host immunity. In this study, each of the six identified immu...

  1. Evidence for Early Local Viral Replication and Local Production of Antiviral Immunity upon Mucosal Simian-Human Immunodeficiency Virus SHIV89.6 Infection in Macaca nemestrina

    PubMed Central

    Ambrose, Zandrea; Larsen, Kay; Thompson, Jannelle; Stevens, Yvonne; Finn, Eric; Hu, Shiu-Lok; Bosch, Marnix L.

    2001-01-01

    Transmission of human immunodeficiency virus type 1 (HIV-1) is largely a result of heterosexual exposure, leading many investigators to evaluate mucosal vaccines for protection against intravaginal (i.vag.) transmission in macaque models of AIDS. Relatively little is known, however, about the dynamics of viral replication and the ensuing immune response following mucosal infection. We have utilized a simian-human immunodeficiency virus (SHIV) to study the differences in viremia, CD4 T-cell percentages, and mucosal and systemic anti-SHIV humoral and cellular immune responses during primary infection of animals infected either intravenously (i.v.) or i.vag. Positive viral cocultures, peripheral blood mononuclear cell viral load peaks, and CD4 cell declines were delayed by 1 week in the i.vag. inoculated animals compared to the animals infected i.v., demonstrating delayed viral spreading to the periphery. In contrast, mucosal anti-SHIV antibody levels were greater in magnitude and arose more rapidly and mucosal CD8+ T-cell responses were enhanced in the i.vag. group animals, whereas both the magnitudes and times of onset of systemic immune responses for the animals in the two groups did not differ. These observations demonstrate that compartmentalization of viral replication and induction of local antiviral immunity occur in the genital tract early after i.vag. but not i.v. inoculation. Induction of mucosal immunity to target this local, contained replication should be a goal in HIV vaccine development. PMID:11507204

  2. PKR Activation Favors Infectious Pancreatic Necrosis Virus Replication in Infected Cells

    PubMed Central

    Gamil, Amr A.A.; Xu, Cheng; Mutoloki, Stephen; Evensen, Øystein

    2016-01-01

    The double-stranded RNA-activated protein kinase R (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, in general, PKR inhibits or promotes viral replication, but PKR-IPNV interaction has not been previously studied. We investigated the involvement of PKR during infectious pancreatic necrosis virus (IPNV) infection using a custom-made rabbit antiserum and the PKR inhibitor C16. Reactivity of the antiserum to PKR in CHSE-214 cells was confirmed after IFNα treatment giving an increased protein level. IPNV infection alone did not give increased PKR levels by Western blot, while pre-treatment with PKR inhibitor before IPNV infection gave decreased eukaryotic initiation factor 2-alpha (eIF2α) phosphorylation. This suggests that PKR, despite not being upregulated, is involved in eIF2α phosphorylation during IPNV infection. PKR inhibitor pre-treatment resulted in decreased virus titers, extra- and intracellularly, concomitant with reduction of cells with compromised membranes in IPNV-permissive cell lines. These findings suggest that IPNV uses PKR activation to promote virus replication in infected cells. PMID:27338445

  3. PKR Activation Favors Infectious Pancreatic Necrosis Virus Replication in Infected Cells.

    PubMed

    Gamil, Amr A A; Xu, Cheng; Mutoloki, Stephen; Evensen, Øystein

    2016-01-01

    The double-stranded RNA-activated protein kinase R (PKR) is a Type I interferon (IFN) stimulated gene that has important biological and immunological functions. In viral infections, in general, PKR inhibits or promotes viral replication, but PKR-IPNV interaction has not been previously studied. We investigated the involvement of PKR during infectious pancreatic necrosis virus (IPNV) infection using a custom-made rabbit antiserum and the PKR inhibitor C16. Reactivity of the antiserum to PKR in CHSE-214 cells was confirmed after IFNα treatment giving an increased protein level. IPNV infection alone did not give increased PKR levels by Western blot, while pre-treatment with PKR inhibitor before IPNV infection gave decreased eukaryotic initiation factor 2-alpha (eIF2α) phosphorylation. This suggests that PKR, despite not being upregulated, is involved in eIF2α phosphorylation during IPNV infection. PKR inhibitor pre-treatment resulted in decreased virus titers, extra- and intracellularly, concomitant with reduction of cells with compromised membranes in IPNV-permissive cell lines. These findings suggest that IPNV uses PKR activation to promote virus replication in infected cells. PMID:27338445

  4. Evolution of Phenotypic Drug Susceptibility and Viral Replication Capacity during Long-Term Virologic Failure of Protease Inhibitor Therapy in Human Immunodeficiency Virus-Infected Adults

    PubMed Central

    Barbour, Jason D.; Wrin, Terri; Grant, Robert M.; Martin, Jeffrey N.; Segal, Mark R.; Petropoulos, Christos J.; Deeks, Steven G.

    2002-01-01

    Continued use of antiretroviral therapy despite the emergence of drug-resistant human immunodeficiency virus (HIV) has been associated with the durable maintenance of plasma HIV RNA levels below pretherapy levels. The factors that may account for this partial control of viral replication were assessed in a longitudinal observational study of 20 HIV-infected adults who remained on a stable protease inhibitor-based regimen despite ongoing viral replication (plasma HIV RNA levels consistently >500 copies/ml). Longitudinal plasma samples (n = 248) were assayed for drug susceptibility and viral replication capacity (measured by using a single-cycle recombinant-virus assay). The initial treatment-mediated decrease in plasma viremia was directly proportional to the reduction in replicative capacity (P = 0.01). Early virologic rebound was associated the emergence of a virus population exhibiting increased protease inhibitor phenotypic resistance, while replicative capacity remained low. During long-term virologic failure, plasma HIV RNA levels often remained stable or increased slowly, while phenotypic resistance continued to increase and replicative capacity decreased slowly. The emergence of primary genotypic mutations within protease (particularly V82A, I84V, and L90M) was temporally associated with increasing phenotypic resistance and decreasing replicative capacity, while the emergence of secondary mutations within protease was associated with more-gradual changes in both phenotypic resistance and replicative capacity. We conclude that HIV may be constrained in its ability to become both highly resistant and highly fit and that this may contribute to the continued partial suppression of plasma HIV RNA levels that is observed in some patients with drug-resistant viremia. PMID:12368352

  5. Activation of the Epstein-Barr virus replicative cycle by human herpesvirus 6.

    PubMed Central

    Flamand, L; Stefanescu, I; Ablashi, D V; Menezes, J

    1993-01-01

    One common attribute of herpesviruses is the ability to establish latent, life-long infections. The role of virus-virus interaction in viral reactivation between or among herpesviruses has not been studied. Preliminary experiments in our laboratory had indicated that infection of Epstein-Barr virus (EBV) genome-positive human lymphoid cell lines with human herpesvirus 6 (HHV-6) results in EBV reactivation in these cells. To further our knowledge of this complex phenomenon, we investigated the effect of HHV-6 infection on expression of the viral lytic cycle proteins of EBV. Our results indicate that HHV-6 upregulates, by up to 10-fold, expression of the immediate-early Zebra antigen and the diffuse and restricted (85 kDa) early antigens (EA-D and EA-R, respectively) in both EBV producer and nonproducer cell lines (i.e., P3HR1, Akata, and Raji). Maximal EA-D induction was observed at 72 h post-HHV-6 infection. Furthermore, expression of late EBV gene products, namely, the viral capsid antigen (125 kDa) and viral membrane glycoprotein gp350, was also increased in EBV producer cells (P3HR1 and Akata) following infection by HHV-6. By using dual-color membrane immunofluorescence, it was found that most of the cells expressing viral membrane glycoprotein gp350 were also positive for HHV-6 antigens, suggesting a direct effect of HHV-6 replication on induction of the EBV replicative cycle. No expression of late EBV antigens was observed in Raji cells following infection by HHV-6, implying a lack of functional complementation between the deleted form of EBV found in Raji cells and the superinfecting HHV-6. The susceptibility of the cell lines to infection by HHV-6 correlated with increased expression of various EBV proteins in that B95-8 cells, which are not susceptible to HHV-6 infection, did not show an increase in expression of EBV antigens following treatment with HHV-6. Moreover, UV light-irradiated or heat-inactivated HHV-6 had no upregulating effect on the Zebra

  6. The X gene of adeno-associated virus 2 (AAV2) is involved in viral DNA replication.

    PubMed

    Cao, Maohua; You, Hong; Hermonat, Paul L

    2014-01-01

    Adeno-associated virus (AAV) (type 2) is a popular human gene therapy vector with a long active transgene expression period and no reported vector-induced adverse reactions. Yet the basic molecular biology of this virus has not been fully addressed. One potential gene at the far 3' end of the AAV2 genome, previously referred to as X (nt 3929 to 4393), overlapping the 3' end of the cap gene, has never been characterized, although we did previously identify a promoter just up-stream (p81). Computer analysis suggested that X was involved in replication and transcription. The X protein was identified during active AAV2 replication using a polyclonal antibody against a peptide starting at amino acid 98. Reagents for the study of X included an AAV2 deletion mutant (dl78-91), a triple nucleotide substitution mutant that destroys all three 5' AUG-initiation products of X, with no effect on the cap coding sequence, and X-positive-293 cell lines. Here, we found that X up-regulated AAV2 DNA replication in differentiating keratinocytes (without helper virus, autonomous replication) and in various forms of 293 cell-based assays with help from wild type adenovirus type 5 (wt Ad5) or Ad5 helper plasmid (pHelper). The strongest contribution by X was seen in increasing wt AAV2 DNA replication in keratinocytes and dl78-91 in Ad5-infected X-positive-293 cell lines (both having multi-fold effects). Mutating the X gene in pAAV-RC (pAAV-RC-3Xneg) yielded approximately a ∼33% reduction in recombinant AAV vector DNA replication and virion production, but a larger effect was seen when using this same X-knockout AAV helper plasmid in X-positive-293 cell lines versus normal 293 cells (again, multi-fold). Taken together these data strongly suggest that AAV2 X encodes a protein involved in the AAV life cycle, particularly in increasing AAV2 DNA replication, and suggests that further studies are warranted.

  7. Role of primary constitutive phosphorylation of Sendai virus P and V proteins in viral replication and pathogenesis.

    PubMed

    Hu, C J; Kato, A; Bowman, M C; Kiyotani, K; Yoshida, T; Moyer, S A; Nagai, Y; Gupta, K C

    1999-10-10

    Functional analysis of the primary constitutive phosphorylation of Sendai virus P and V proteins was performed using both in vitro and in vivo systems. Sendai virus minigenome transcription and replication in transfected cells were not significantly affected in the presence of primary phosphorylation deficient P protein (S249A, S249D, P250A) as measured by either the luciferase activity or the Northern blot analysis. Similarly, recombinant Sendai viruses lacking the primary phosphorylation in P grew to titers close to the wild-type virus in cell cultures and in the natural host of Sendai virus, the mouse. Mutant viruses showed no altered pathogenesis in mice lungs. Oligomerization of P by binding WT P or mutant P to GST-P (WT) Sepharose beads revealed that the primary phosphorylation was not crucial for P protein oligomerization. Similar to P protein primary phosphorylation, the V protein primary phosphorylation at serine249 was not essential for minigenome transcription and replication, as both WT and mutant V proteins were found equally inhibitory to the minigenome replication. These results show that the primary phosphorylation of P protein has no essential role in Sendai virus transcription, replication, and pathogenesis.

  8. Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein.

    PubMed

    van der Schaar, H M; Melia, C E; van Bruggen, J A C; Strating, J R P M; van Geenen, M E D; Koster, A J; Bárcena, M; van Kuppeveld, F J M

    2016-01-01

    Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for

  9. Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein

    PubMed Central

    van der Schaar, H. M.; Melia, C. E.; van Bruggen, J. A. C.; Strating, J. R. P. M.; van Geenen, M. E. D.; Koster, A. J.; Bárcena, M.

    2016-01-01

    ABSTRACT Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition

  10. Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein.

    PubMed

    van der Schaar, H M; Melia, C E; van Bruggen, J A C; Strating, J R P M; van Geenen, M E D; Koster, A J; Bárcena, M; van Kuppeveld, F J M

    2016-01-01

    Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for

  11. Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome.

    PubMed

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

    2011-12-01

    Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general

  12. Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei.

    PubMed Central

    Brison, O; Kedinger, C; Wilhelm, J

    1977-01-01

    When HeLa cell nuclei, isolated 17 h after infection with adenovirus type 2 (Ad2), were extracted with 200 mM ammonium sulfate, Ad2 nucleoprotein complexes were selectively released. These complexes contained a DNA polymerase activity that corresponded to DNA polymerase molecules actively engaged in Ad2 DNA replication. Under our high-salt (200 mM ammonium sulfate) incubation conditions, where no reinitiation occurred, full-length Ad2 DNA chains were synthesized by elongation of chains that had been initiated in vivo. This conclusion was further supported by density labeling experiments indicating that the in vitro DNA synthesis was semiconservative. Evidence is presented suggesting that at least part of the DNA polymerase molecules engaged in Ad2 DNA replication belong to the gamma class. PMID:916022

  13. Love the one you’re with: replicate viral adaptations converge on the same phenotypic change

    PubMed Central

    Nagel, Anna C.; Scott, LuAnn; Settles, Matt; Wichman, Holly A.

    2016-01-01

    Parallelism is important because it reveals how inherently stochastic adaptation is. Even as we come to better understand evolutionary forces, stochasticity limits how well we can predict evolutionary outcomes. Here we sought to quantify parallelism and some of its underlying causes by adapting a bacteriophage (ID11) with nine different first-step mutations, each with eight-fold replication, for 100 passages. This was followed by whole-genome sequencing five isolates from each endpoint. A large amount of variation arose—281 mutational events occurred representing 112 unique mutations. At least 41% of the mutations and 77% of the events were adaptive. Within wells, populations generally experienced complex interference dynamics. The genome locations and counts of mutations were highly uneven: mutations were concentrated in two regulatory elements and three genes and, while 103 of the 112 (92%) of the mutations were observed in ≤4 wells, a few mutations arose many times. 91% of the wells and 81% of the isolates had a mutation in the D-promoter. Parallelism was moderate compared to previous experiments with this system. On average, wells shared 27% of their mutations at the DNA level and 38% when the definition of parallel change is expanded to include the same regulatory feature or residue. About half of the parallelism came from D-promoter mutations. Background had a small but significant effect on parallelism. Similarly, an analyses of epistasis between mutations and their ancestral background was significant, but the result was mostly driven by four individual mutations. A second analysis of epistasis focused on de novo mutations revealed that no isolate ever had more than one D-promoter mutation and that 56 of the 65 isolates lacking a D-promoter mutation had a mutation in genes D and/or E. We assayed time to lysis in four of these mutually exclusive mutations (the two most frequent D-promoter and two in gene D) across four genetic backgrounds. In all cases

  14. Love the one you're with: replicate viral adaptations converge on the same phenotypic change.

    PubMed

    Miller, Craig R; Nagel, Anna C; Scott, LuAnn; Settles, Matt; Joyce, Paul; Wichman, Holly A

    2016-01-01

    Parallelism is important because it reveals how inherently stochastic adaptation is. Even as we come to better understand evolutionary forces, stochasticity limits how well we can predict evolutionary outcomes. Here we sought to quantify parallelism and some of its underlying causes by adapting a bacteriophage (ID11) with nine different first-step mutations, each with eight-fold replication, for 100 passages. This was followed by whole-genome sequencing five isolates from each endpoint. A large amount of variation arose-281 mutational events occurred representing 112 unique mutations. At least 41% of the mutations and 77% of the events were adaptive. Within wells, populations generally experienced complex interference dynamics. The genome locations and counts of mutations were highly uneven: mutations were concentrated in two regulatory elements and three genes and, while 103 of the 112 (92%) of the mutations were observed in ≤4 wells, a few mutations arose many times. 91% of the wells and 81% of the isolates had a mutation in the D-promoter. Parallelism was moderate compared to previous experiments with this system. On average, wells shared 27% of their mutations at the DNA level and 38% when the definition of parallel change is expanded to include the same regulatory feature or residue. About half of the parallelism came from D-promoter mutations. Background had a small but significant effect on parallelism. Similarly, an analyses of epistasis between mutations and their ancestral background was significant, but the result was mostly driven by four individual mutations. A second analysis of epistasis focused on de novo mutations revealed that no isolate ever had more than one D-promoter mutation and that 56 of the 65 isolates lacking a D-promoter mutation had a mutation in genes D and/or E. We assayed time to lysis in four of these mutually exclusive mutations (the two most frequent D-promoter and two in gene D) across four genetic backgrounds. In all cases

  15. Timeless links replication termination to mitotic kinase activation.

    PubMed

    Dheekollu, Jayaraju; Wiedmer, Andreas; Hayden, James; Speicher, David; Gotter, Anthony L; Yen, Tim; Lieberman, Paul M

    2011-01-01

    The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication. PMID:21573113

  16. Timeless links replication termination to mitotic kinase activation.

    PubMed

    Dheekollu, Jayaraju; Wiedmer, Andreas; Hayden, James; Speicher, David; Gotter, Anthony L; Yen, Tim; Lieberman, Paul M

    2011-05-06

    The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

  17. Inhibition of Coxsackievirus B3 Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral Positive Strand

    PubMed Central

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

    2005-01-01

    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. PMID:15681418

  18. Viral Replication, Persistence in Water and Genetic Characterization of Two Influenza A Viruses Isolated from Surface Lake Water

    PubMed Central

    Lebarbenchon, Camille; Yang, My; Keeler, Shamus P.; Ramakrishnan, Muthannan A.; Brown, Justin D.; Stallknecht, David E.; Sreevatsan, Srinand

    2011-01-01

    Water-borne transmission has been suggested as an important transmission mechanism for Influenza A (IA) viruses in wild duck populations; however, relatively few studies have attempted to detect IA viruses from aquatic habitats. Water-isolated viruses have rarely been genetically characterized and evaluation for persistence in water and infectivity in natural hosts has never been documented. In this study, we focused on two IA viruses (H3N8 and H4N6 subtypes) isolated from surface lake water in Minnesota, USA. We investigated the relative prevalence of the two virus subtypes in wild duck populations at the sampling site and their genetic relatedness to IA viruses isolated in wild waterbirds in North America. Viral persistence under different laboratory conditions (temperature and pH) and replication in experimentally infected Mallards (Anas platyrhynchos) were also characterized. Both viruses were the most prevalent subtype one year following their isolation in lake water. The viruses persisted in water for an extended time period at constant temperature (several weeks) but infectivity rapidly reduced under multiple freeze-thaw cycles. Furthermore, the two isolates efficiently replicated in Mallards. The complete genome characterization supported that these isolates originated from genetic reassortments with other IA viruses circulating in wild duck populations during the year of sampling. Based on phylogenetic analyses, we couldn't identify genetically similar viruses in duck populations in the years following their isolation from lake water. Our study supports the role for water-borne transmission for IA viruses but also highlights that additional field and experimental studies are required to support inter-annual persistence in aquatic habitats. PMID:22028909

  19. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins.

    PubMed

    Zhao, Chen; Sridharan, Haripriya; Chen, Ran; Baker, Darren P; Wang, Shanshan; Krug, Robert M

    2016-01-01

    The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP. PMID:27587337

  20. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins.

    PubMed

    Zhao, Chen; Sridharan, Haripriya; Chen, Ran; Baker, Darren P; Wang, Shanshan; Krug, Robert M

    2016-01-01

    The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP.

  1. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins

    PubMed Central

    Zhao, Chen; Sridharan, Haripriya; Chen, Ran; Baker, Darren P.; Wang, Shanshan; Krug, Robert M.

    2016-01-01

    The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP. PMID:27587337

  2. Sustained inhibition of hepatitis B virus replication in vivo using RNAi-activating lentiviruses.

    PubMed

    Ivacik, D; Ely, A; Ferry, N; Arbuthnot, P

    2015-02-01

    Chronic infection with hepatitis B virus (HBV) puts individuals at high risk for complicating cirrhosis and liver cancer, but available treatment to counter the virus rarely eliminates infection. Although harnessing RNA interference (RNAi) to silence HBV genes has shown the potential, achieving efficient and durable silencing of viral genes remains an important goal. Here we report on the propagation of lentiviral vectors (LVs) that successfully deliver HBV-targeting RNAi activators to liver cells. Mono- and tricistronic artificial primary microRNAs (pri-miRs) derived from pri-miR-31, placed under transcriptional control of the liver-specific modified murine transthyretin (mTTR) promoter, caused efficient inhibition of HBV replication markers. The tricistronic cassette was capable of silencing a mutant viral target and the effects were observed without disrupting the function of an endogenous miR (miR-16). The mTTR promoter stably expressed a reporter transgene in mouse livers over a study period of 1 year. Good silencing of HBV genes, without evidence of toxicity, was demonstrated following intravenous injection of LVs into neonatal HBV transgenic mice. Collectively, these data indicate that LVs may achieve sustained inhibition of HBV replication that is appealing for their therapeutic use.

  3. Rotavirus VP2 core shell regions critical for viral polymerase activation.

    PubMed

    McDonald, Sarah M; Patton, John T

    2011-04-01

    The innermost VP2 core shell of the triple-layered, icosahedral rotavirus particle surrounds the viral genome and RNA processing enzymes, including the RNA-dependent RNA polymerase (VP1). In addition to anchoring VP1 within the core, VP2 is also an essential cofactor that triggers the polymerase to initiate double-stranded RNA (dsRNA) synthesis using packaged plus-strand RNA templates. The VP2 requirement effectively couples packaging with genome replication and ensures that VP1 makes dsRNA only within an assembling previrion particle. However, the mechanism by which the rotavirus core shell protein activates the viral polymerase remains very poorly understood. In the current study, we sought to elucidate VP2 regions critical for VP1-mediated in vitro dsRNA synthesis. By comparing the functions of proteins from several different rotaviruses, we found that polymerase activation by the core shell protein is specific. Through truncation and chimera mutagenesis, we demonstrate that the VP2 amino terminus, which forms a decameric, internal hub underneath each 5-fold axis, plays an important but nonspecific role in VP1 activation. Our results indicate that the VP2 residues correlating with polymerase activation specificity are located on the inner face of the core shell, distinct from the amino terminus. Based on these findings, we predict that several regions of VP2 engage the polymerase during the concerted processes of rotavirus core assembly and genome replication.

  4. Resveratrol enhances HBV replication through activating Sirt1-PGC-1α-PPARα pathway

    PubMed Central

    Shi, Yixian; Li, Yongjun; Huang, Chenjie; Ying, Lixiong; Xue, Jihua; Wu, Haicong; Chen, Zhi; Yang, Zhenggang

    2016-01-01

    The population of hepatitis B combined with a number of metabolic disorders is increasing significantly. Resveratrol (RSV) has been used as a preclinical drug for the treatment of the metabolic disorders. However, the impact of RSV on HBV replication remains unknown. In this study, the HBV-expressing hepatocelluar carcinoma cell line and mouse model created by hydrodynamic injection of viral DNA were used. We found that RSV activates Sirt1, which in turn deacetylates PGC-1α and subsequently increases the transcriptional activity of PPARα, leading to the enhanced HBV transcription and replication in vitro and in vivo. In addition, we found that this pathway is also required for fasting-induced HBV transcription. Taken together, this study identifies that RSV enhances HBV transcription and replication especially acting on the core promoter, which depends on Sirt1-PGC-1α-PPARα pathway. We conclude that RSV may exacerbate the progression of hepatitis B and that patients with hepatitis B infection should be cautious taking RSV as a dietary supplement. PMID:27098390

  5. Differential responses of Africanized and European honey bees (Apis mellifera) to viral replication following mechanical transmission or Varroa destructor parasitism.

    PubMed

    Hamiduzzaman, Mollah Md; Guzman-Novoa, Ernesto; Goodwin, Paul H; Reyes-Quintana, Mariana; Koleoglu, Gun; Correa-Benítez, Adriana; Petukhova, Tatiana

    2015-03-01

    For the first time, adults and brood of Africanized and European honey bees (Apis mellifera) were compared for relative virus levels over 48 h following Varroa destructor parasitism or injection of V. destructor homogenate. Rates of increase of deformed wing virus (DWV) for Africanized versus European bees were temporarily lowered for 12h with parasitism and sustainably lowered over the entire experiment (48 h) with homogenate injection in adults. The rates were also temporarily lowered for 24h with parasitism but were not affected by homogenate injection in brood. Rates of increase of black queen cell virus (BQCV) for Africanized versus European bees were similar with parasitism but sustainably lowered over the entire experiment with homogenate injection in adults and were similar for parasitism and homogenate injection in brood. Analyses of sac brood bee virus and Israeli acute paralysis virus were limited as detection did not occur after both homogenate injection and parasitism treatment, or levels were not significantly higher than those following control buffer injection. Lower rates of replication of DWV and BQCV in Africanized bees shows that they may have greater viral resistance, at least early after treatment.

  6. Differential responses of Africanized and European honey bees (Apis mellifera) to viral replication following mechanical transmission or Varroa destructor parasitism.

    PubMed

    Hamiduzzaman, Mollah Md; Guzman-Novoa, Ernesto; Goodwin, Paul H; Reyes-Quintana, Mariana; Koleoglu, Gun; Correa-Benítez, Adriana; Petukhova, Tatiana

    2015-03-01

    For the first time, adults and brood of Africanized and European honey bees (Apis mellifera) were compared for relative virus levels over 48 h following Varroa destructor parasitism or injection of V. destructor homogenate. Rates of increase of deformed wing virus (DWV) for Africanized versus European bees were temporarily lowered for 12h with parasitism and sustainably lowered over the entire experiment (48 h) with homogenate injection in adults. The rates were also temporarily lowered for 24h with parasitism but were not affected by homogenate injection in brood. Rates of increase of black queen cell virus (BQCV) for Africanized versus European bees were similar with parasitism but sustainably lowered over the entire experiment with homogenate injection in adults and were similar for parasitism and homogenate injection in brood. Analyses of sac brood bee virus and Israeli acute paralysis virus were limited as detection did not occur after both homogenate injection and parasitism treatment, or levels were not significantly higher than those following control buffer injection. Lower rates of replication of DWV and BQCV in Africanized bees shows that they may have greater viral resistance, at least early after treatment. PMID:25527405

  7. Targeting human respiratory syncytial virus transcription anti-termination factor M2-1 to inhibit in vivo viral replication

    PubMed Central

    Bailly, B.; Richard, C.-A.; Sharma, G.; Wang, L.; Johansen, L.; Cao, J.; Pendharkar, V.; Sharma, D.-C.; Galloux, M.; Wang, Y.; Cui, R.; Zou, G.; Guillon, P.; von Itzstein, M.; Eléouët, J.-F.; Altmeyer, R.

    2016-01-01

    Human respiratory syncytial virus (hRSV) is a leading cause of acute lower respiratory tract infection in infants, elderly and immunocompromised individuals. To date, no specific antiviral drug is available to treat or prevent this disease. Here, we report that the Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication. Cyclopamine inhibits hRSV through a novel, Smo-independent mechanism. It specifically impairs the function of the hRSV RNA-dependent RNA polymerase complex notably by reducing expression levels of the viral anti-termination factor M2-1. The relevance of these findings is corroborated by the demonstration that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine in vitro and that cyclopamine is able to reduce virus titers in a mouse model of hRSV infection. The results of our study open a novel avenue for the development of future therapies against hRSV infection. PMID:27194388

  8. Intracellular membrane association of the N-terminal domain of classical swine fever virus NS4B determines viral genome replication and virulence.

    PubMed

    Tamura, Tomokazu; Ruggli, Nicolas; Nagashima, Naofumi; Okamatsu, Masatoshi; Igarashi, Manabu; Mine, Junki; Hofmann, Martin A; Liniger, Matthias; Summerfield, Artur; Kida, Hiroshi; Sakoda, Yoshihiro

    2015-09-01

    Classical swine fever virus (CSFV) causes a highly contagious disease in pigs that can range from a severe haemorrhagic fever to a nearly unapparent disease, depending on the virulence of the virus strain. Little is known about the viral molecular determinants of CSFV virulence. The nonstructural protein NS4B is essential for viral replication. However, the roles of CSFV NS4B in viral genome replication and pathogenesis have not yet been elucidated. NS4B of the GPE-  vaccine strain and of the highly virulent Eystrup strain differ by a total of seven amino acid residues, two of which are located in the predicted trans-membrane domains of NS4B and were described previously to relate to virulence, and five residues clustering in the N-terminal part. In the present study, we examined the potential role of these five amino acids in modulating genome replication and determining pathogenicity in pigs. A chimeric low virulent GPE- -derived virus carrying the complete Eystrup NS4B showed enhanced pathogenicity in pigs. The in vitro replication efficiency of the NS4B chimeric GPE-  replicon was significantly higher than that of the replicon carrying only the two Eystrup-specific amino acids in NS4B. In silico and in vitro data suggest that the N-terminal part of NS4B forms an amphipathic α-helix structure. The N-terminal NS4B with these five amino acid residues is associated with the intracellular membranes. Taken together, this is the first gain-of-function study showing that the N-terminal domain of NS4B can determine CSFV genome replication in cell culture and viral pathogenicity in pigs. PMID:26018962

  9. Two single amino acid substitutions in the intervening region of Newcastle disease virus HN protein attenuate viral replication and pathogenicity

    PubMed Central

    Liu, Bin; Ji, Yanhong; Lin, Zhongqing; Fu, Yuguang; Muhammad Dafallah, Rihab; Zhu, Qiyun

    2015-01-01

    Among the proteins encoded by Newcastle disease virus (NDV), the attachment protein (HN) is an important determinant of virulence and pathogenicity. HN has been molecularly characterized at the protein level; however, the relationship between the molecular character of HN and the animal pathotype it causes has not been well explored. Here, we revisited the intervening region (IR) of the HN stalk and extended the known biological functions of HN. Three distinct substitutions (A89Q, P93A, and L94A) in the IR of genotype VII NDV (G7 strain) HN protein were analyzed. The A89Q and L94A mutations weakened the fusion promotion activity of HN to 44% and 41% of that of wild type, respectively, whereas P93A decreased the neuraminidase activity to 21% of the parental level. At the virus level, P93A and L94A-bearing viruses displayed impaired receptor recognition ability, neuraminidase activity, and fusion-promoting activity, all of which led to virus attenuation. In addition, the L94A-mutated virus showed a dramatic decline in replication and was attenuated in cells and in chickens. Our data demonstrate that the HN biological activities and functions modulated by these specific amino acids in the IR are associated with NDV replication and pathogenicity. PMID:26267791

  10. The presence of tomato leaf curl Kerala virus AC3 protein enhances viral DNA replication and modulates virus induced gene-silencing mechanism in tomato plants

    PubMed Central

    2011-01-01

    Background Geminiviruses encode few viral proteins. Most of the geminiviral proteins are multifunctional and influence various host cellular processes for the successful viral infection. Though few viral proteins like AC1 and AC2 are well characterized for their multiple functions, role of AC3 in the successful viral infection has not been investigated in detail. Results We performed phage display analysis with the purified recombinant AC3 protein with Maltose Binding Protein as fusion tag (MBP-AC3). Putative AC3 interacting peptides identified through phage display were observed to be homologous to peptides of proteins from various metabolisms. We grouped these putative AC3 interacting peptides according to the known metabolic function of the homologous peptide containing proteins. In order to check if AC3 influences any of these particular metabolic pathways, we designed vectors for assaying DNA replication and virus induced gene-silencing of host gene PCNA. Investigation with these vectors indicated that AC3 enhances viral replication in the host plant tomato. In the PCNA gene-silencing experiment, we observed that the presence of functional AC3 ORF strongly manifested the stunted phenotype associated with the virus induced gene-silencing of PCNA in tomato plants. Conclusions Through the phage display analysis proteins from various metabolic pathways were identified as putative AC3 interacting proteins. By utilizing the vectors developed, we could analyze the role of AC3 in viral DNA replication and host gene-silencing. Our studies indicate that AC3 is also a multifunctional protein. PMID:21496351

  11. Productive replication of nephropathogenic infectious bronchitis virus in peripheral blood monocytic cells, a strategy for viral dissemination and kidney infection in chickens.

    PubMed

    Reddy, Vishwanatha R A P; Trus, Ivan; Desmarets, Lowiese M B; Li, Yewei; Theuns, Sebastiaan; Nauwynck, Hans J

    2016-01-01

    In the present study, the replication kinetics of nephropathogenic (B1648) and respiratory (Massachusetts-M41) IBV strains were compared in vitro in respiratory mucosa explants and blood monocytes (KUL01(+) cells), and in vivo in chickens to understand why some IBV strains have a kidney tropism. B1648 was replicating somewhat better than M41 in the epithelium of the respiratory mucosa explants and used more KUL01(+) cells to penetrate the deeper layers of the respiratory tract. B1648 was productively replicating in KUL01(+) monocytic cells in contrast with M41. In B1648 inoculated animals, 10(2.7-6.8) viral RNA copies/100 mg were detected in tracheal secretions at 2, 4, 6, 8, 10 and 12 days post inoculation (dpi), 10(2.4-4.5) viral RNA copies/mL in plasma at 2, 4, 6, 8, 10 and 12 dpi and 10(1.8-4.4) viral RNA copies/10(6) mononuclear cells in blood at 2, 4, 6 and 8 dpi. In M41 inoculated animals, 10(2.6-7.0) viral RNA copies/100 mg were detected in tracheal secretions at 2, 4, 6, 8 and 10 dpi, but viral RNA was not demonstrated in plasma and mononuclear cells (except in one chicken at 6 dpi). Infectious virus was detected only in plasma and mononuclear cells of the B1648 group. At euthanasia (12 dpi), viral RNA and antigen positive cells were detected in lungs, liver, spleen and kidneys of only the B1648 group and in tracheas of both the B1648 and M41 group. In conclusion, only B1648 can easily disseminate to internal organs via a cell-free and -associated viremia with KUL01(+) cells as important carrier cells. PMID:27412035

  12. HCV Infection Induces Autocrine Interferon Signaling by Human Liver Endothelial Cell and Release of Exosomes, Which Inhibits Viral Replication

    PubMed Central

    Giugliano, Silvia; Kriss, Michael; Golden-Mason, Lucy; Dobrinskikh, Evgenia; Stone, Amy E.L.; Soto-Gutierrez, Alejandro; Mitchell, Angela; Khetani, Salman R.; Yamane, Daisuke; Stoddard, Mark; Li, Hui; Shaw, George M.; Edwards, Michael G.; Lemon, Stanley M.; Gale, Michael; Shah, Vijay H.; Rosen, Hugo R.

    2014-01-01

    Background & Aims Liver sinusoidal endothelial cells (LSECs) make up a large proportion of the non-parenchymal cells in the liver. LSECs are involved in induction of immune tolerance, but little is known about their functions during hepatitis C virus (HCV) infection. Methods Primary human LSECs (HLSECs) and immortalized liver endothelial cells (TMNK-1) were exposed to various forms of HCV, including full-length transmitted/founder virus, sucrose-purified Japanese Fulminant Hepatitis-1 (JFH-1), a virus encoding a luciferase reporter, and the HCV-specific pathogen-associated molecular pattern molecules. Cells were analyzed by confocal immunofluorescence, immunohistochemical, and PCR assays. Results HLSECs internalized HCV, independent of cell–cell contacts; HCV RNA was translated but not replicated. Through pattern recognition receptors (TLR7 and retinoic acid inducible gene 1), HCV RNA induced consistent and broad transcription of multiple interferons (IFNs); supernatants from primary HLSECs transfected with HCV-specific pathogen-associated molecular pattern molecules increased induction of IFNs and IFN-stimulated genes in HLSECs. Recombinant type I and type III IFNs strongly up-regulated HLSEC transcription of interferon λ 3 (IFNL3) and viperin (RSAD2), which inhibit replication of HCV. Compared to CD8+ T cells, HLSECs suppressed HCV replication within Huh7.5.1 cells, also inducing IFN-stimulated genes in co-culture. Conditioned media from IFN-stimulated HLSECs induced expression of antiviral genes by uninfected primary human hepatocytes. Exosomes, derived from HLSECs following stimulation with either type I or type III IFNs, controlled HCV replication in a dose-dependent manner. Conclusions Cultured HLSECs produce factors that mediate immunity against HCV. HLSECs induce self-amplifying IFN-mediated responses and release of exosomes with antiviral activity. PMID:25447848

  13. Kaposi's Sarcoma Associated Herpesvirus Tegument Protein ORF75 Is Essential for Viral Lytic Replication and Plays a Critical Role in the Antagonization of ND10-Instituted Intrinsic Immunity

    PubMed Central

    Full, Florian; Jungnickl, Doris; Reuter, Nina; Bogner, Elke; Brulois, Kevin; Scholz, Brigitte; Stürzl, Michael; Myoung, Jinjong; Jung, Jae U.; Stamminger, Thomas; Ensser, Armin

    2014-01-01

    Nuclear domain 10 (ND10) components are restriction factors that inhibit herpesviral replication. Effector proteins of different herpesviruses can antagonize this restriction by a variety of strategies, including degradation or relocalization of ND10 proteins. We investigated the interplay of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) infection and cellular defense by nuclear domain 10 (ND10) components. Knock-down experiments in primary human cells show that KSHV-infection is restricted by the ND10 components PML and Sp100, but not by ATRX. After KSHV infection, ATRX is efficiently depleted and Daxx is dispersed from ND10, indicating that these two ND10 components can be antagonized by KSHV. We then identified the ORF75 tegument protein of KSHV as the viral factor that induces the disappearance of ATRX and relocalization of Daxx. ORF75 belongs to a viral protein family (viral FGARATs) that has homologous proteins in all gamma-herpesviruses. Isolated expression of ORF75 in primary cells induces a relocalization of PML and dispersal of Sp100, indicating that this viral effector protein is able to influence multiple ND10 components. Moreover, by constructing a KSHV mutant harboring a stop codon at the beginning of ORF75, we could demonstrate that ORF75 is absolutely essential for viral replication and the initiation of viral immediate-early gene expression. Using recombinant viruses either carrying Flag- or YFP-tagged variants of ORF75, we could further corroborate the role of ORF75 in the antagonization of ND10-mediated intrinsic immunity, and show that it is independent of the PML antagonist vIRF3. Members of the viral FGARAT family target different ND10 components, suggesting that the ND10 targets of viral FGARAT proteins have diversified during evolution. We assume that overcoming ND10 intrinsic defense constitutes a critical event in the replication of all herpesviruses; on the other hand, restriction of herpesviral replication by ND10 components may also

  14. 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

  15. Granulocyte-Macrophage Colony-Stimulating Factor Expressed by Recombinant Respiratory Syncytial Virus Attenuates Viral Replication and Increases the Level of Pulmonary Antigen-Presenting Cells

    PubMed Central

    Bukreyev, Alexander; Belyakov, Igor M.; Berzofsky, Jay A.; Murphy, Brian R.; Collins, Peter L.

    2001-01-01

    An obstacle to developing a vaccine against human respiratory syncytial virus (RSV) is that natural infection typically does not confer solid immunity to reinfection. To investigate methods to augment the immune response, recombinant RSV (rRSV) was constructed that expresses murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) from a transcription cassette inserted into the G-F intergenic region. Replication of rRSV/mGM-CSF in the upper and lower respiratory tracts of BALB/c mice was reduced 23- to 74- and 5- to 588-fold, respectively, compared to that of the parental rRSV. Despite this strong attenuation of replication, the level of RSV-specific serum antibodies induced by rRSV/mGM-CSF was comparable to, or marginally higher than, that of the parental rRSV. The induction of RSV-specific CD8+ cytotoxic T cells was moderately reduced during the initial infection, which might be a consequence of reduced antigen expression. Mice infected with rRSV/mGM-CSF had elevated levels of pulmonary mRNA for gamma interferon (IFN-γ) and interleukin 12 (IL-12) p40 compared to animals infected by wild-type rRSV. Elevated synthesis of IFN-γ could account for the restriction of RSV replication, as was observed previously with an IFN-γ-expressing rRSV. The accumulation of total pulmonary mononuclear cells and total CD4+ T lymphocytes was accelerated in animals infected with rRSV/mGM-CSF compared to that in animals infected with the control virus, and the level of IFN-γ-positive or IL-4-positive pulmonary CD4+ cells was elevated approximately twofold. The number of pulmonary lymphoid and myeloid dendritic cells and macrophages was increased up to fourfold in mice infected with rRSV/mGM-CSF compared to those infected with the parental rRSV, and the mean expression of major histocompatibility complex class II molecules, a marker of activation, was significantly increased in the two subsets of dendritic cells. Enhanced antigen presentation likely accounts for the

  16. Compartmentalization of prokaryotic DNA replication.

    PubMed

    Bravo, Alicia; Serrano-Heras, Gemma; Salas, Margarita

    2005-01-01

    It becomes now apparent that prokaryotic DNA replication takes place at specific intracellular locations. Early studies indicated that chromosomal DNA replication, as well as plasmid and viral DNA replication, occurs in close association with the bacterial membrane. Moreover, over the last several years, it has been shown that some replication proteins and specific DNA sequences are localized to particular subcellular regions in bacteria, supporting the existence of replication compartments. Although the mechanisms underlying compartmentalization of prokaryotic DNA replication are largely unknown, the docking of replication factors to large organizing structures may be important for the assembly of active replication complexes. In this article, we review the current state of this subject in two bacterial species, Escherichia coli and Bacillus subtilis, focusing our attention in both chromosomal and extrachromosomal DNA replication. A comparison with eukaryotic systems is also presented.

  17. Residues in human respiratory syncytial virus P protein that are essential for its activity on RNA viral synthesis.

    PubMed

    Asenjo, Ana; Mendieta, Jesús; Gómez-Puertas, Paulino; Villanueva, Nieves

    2008-03-01

    Human respiratory syncytial virus (HRSV) P protein, 241 amino acid long, is a structural homotetrameric phosphoprotein. Viral transcription and replication processes are dependent on functional P protein interactions inside viral ribonucleoprotein complexes (RNPs). Binding capacity to RNPs proteins and transcription and replication complementation analyses, using inactive P protein variants, have identified residues essential for functional interactions with itself, L, N and M2-1 proteins. P protein may establish some of these interactions as monomer, but efficient viral transcription and replication requires P protein oligomerization through the central region of the molecule. A structurally stable three-dimensional model has been generated in silico for this region (residues 98-158). Our analysis has indicated that P protein residues L135, D139, E140 and L142 are involved in homotetramerization. Additionally, the residues D136, S156, T160 and E179 appear to be essential for P protein activity on viral RNA synthesis and very high turnover phosphorylation at S143, T160 and T210 could regulate it. Thus, compounds targeted to those of these residues, located in the modeled three-dimensional structure, could have specific anti-HRSV effect.

  18. An A14U Substitution in the 3′ Noncoding Region of the M Segment of Viral RNA Supports Replication of Influenza Virus with an NS1 Deletion by Modulating Alternative Splicing of M Segment mRNAs

    PubMed Central

    Zheng, Min; Wang, Pui; Song, Wenjun; Lau, Siu-Ying; Liu, Siwen; Huang, Xiaofeng; Mok, Bobo Wing-Yee; Liu, Yen-Chin; Chen, Yixin

    2015-01-01

    ABSTRACT The NS1 protein of influenza virus has multiple functions and is a determinant of virulence. Influenza viruses with NS1 deletions (DelNS1 influenza viruses) are a useful tool for studying virus replication and can serve as effective live attenuated vaccines, but deletion of NS1 severely diminishes virus replication, hampering functional studies and vaccine production. We found that WSN-DelNS1 viruses passaged in cells consistently adapted to gain an A14U substitution in the 3′ noncoding region of the M segment of viral RNA (vRNA) which restored replicative ability. DelNS1-M-A14U viruses cannot inhibit interferon expression in virus infected-cells, providing an essential model for studying virus replication in the absence of the NS1 protein. Characterization of DelNS1-M-A14U virus showed that the lack of NS1 has no apparent effect on expression of other viral proteins, with the exception of M mRNAs. Expression of the M transcripts, M1, M2, mRNA3, and mRNA4, is regulated by alternative splicing. The A14U substitution changes the splicing donor site consensus sequence of mRNA3, altering expression of M transcripts, with M2 expression significantly increased and mRNA3 markedly suppressed in DelNS1-M-A14U, but not DelNS1-M-WT, virus-infected cells. Further analysis revealed that the A14U substitution also affects promoter function during replication of the viral genome. The M-A14U mutation increases M vRNA synthesis in DelNS1 virus infection and enhances alternative splicing of M2 mRNA in the absence of other viral proteins. The findings demonstrate that NS1 is directly involved in influenza virus replication through modulation of alternative splicing of M transcripts and provide strategic information important to construction of vaccine strains with NS1 deletions. IMPORTANCE Nonstructural protein (NS1) of influenza virus has multiple functions. Besides its role in antagonizing host antiviral activity, NS1 is also believed to be involved in regulating virus

  19. 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 Central

    Rattay, Stephanie; Trilling, Mirko; Megger, Dominik A.; Sitek, Barbara; Meyer, Helmut E.; Hengel, Hartmut

    2015-01-01

    ABSTRACT 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. IMPORTANCE 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

  20. Discordant Impact of HLA on Viral Replicative Capacity and Disease Progression in Pediatric and Adult HIV Infection.

    PubMed

    Adland, Emily; Paioni, Paolo; Thobakgale, Christina; Laker, Leana; Mori, Luisa; Muenchhoff, Maximilian; Csala, Anna; Clapson, Margaret; Flynn, Jacquie; Novelli, Vas; Hurst, Jacob; Naidoo, Vanessa; Shapiro, Roger; Huang, Kuan-Hsiang Gary; Frater, John; Prendergast, Andrew; Prado, Julia G; Ndung'u, Thumbi; Walker, Bruce D; Carrington, Mary; Jooste, Pieter; Goulder, Philip J R

    2015-06-01

    HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p<0.0001), but not in children. Similarly, in adults, but not in children, VRCs were significantly higher in subjects expressing the disease-susceptible alleles HLA-B*18:01/45:01/58:02 (p = 0.007). Irrespective of the subject, VRCs were strongly correlated with the number of Gag CD8+ T-cell escape mutants driven by HLA-B*57/58:01/81:01 present in each virus (p = 0.0002). In contrast to the impact of VRC common to progression in adults and children, the HLA effects on disease outcome, that are substantial in adults, are small and statistically insignificant in infected children. These data further highlight the important role that VRC plays both in adult and pediatric progression, and demonstrate that HLA-independent factors, yet to be fully defined, are predominantly responsible for pediatric non-progression.

  1. HMGB1 Promotes Hepatitis C Virus Replication by Interaction with Stem-Loop 4 in the Viral 5′ Untranslated Region

    PubMed Central

    Yu, Rong; Yang, Darong; Lei, Shaohua; Wang, Xiaohong; Meng, Xianghe; Xue, Binbin

    2015-01-01

    ABSTRACT High-mobility group box 1 (HMGB1) protein is a highly conserved nuclear protein involved in multiple human diseases, including infectious diseases, immune disorders, metabolic disorders, and cancer. HMGB1 is comprised of two tandem HMG boxes (the A box and the B box) containing DNA-binding domains and an acidic C-terminal peptide. It has been reported that HMGB1 enhances viral replication by binding to viral proteins. However, its role in hepatitis C virus (HCV) replication is unknown. Here, we show that HMGB1 promoted HCV replication but had no effect on HCV translation. RNA immunoprecipitation experiments indicated that the positive strand, not the negative strand, of HCV RNA interacted with HMGB1. HCV infection triggered HMGB1 protein translocation from the nucleus to the cytoplasm, in which it interacted with the HCV genome. Moreover, the A box of HMGB1 is the pivotal domain to interact with stem-loop 4 (SL4) of the HCV 5′ untranslated region. Deletion of the HMGB1 A box abrogated the enhancement of HCV replication by HMGB1. Our data suggested that HMGB1 serves as a proviral factor of HCV to facilitate viral replication in hepatocytes by interaction with the HCV genome. IMPORTANCE Hepatitis C virus (HCV) is a major global health threat, affecting more than 170 million people infection worldwide. These patients are at high risk of developing severe liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Currently, no vaccine is available. Many host factors may be implicated in the pathogenesis of HCV-related diseases. In this study, we found a novel HCV RNA-binding protein, HMGB1, that promotes HCV RNA replication. Moreover, SL4 in the 5′ untranslated region of the HCV genome is the key region for HMGB1 binding, and the A box of HMGB1 protein is the functional domain to interact with HCV RNA and enhance viral replication. HMGB1 appears to play an important role in HCV-related diseases, and further investigation is

  2. Cholesterol-Enriched Domain Formation Induced by Viral-Encoded, Membrane-Active Amphipathic Peptide.

    PubMed

    Hanson, Joshua M; Gettel, Douglas L; Tabaei, Seyed R; Jackman, Joshua; Kim, Min Chul; Sasaki, Darryl Y; Groves, Jay T; Liedberg, Bo; Cho, Nam-Joon; Parikh, Atul N

    2016-01-01

    The α-helical (AH) domain of the hepatitis C virus nonstructural protein NS5A, anchored at the cytoplasmic leaflet of the endoplasmic reticulum, plays a role in viral replication. However, the peptides derived from this domain also exhibit remarkably broad-spectrum virocidal activity, raising questions about their modes of membrane association. Here, using giant lipid vesicles, we show that the AH peptide discriminates between membrane compositions. In cholesterol-containing membranes, peptide binding induces microdomain formation. By contrast, cholesterol-depleted membranes undergo global softening at elevated peptide concentrations. Furthermore, in mixed populations, the presence of ∼100 nm vesicles of viral dimensions suppresses these peptide-induced perturbations in giant unilamellar vesicles, suggesting size-dependent membrane association. These synergistic composition- and size-dependent interactions explain, in part, how the AH domain might on the one hand segregate molecules needed for viral assembly and on the other hand furnish peptides that exhibit broad-spectrum virocidal activity.

  3. Mutations Designed by Ensemble Defect to Misfold Conserved RNA Structures of Influenza A Segments 7 and 8 Affect Splicing and Attenuate Viral Replication in Cell Culture

    PubMed Central

    Jiang, Tian; Nogales, Aitor; Baker, Steven F; Martinez-Sobrido, Luis; Turner, Douglas H

    2016-01-01

    Influenza A virus is a significant public health threat, but little is understood about the viral RNA structure and function. Current vaccines and therapeutic options to control influenza A virus infections are mostly protein-centric and of limited effectiveness. Here, we report using an ensemble defect approach to design mutations to misfold regions of conserved mRNA structures in influenza A virus segments 7 and 8. Influenza A mutant viruses inhibit pre-mRNA splicing and attenuate viral replication in cell culture, thus providing evidence for functions of the targeted regions. Targeting these influenza A viral RNA regions provides new possibilities for designing vaccines and therapeutics against this important human respiratory pathogen. The results also demonstrate that the ensemble defect approach is an efficient way to test for function of RNA sequences. PMID:27272307

  4. Low-Replicating Viruses and Strong Anti-Viral Immune Response Associated with Prolonged Disease Control in a Superinfected HIV-1 LTNP Elite Controller

    PubMed Central

    Pernas, María; Casado, Concepción; Arcones, Carolina; Llano, Anuska; Sánchez-Merino, Víctor; Mothe, Beatriz; Vicario, José L.; Grau, Eulalia; Ruiz, Lidia; Sánchez, Jorge; Telenti, Amalio; Yuste, Eloísa; Brander, Christian; Galíndez, Cecilio López-

    2012-01-01

    Objective To study the causes for the lack of clinical progression in a superinfected HIV-1 LTNP elite controller patient. Methodology and Principal Findings We studied host genetic, virological and immunological factors associated with viral control in a SI long term non progressor elite controller (LTNP-EC). The individual contained both viruses and maintained undetectable viral loads for >20 years and he did not express any of the described host genetic polymorphisms associated with viral control. None of four full-length gp160 recombinants derived from the LTNP-EC replicated in heterologous peripheral blood mononuclear cells. CTL responses after SI were maintained in two samples separated by 9 years and they were higher in breadth and magnitude than responses seen in most of 250 treatment naïve patients and also 25 controller subjects. The LTNP-EC showed a neutralization response, against 4 of the 6 viruses analyzed, superior to other ECs. Conclusions The study demonstrated that a strong and sustained cellular and humoral immune response and low replicating viruses are associated with viral control in the superinfected LTNP-EC. PMID:22384103

  5. BK Polyomavirus Replication in Renal Tubular Epithelial Cells Is Inhibited by Sirolimus, but Activated by Tacrolimus Through a Pathway Involving FKBP‐12

    PubMed Central

    Yakhontova, K.; Lu, M.; Manzetti, J.

    2015-01-01

    BK polyomavirus (BKPyV) replication causes nephropathy and premature kidney transplant failure. Insufficient BKPyV‐specific T cell control is regarded as a key mechanism, but direct effects of immunosuppressive drugs on BKPyV replication might play an additional role. We compared the effects of mammalian target of rapamycin (mTOR)‐ and calcineurin‐inhibitors on BKPyV replication in primary human renal tubular epithelial cells. Sirolimus impaired BKPyV replication with a 90% inhibitory concentration of 4 ng/mL by interfering with mTOR–SP6‐kinase activation. Sirolimus inhibition was rapid and effective up to 24 h postinfection during viral early gene expression, but not thereafter, during viral late gene expression. The mTORC‐1 kinase inhibitor torin‐1 showed a similar inhibition profile, supporting the notion that early steps of BKPyV replication depend on mTOR activity. Cyclosporine A also inhibited BKPyV replication, while tacrolimus activated BKPyV replication and reversed sirolimus inhibition. FK binding protein 12kda (FKBP‐12) siRNA knockdown abrogated sirolimus inhibition and increased BKPyV replication similar to adding tacrolimus. Thus, sirolimus and tacrolimus exert opposite effects on BKPyV replication in renal tubular epithelial cells by a mechanism involving FKBP‐12 as common target. Immunosuppressive drugs may therefore contribute directly to the risk of BKPyV replication and nephropathy besides suppressing T cell functions. The data provide rationales for clinical trials aiming at reducing the risk of BKPyV replication and disease in kidney transplantation. PMID:26639422

  6. Hepatitis C Virus Co-Opts Ras-GTPase-Activating Protein-Binding Protein 1 for Its Genome Replication

    PubMed Central

    Yi, Zhigang; Pan, Tingting; Wu, Xianfang; Song, Wuhui; Wang, Shanshan; Xu, Yan; Rice, Charles M.; MacDonald, Margaret R.; Yuan, Zhenghong

    2011-01-01

    We recently reported that Ras-GTPase-activating protein-binding protein 1 (G3BP1) interacts with hepatitis C virus (HCV) nonstructural protein (NS)5B and the 5′ end of the HCV minus-strand RNA. In the current study we confirmed these observations using immunoprecipitation and RNA pulldown assays, suggesting that G3BP1 might be an HCV replication complex (RC) component. In replicon cells, transfected G3BP1 interacts with multiple HCV nonstructural proteins. Using immunostaining and confocal microscopy, we demonstrate that G3BP1 is colocalized with HCV RCs in replicon cells. Small interfering RNA (siRNA)-mediated knockdown of G3BP1 moderately reduces established HCV RNA replication in HCV replicon cells and dramatically reduces HCV replication-dependent colony formation and cell-culture-produced HCV (HCVcc) infection. In contrast, knockdown of G3BP2 has no effect on HCVcc infection. Transient replication experiments show that G3BP1 is involved in HCV genome amplification. Thus, G3BP1 is associated with HCV RCs and may be co-opted as a functional RC component for viral replication. These findings may facilitate understanding of the molecular mechanisms of HCV genome replication. PMID:21561913

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

    PubMed

    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

  8. 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

  9. Massive Activation of Archaeal Defense Genes during Viral Infection

    PubMed Central

    Voet, Marleen; Sismeiro, Odile; Dillies, Marie-Agnes; Jagla, Bernd; Coppée, Jean-Yves; Sezonov, Guennadi; Forterre, Patrick; van der Oost, John; Lavigne, Rob

    2013-01-01

    Archaeal viruses display unusually high genetic and morphological diversity. Studies of these viruses proved to be instrumental for the expansion of knowledge on viral diversity and evolution. The Sulfolobus islandicus rod-shaped virus 2 (SIRV2) is a model to study virus-host interactions in Archaea. It is a lytic virus that exploits a unique egress mechanism based on the formation of remarkable pyramidal structures on the host cell envelope. Using whole-transcriptome sequencing, we present here a global map defining host and viral gene expression during the infection cycle of SIRV2 in its hyperthermophilic host S. islandicus LAL14/1. This information was used, in combination with a yeast two-hybrid analysis of SIRV2 protein interactions, to advance current understanding of viral gene functions. As a consequence of SIRV2 infection, transcription of more than one-third of S. islandicus genes was differentially regulated. While expression of genes involved in cell division decreased, those genes playing a role in antiviral defense were activated on a large scale. Expression of genes belonging to toxin-antitoxin and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems was specifically pronounced. The observed different degree of activation of various CRISPR-Cas systems highlights the specialized functions they perform. The information on individual gene expression and activation of antiviral defense systems is expected to aid future studies aimed at detailed understanding of the functions and interplay of these systems in vivo. PMID:23698312

  10. The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin

    PubMed Central

    Dimude, Juachi U.; Stockum, Anna; Midgley-Smith, Sarah L.; Upton, Amy L.; Foster, Helen A.; Khan, Arshad; Saunders, Nigel J.; Retkute, Renata

    2015-01-01

    ABSTRACT Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli, the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid metabolism. In cells lacking RNase HI, replication initiates independently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI, origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics, and, ultimately, cell viability. PMID:26530381

  11. Novel antiviral activity of bromocriptine against dengue virus replication.

    PubMed

    Kato, Fumihiro; Ishida, Yuki; Oishi, Shinya; Fujii, Nobutaka; Watanabe, Satoru; Vasudevan, Subhash G; Tajima, Shigeru; Takasaki, Tomohiko; Suzuki, Youichi; Ichiyama, Koji; Yamamoto, Naoki; Yoshii, Kentaro; Takashima, Ikuo; Kobayashi, Takeshi; Miura, Tomoyuki; Igarashi, Tatsuhiko; Hishiki, Takayuki

    2016-07-01

    Dengue virus (DENV) infectious disease is a major public health problem worldwide; however, licensed vaccines or specific antiviral drugs against this infection are not available. To identify novel anti-DENV compounds, we screened 1280 pharmacologically active compounds using focus reduction assay. Bromocriptine (BRC) was found to have potent anti-DENV activity and low cytotoxicity (half maximal effective concentration [EC50], 0.8-1.6 μM; and half maximal cytotoxicity concentration [CC50], 53.6 μM). Time-of-drug-addition and time-of-drug-elimination assays suggested that BRC inhibits translation and/or replication steps in the DENV life cycle. A subgenomic replicon system was used to verify that BRC restricts RNA replication step. Furthermore, a single amino acid substitution (N374H) was detected in the NS3 protein that conferred resistance to BRC. In summary, BRC was found to be a novel DENV inhibitor and a potential candidate for the treatment of DENV infectious disease.

  12. Host Acyl Coenzyme A Binding Protein Regulates Replication Complex Assembly and Activity of a Positive-Strand RNA Virus

    PubMed Central

    Zhang, Jiantao; Diaz, Arturo; Mao, Lan; Ahlquist, Paul

    2012-01-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly. PMID:22345450

  13. Host acyl coenzyme A binding protein regulates replication complex assembly and activity of a positive-strand RNA virus.

    PubMed

    Zhang, Jiantao; Diaz, Arturo; Mao, Lan; Ahlquist, Paul; Wang, Xiaofeng

    2012-05-01

    All positive-strand RNA viruses reorganize host intracellular membranes to assemble their replication complexes. Similarly, brome mosaic virus (BMV) induces two alternate forms of membrane-bound RNA replication complexes: vesicular spherules and stacks of appressed double-membrane layers. The mechanisms by which these membrane rearrangements are induced, however, remain unclear. We report here that host ACB1-encoded acyl coenzyme A (acyl-CoA) binding protein (ACBP) is required for the assembly and activity of both BMV RNA replication complexes. ACBP is highly conserved among eukaryotes, specifically binds to long-chain fatty acyl-CoA, and promotes general lipid synthesis. Deleting ACB1 inhibited BMV RNA replication up to 30-fold and resulted in formation of spherules that were ∼50% smaller but ∼4-fold more abundant than those in wild-type (wt) cells, consistent with the idea that BMV 1a invaginates and maintains viral spherules by coating the inner spherule membrane. Furthermore, smaller and more frequent spherules were preferentially formed under conditions that induce layer formation in wt cells. Conversely, cellular karmella structures, which are arrays of endoplasmic reticulum (ER) membranes formed upon overexpression of certain cellular ER membrane proteins, were formed normally, indicating a selective inhibition of 1a-induced membrane rearrangements. Restoring altered lipid composition largely complemented the BMV RNA replication defect, suggesting that ACBP was required for maintaining lipid homeostasis. Smaller and more frequent spherules are also induced by 1a mutants with specific substitutions in a membrane-anchoring amphipathic α-helix, implying that the 1a-lipid interactions play critical roles in viral replication complex assembly.

  14. Loss of Smu1 function de-represses DNA replication and over-activates ATR-dependent replication checkpoint.

    PubMed

    Ren, Laifeng; Liu, Yao; Guo, Liandi; Wang, Haibin; Ma, Lei; Zeng, Ming; Shao, Xin; Yang, Chunlei; Tang, Yaxiong; Wang, Lei; Liu, Cong; Li, Mingyuan

    2013-06-28

    Smu1 is an evolutionarily conserved gene that encodes a member of the WD40-repeat protein family. Disruption of Smu1 function leads to multiple cellular defects including chromosomal instability, aberrant DNA replication and alternative RNA splicing events. In this paper, we show that Smu1 is a chromatin-bound protein that functions as a negative regulator of DNA replication. Knockdown of Smu1 gene expression promotes excessive incorporation of dNTP analogue, implicating the acceleration of DNA synthesis. Smu1-silenced cells show an excessive activation of replication checkpoint in response to ultraviolate (UV) or hydroxyurea treatment, indicating that abnormal stimulation of DNA replication leads to instability of genomic structure. Hence, we propose that Smu1 participates in the protection of genomic integrity by negatively regulating the process of DNA synthesis. PMID:23727573

  15. The early region 1B 55-kilodalton oncoprotein of adenovirus relieves growth restrictions imposed on viral replication by the cell cycle.

    PubMed Central

    Goodrum, F D; Ornelles, D A

    1997-01-01

    The E1B 55-kDa oncoprotein of adenovirus enables the virus to overcome restrictions imposed on viral replication by the cell cycle. Approximately 20% of HeLa cells infected with an E1B 55-kDa mutant adenovirus produced virus when evaluated by electron microscopy or by assays for infectious centers. By contrast, all HeLa cells infected with a wild-type adenovirus produced virus. The yield of E1B mutant virus from randomly cycling HeLa cells correlated with the fraction of cells in S phase at the time of infection. In synchronously growing HeLa cells, approximately 75% of the cells infected during S phase with the E1B mutant virus produced virus, whereas only 10% of the cells infected during G1 produced virus. The yield of E1B mutant virus from HeLa cells infected during S phase was sevenfold greater than that of cells infected during G1 and threefold greater than that of cells infected during asynchronous growth. Cells infected during S phase with the E1B mutant virus exhibited severe cytopathic effects, whereas cells infected with the E1B mutant virus during G1 exhibited a mild cytopathic effect. Viral DNA synthesis appeared independent of the cell cycle because equivalent amounts of viral DNA were synthesized in cells infected with either wild-type or E1B mutant virus. The inability of the E1B mutant virus to replicate was not mediated by the status of p53. These results define a novel property of the large tumor antigen of adenovirus in relieving growth restrictions imposed on viral replication by the cell cycle. PMID:8985383

  16. Specific induction of endogenous viral restriction factors using CRISPR/Cas-derived transcriptional activators

    PubMed Central

    Bogerd, Hal P.; Kornepati, Anand V. R.; Marshall, Joy B.; Kennedy, Edward M.; Cullen, Bryan R.

    2015-01-01

    Whereas several mammalian proteins can restrict the replication of HIV-1 and other viruses, these are often not expressed in relevant target cells. A potential method to inhibit viral replication might therefore be to use synthetic transcription factors to induce restriction factor expression. In particular, mutants of the RNA-guided DNA binding protein Cas9 that have lost their DNA cleavage activity could be used to recruit transcription activation domains to specific promoters. However, initial experiments revealed only weak activation unless multiple promoter-specific single guide RNAs (sgRNAs) were used. Recently, the recruitment of multiple transcription activation domains by a single sgRNA, modified to contain MS2-derived stem loops that recruit fusion proteins consisting of the MS2 coat protein linked to transcription activation domains, was reported to induce otherwise silent cellular genes. Here, we demonstrate that such “synergistic activation mediators” can induce the expression of two restriction factors, APOBEC3G (A3G) and APOBEC3B (A3B), in human cells that normally lack these proteins. We observed modest activation of endogenous A3G or A3B expression using single sgRNAs but high expression when two sgRNAs were used. Whereas the induced A3G and A3B proteins both blocked infection by an HIV-1 variant lacking a functional vif gene by inducing extensive dC-to-dU editing, only the induced A3B protein inhibited wild-type HIV-1. These data demonstrate that Cas9-derived transcriptional activators have the potential to be used for screens for endogenous genes that affect virus replication and raise the possibility that synthetic transcription factors might prove clinically useful if efficient delivery mechanisms could be developed. PMID:26668372

  17. Viral infection

    PubMed Central

    Puigdomènech, Isabel; de Armas-Rillo, Laura; Machado, José-David

    2011-01-01

    Viruses have developed different survival strategies in host cells by crossing cell-membrane compartments, during different steps of their viral life cycle. In fact, the non-regenerative viral membrane of enveloped viruses needs to encounter the dynamic cell-host membrane, during early steps of the infection process, in which both membranes fuse, either at cell-surface or in an endocytic compartment, to promote viral entry and infection. Once inside the cell, many viruses accomplish their replication process through exploiting or modulating membrane traffic, and generating specialized compartments to assure viral replication, viral budding and spreading, which also serve to evade the immune responses against the pathogen. In this review, we have attempted to present some data that highlight the importance of membrane dynamics during viral entry and replicative processes, in order to understand how viruses use and move through different complex and dynamic cell-membrane structures and how they use them to persist. PMID:21966556

  18. Novel Inhibitors of Neurotropic Alphavirus Replication That Improve Host Survival in a Mouse Model of Acute Viral Encephalitis

    PubMed Central

    Sindac, Janice; Yestrepsky, Bryan D.; Barraza, Scott J.; Bolduc, Kyle L.; Blakely, Pennelope K.; Keep, Richard F.; Irani, David N.; Miller, David J.; Larsen, Scott D.

    2012-01-01

    Arboviral encephalitis is a potentially devastating human disease with no approved therapies that target virus replication. We previously discovered a novel class of thieno[3,2-b]pyrrole-based inhibitors active against neurotropic alphaviruses such as western equine encephalitis virus (WEEV) in cultured cells. In this report we describe initial development of these novel antiviral compounds, including bioisosteric replacement of the 4H-thieno[3,2-b]pyrrole core with indole to improve metabolic stability and the introduction of chirality to assess target enantioselectivity. Selected modifications enhanced antiviral activity while maintaining low cytotoxicity, increased stability to microsomal metabolism, and also revealed striking enantiospecific activity in cultured cells. Furthermore, we demonstrate improved outcomes (both symptoms and survival) following treatment with indole analog 9h (CCG-203926) in an in vivo mouse model of alphaviral encephalitis that closely correlate with the enantiospecific in vitro antiviral activity. These results represent a substantial advancement in the early preclinical development of a promising class of novel antiviral drugs against virulent neurotropic alphaviruses. PMID:22428985

  19. Heterogeneous pathological outcomes after experimental pH1N1 influenza infection in ferrets correlate with viral replication and host immune responses in the lung.

    PubMed

    Vidaña, Beatriz; Martínez, Jorge; Martínez-Orellana, Pamela; García Migura, Lourdes; Montoya, María; Martorell, Jaime; Majó, Natàlia

    2014-01-01

    The swine-origin pandemic (p) H1N1 influenza A virus causes mild upper-respiratory tract disease in most human patients. However, some patients developed severe lower-respiratory tract infections with fatal consequences, and the cause of these infections remain unknown. Recently, it has been suggested that different populations have different degrees of susceptibility to pH1N1 strains due to host genetic variations that are associated with inappropriate immune responses against viral genetic characteristics. Here, we tested whether the pathologic patterns of influenza strains that produce different disease outcomes in humans could be reproduced in a ferret model. Our results revealed that the severities of infection did not correspond to particular viral isolate and were not associated with the clinical phenotypes of the corresponding patients. Severe pathological outcomes were associated with higher viral replication, especially in alveolar areas, and with an exacerbated innate cellular immune response that was characterised by substantial phagocytic and cytotoxic cell migration into the lungs. Moreover, detrimental innate cellular responses were linked to the up-regulation of several proinflammatory cytokines and chemokines and the down-regulation of IFNα in the lungs. Additionally, severe lung lesions were associated with greater up-regulations of pro-apoptotic markers and higher levels of apoptotic neutrophils and macrophages. In conclusion, this study confirmed that the clinicopathological outcomes of pH1N1 infection in ferrets were not only due to viral replication abilities but also depended on the hosts' capacities to mount efficient immune responses to control viral infection of the lung.

  20. The Viral Polymerase Inhibitor 7-Deaza-2’-C-Methyladenosine Is a Potent Inhibitor of In Vitro Zika Virus Replication and Delays Disease Progression in a Robust Mouse Infection Model

    PubMed Central

    Zmurko, Joanna; Marques, Rafael E.; Schols, Dominique; Verbeken, Erik; Kaptein, Suzanne J.F.; Neyts, Johan

    2016-01-01

    Zika virus (ZIKV) is an emerging flavivirus typically causing a dengue-like febrile illness, but neurological complications, such as microcephaly in newborns, have potentially been linked to this viral infection. We established a panel of in vitro assays to allow the identification of ZIKV inhibitors and demonstrate that the viral polymerase inhibitor 7-deaza-2’-C-methyladenosine (7DMA) efficiently inhibits replication. Infection of AG129 (IFN-α/β and IFN-γ receptor knock-out) mice with ZIKV resulted in acute neutrophilic encephalitis with viral antigens accumulating in neurons of the brain and spinal cord. Additionally, high levels of viral RNA were detected in the spleen, liver and kidney, and levels of IFN-γ and IL-18 were systematically increased in serum of ZIKV-infected mice. Interestingly, the virus was also detected in testicles of infected mice. In line with its in vitro anti-ZIKV activity, 7DMA reduced viremia and delayed virus-induced morbidity and mortality in infected mice, which also validates this small animal model to assess the in vivo efficacy of novel ZIKV inhibitors. Since AG129 mice can generate an antibody response, and have been used in dengue vaccine studies, the model can also be used to assess the efficacy of ZIKV vaccines.   PMID:27163257

  1. The Viral Polymerase Inhibitor 7-Deaza-2'-C-Methyladenosine Is a Potent Inhibitor of In Vitro Zika Virus Replication and Delays Disease Progression in a Robust Mouse Infection Model.

    PubMed

    Zmurko, Joanna; Marques, Rafael E; Schols, Dominique; Verbeken, Erik; Kaptein, Suzanne J F; Neyts, Johan

    2016-05-01

    Zika virus (ZIKV) is an emerging flavivirus typically causing a dengue-like febrile illness, but neurological complications, such as microcephaly in newborns, have potentially been linked to this viral infection. We established a panel of in vitro assays to allow the identification of ZIKV inhibitors and demonstrate that the viral polymerase inhibitor 7-deaza-2'-C-methyladenosine (7DMA) efficiently inhibits replication. Infection of AG129 (IFN-α/β and IFN-γ receptor knock-out) mice with ZIKV resulted in acute neutrophilic encephalitis with viral antigens accumulating in neurons of the brain and spinal cord. Additionally, high levels of viral RNA were detected in the spleen, liver and kidney, and levels of IFN-γ and IL-18 were systematically increased in serum of ZIKV-infected mice. Interestingly, the virus was also detected in testicles of infected mice. In line with its in vitro anti-ZIKV activity, 7DMA reduced viremia and delayed virus-induced morbidity and mortality in infected mice, which also validates this small animal model to assess the in vivo efficacy of novel ZIKV inhibitors. Since AG129 mice can generate an antibody response, and have been used in dengue vaccine studies, the model can also be used to assess the efficacy of ZIKV vaccines.  .

  2. The Viral Polymerase Inhibitor 7-Deaza-2'-C-Methyladenosine Is a Potent Inhibitor of In Vitro Zika Virus Replication and Delays Disease Progression in a Robust Mouse Infection Model.

    PubMed

    Zmurko, Joanna; Marques, Rafael E; Schols, Dominique; Verbeken, Erik; Kaptein, Suzanne J F; Neyts, Johan

    2016-05-01

    Zika virus (ZIKV) is an emerging flavivirus typically causing a dengue-like febrile illness, but neurological complications, such as microcephaly in newborns, have potentially been linked to this viral infection. We established a panel of in vitro assays to allow the identification of ZIKV inhibitors and demonstrate that the viral polymerase inhibitor 7-deaza-2'-C-methyladenosine (7DMA) efficiently inhibits replication. Infection of AG129 (IFN-α/β and IFN-γ receptor knock-out) mice with ZIKV resulted in acute neutrophilic encephalitis with viral antigens accumulating in neurons of the brain and spinal cord. Additionally, high levels of viral RNA were detected in the spleen, liver and kidney, and levels of IFN-γ and IL-18 were systematically increased in serum of ZIKV-infected mice. Interestingly, the virus was also detected in testicles of infected mice. In line with its in vitro anti-ZIKV activity, 7DMA reduced viremia and delayed virus-induced morbidity and mortality in infected mice, which also validates this small animal model to assess the in vivo efficacy of novel ZIKV inhibitors. Since AG129 mice can generate an antibody response, and have been used in dengue vaccine studies, the model can also be used to assess the efficacy of ZIKV vaccines.  . PMID:27163257

  3. Antiviral Activities of Different Interferon Types and Subtypes against Hepatitis E Virus Replication

    PubMed Central

    Todt, Daniel; François, Catherine; Anggakusuma; Behrendt, Patrick; Engelmann, Michael; Knegendorf, Leonard; Vieyres, Gabrielle; Wedemeyer, Heiner; Hartmann, Rune; Pietschmann, Thomas; Duverlie, Gilles

    2016-01-01

    Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. HEV infections are the common cause of acute hepatitis but can also take chronic courses. Ribavirin is the treatment of choice for most patients, and type I interferon (IFN) has been evaluated in a few infected transplant patients in vivo. In this study, the antiviral effects of different exogenously administered interferons were investigated by using state-of-the-art subgenomic replicon and full-length HEV genome cell culture models. Hepatitis C virus (HCV) subgenomic replicons based on the genotype 2a JFH1 isolate served as the reference. The experiments revealed that HEV RNA replication was inhibited by the application of all types of IFN, including IFN-α (type I), IFN-γ (type II), and IFN-λ3 (type III), but to a far lesser extent than HCV replication. Simultaneous determination of interferon-stimulated gene (ISG) expression levels for all IFN types demonstrated efficient downregulation by HEV. Furthermore, different IFN-α subtypes were also able to block viral replication in combination with ribavirin. The IFN-α subtypes 2a and 2b exerted the strongest antiviral activity against HEV. In conclusion, these data demonstrate for the first time moderate anti-HEV activities of types II and III IFNs and different IFN-α subtypes. As HEV employed a potent anti-interferon mechanism by restricting ISG expression, exogenous application of IFNs as immunotherapy should be carefully assessed. PMID:26787701

  4. Antiviral Activities of Different Interferon Types and Subtypes against Hepatitis E Virus Replication.

    PubMed

    Todt, Daniel; François, Catherine; Anggakusuma; Behrendt, Patrick; Engelmann, Michael; Knegendorf, Leonard; Vieyres, Gabrielle; Wedemeyer, Heiner; Hartmann, Rune; Pietschmann, Thomas; Duverlie, Gilles; Steinmann, Eike

    2016-04-01

    Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genusOrthohepevirusin the familyHepeviridae HEV infections are the common cause of acute hepatitis but can also take chronic courses. Ribavirin is the treatment of choice for most patients, and type I interferon (IFN) has been evaluated in a few infected transplant patientsin vivo In this study, the antiviral effects of different exogenously administered interferons were investigated by using state-of-the-art subgenomic replicon and full-length HEV genome cell culture models. Hepatitis C virus (HCV) subgenomic replicons based on the genotype 2a JFH1 isolate served as the reference. The experiments revealed that HEV RNA replication was inhibited by the application of all types of IFN, including IFN-α (type I), IFN-γ (type II), and IFN-λ3 (type III), but to a far lesser extent than HCV replication. Simultaneous determination of interferon-stimulated gene (ISG) expression levels for all IFN types demonstrated efficient downregulation by HEV. Furthermore, different IFN-α subtypes were also able to block viral replication in combination with ribavirin. The IFN-α subtypes 2a and 2b exerted the strongest antiviral activity against HEV. In conclusion, these data demonstrate for the first time moderate anti-HEV activities of types II and III IFNs and different IFN-α subtypes. As HEV employed a potent anti-interferon mechanism by restricting ISG expression, exogenous application of IFNs as immunotherapy should be carefully assessed. PMID:26787701

  5. A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication.

    PubMed

    Simeon, Rudo L; Chen, Zhilei

    2013-01-01

    Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1's anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides. PMID:24391867

  6. A Screen for Genetic Suppressor Elements of Hepatitis C Virus Identifies a Supercharged Protein Inhibitor of Viral Replication

    PubMed Central

    Simeon, Rudo L.; Chen, Zhilei

    2013-01-01

    Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1’s anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides. PMID:24391867

  7. A screen for genetic suppressor elements of hepatitis C virus identifies a supercharged protein inhibitor of viral replication.

    PubMed

    Simeon, Rudo L; Chen, Zhilei

    2013-01-01

    Genetic suppressor elements (GSEs) are biomolecules derived from a gene or genome of interest that act as transdominant inhibitors of biological functions presumably by disruption of critical biological interfaces. We exploited a cell death reporter cell line for hepatitis C virus (HCV) infection, n4mBid, to develop an iterative selection/enrichment strategy for the identification of anti-HCV GSEs. Using this approach, a library of fragments of an HCV genome was screened for sequences that suppress HCV infection. A 244 amino acid gene fragment, B1, was strongly enriched after 5 rounds of selection. B1 derives from a single-base frameshift of the enhanced green fluorescent protein (eGFP) which was used as a filler during fragment cloning. B1 has a very high net positive charge of 43 at neutral pH and a high charge-to-mass (kDa) ratio of 1.5. We show that B1 expression specifically inhibits HCV replication. In addition, five highly positively charged B1 fragments produced from progressive truncation at the C-terminus all retain the ability to inhibit HCV, suggesting that a high positive charge, rather than a particular motif in B1, likely accounts for B1's anti-HCV activity. Another supercharged protein, +36GFP, was also found to strongly inhibit HCV replication when added to cells at the time of infection. This study reports a new methodology for HCV inhibitor screening and points to the anti-HCV potential of positively charged proteins/peptides.

  8. Silencing of neurotropic flavivirus replication in the central nervous system by combining multiple microRNA target insertions in two distinct viral genome regions

    PubMed Central

    Teterina, Natalya L.; Liu, Guangping; Maximova, Olga A.; Pletnev, Alexander G.

    2014-01-01

    In recent years, microRNA-targeting has become an effective strategy for selective control of tissue-tropism and pathogenesis of both DNA and RNA viruses. Here, using a neurotropic flavivirus as a model, we demonstrate that simultaneous miRNA targeting of the viral genome in the open reading frame and 3′-noncoding regions for brain-expressed miRNAs had an additive effect and produced a more potent attenuation of the virus compared to separate targeting of those regions. Multiple miRNA co-targeting of these two distantly located regions completely abolished the virus neurotropism as no viral replication was detected in the developing brain of neonatal mice. Furthermore, no viral antigens were detected in neurons, and neuronal integrity in the brain of mice was well preserved. This miRNA co-targeting approach can be adapted for other viruses in order to minimize their replication in a cell- or tissue-type specific manner, but most importantly, to prevent virus escape from miRNA-mediated silencing. PMID:24889244

  9. Ribonuclease H activities associated with viral reverse transcriptases are endonucleases.

    PubMed Central

    Krug, M S; Berger, S L

    1989-01-01

    A series of test substrates have been synthesized to establish the effect of termini on the putative exoribonuclease H activity of reverse transcriptase. Recombinant reverse transcriptase from human immunodeficiency virus, natural enzyme from avian myeloblastosis virus, and a known endonuclease, Escherichia coli ribonuclease H, cleaved relaxed, circular, covalently closed plasmids in which 770 consecutive residues of one strand were ribonucleotides. The avian enzyme also deadenylated capped globin mRNA with a covalently attached oligo(dT) tail at the 3' end. These results resolve a long-standing controversy--that the viral enzymes are obligatory exonucleases in vitro, based on their failure to cleave certain substrates for E. coli ribonuclease H, including circular poly(A).linear poly(T) and ribonucleotide-substituted supercoiled plasmids, but resemble endonucleases in vivo, based on their ability to degrade RNA in complex DNA.RNA hybrids. The data strongly suggest that the viral enzymes are endonucleases with exquisite sensitivity to the conformation of heteroduplexes. Inhibition of viral, but not cellular, ribonuclease H with ribonucleoside-vanadyl complexes further distinguishes these enzymes. Images PMID:2471188

  10. The Rac1 inhibitor NSC23766 exerts anti-influenza virus properties by affecting the viral polymerase complex activity.

    PubMed

    Dierkes, Rüdiger; Warnking, Kathrin; Liedmann, Swantje; Seyer, Roman; Ludwig, Stephan; Ehrhardt, Christina

    2014-01-01

    The frequent emergence of new influenza viruses in the human population underlines the urgent need for antiviral therapeutics in addition to the preventative vaccination against the seasonal flu. To circumvent the development of resistance, recent antiviral approaches target cellular proteins needed by the virus for efficient replication. We investigated the contribution of the small GTPase Rac1 to the replication of influenza viruses. Inhibition of Rac1 by NSC23766 resulted in impaired replication of a wide variety of influenza viruses, including a human virus strain of the pandemic from 2009 as well as highly pathogenic avian virus strains. Furthermore, we identified a crucial role of Rac1 for the activity of the viral polymerase complex. The antiviral potential of NSC23766 was confirmed in mouse experiments, identifying Rac1 as a new cellular target for therapeutic treatment of influenza virus infections.

  11. Human cytomegalovirus microRNA miR-US25-1-5p inhibits viral replication by targeting multiple cellular genes during infection.

    PubMed

    Jiang, Shujuan; Qi, Ying; He, Rong; Huang, Yujing; Liu, Zhongyang; Ma, Yanping; Guo, Xin; Shao, Yaozhong; Sun, Zhengrong; Ruan, Qiang

    2015-10-01

    MicroRNAs (miRNAs) play important roles in regulating various cellular processes in plants, animals, and viruses. This mechanism is also utilized by human cytomegalovirus (HCMV) in the process of infection and pathogenesis. The HCMV-encoded miRNA, hcmv-miR-US25-1-5p, was highly expressed during lytic and latent infections, and was found to inhibit viral replication. Identification of functional target genes of this microRNA is important in that it will enable a better understanding of the function of hcmv-miR-US25-1-5p during HCMV infection. In the present study, 35 putative cellular transcript targets of hcmv-miR-US25-1-5p were identified. Down-regulation of the targets YWHAE, UBB, NPM1, and HSP90AA1 by hcmv-miR-US25-1-5p was validated by luciferase reporter assay and Western blot analysis. In addition, we showed that hcmv-miR-US25-1-5p could inhibit viral replication by interacting with these targets, the existence of which may impact virus replication directly or indirectly.

  12. Viral RNA but no evidence of replication can be detected in the peripheral blood mononuclear cells of hepatitis E virus infected patients

    PubMed Central

    Ippagunta, Sirish Kumar; Naik, Sita; Jameel, Shahid; KN, Sudha Ramana; Aggarwal, Rakesh

    2010-01-01

    SUMMARY Hepatitis E virus (HEV) infection is an important cause of acute viral hepatitis in several developing countries, but has recently been shown to cause chronic hepatitis in immunosuppressed persons. Other hepatotropic viruses that cause chronic infection have been shown to infect peripheral blood mononuclear cells (PBMCs) and to persist in those cells. We therefore decided to look for evidence of replication of HEV in PBMCs obtained from patients with acute hepatitis E, using strand-specific assays for positive and negative HEV RNA. Of the 44 patients with acute hepatitis E during an outbreak in India, including 27 with detectable IgM anti-HEV and 19 with detectable serum HEV RNA, 11 had detectable HEV RNA in their PBMCs. However, of the six PBMC specimens with strong HEV RNA signal, none had detectable negative-strand-HEV RNA, a marker of viral replication. These findings indicate the presence of HEV RNA but the absence of its replication in PBMCs from patients with acute hepatitis E. PMID:20659304

  13. Roles of Epstein-Barr virus BGLF3.5 gene and two upstream open reading frames in lytic viral replication in HEK293 cells.

    PubMed

    Watanabe, Takahiro; Fuse, Kenshiro; Takano, Takahiro; Narita, Yohei; Goshima, Fumi; Kimura, Hiroshi; Murata, Takayuki

    2015-09-01

    The Epstein-Barr virus (EBV) predominantly establishes a latent infection in B lymphocytes, but a small percentage of infected cells switch from the latent state to the lytic cycle, leading to potent viral DNA replication and progeny viruses production. We here focused on a lytic gene BGLF3.5, and first established BGLF3.5 mutants by marker cassette insertion. Unexpectedly, this insertion mutant failed to produce BGLF4 protein and thus progeny production was severely inhibited. Then we carefully made two point mutant viruses (stop codon insertion or frame-shift mutation) and found that BGLF3.5 is not essential for EBV lytic replication processes, such as viral gene expression, DNA replication, or progeny production in the HEK293 cells although its homolog in murine gammaherpesvirus 68 (MHV-68) was reported to be essential. In addition, we examined the roles of two short, upstream open reading frames within the 5'UTR of BGLF3.5 gene in translation of BGLF4.

  14. Antiviral Activity of Hatay Propolis Against Replication of Herpes Simplex Virus Type 1 and Type 2

    PubMed Central

    Yildirim, Ayse; Duran, Gulay Gulbol; Duran, Nizami; Jenedi, Kemal; Bolgul, Behiye Sezgin; Miraloglu, Meral; Muz, Mustafa

    2016-01-01

    Background Propolis is a bee product widely used in folk medicine and possessing many pharmacological properties. In this study we aimed to investigate: i) the antiviral activities of Hatay propolis samples against HSV-1 and HSV-2 in HEp-2 cell line, and ii) the presence of the synergistic effects of propolis with acyclovir against these viruses. Material/Methods All experiments were carried out in HEp-2 cell cultures. Proliferation assays were performed in 24-well flat bottom microplates. We inoculated 1×105 cells per ml and RPMI 1640 medium with 10% fetal calf serum into each well. Studies to determine cytotoxic effect were performed. To investigate the presence of antiviral activity of propolis samples, different concentrations of propolis (3200, 1600, 800, 400, 200, 100, 75, 50, and 25 μg/mL) were added into the culture medium. The amplifications of HSV-1 and HSV-2 DNA were performed by real-time PCR method. Acyclovir (Sigma, USA) was chosen as a positive control. Cell morphology was evaluated by scanning electron microscopy (SEM). Results The replication of HSV-1 and HSV-2 was significantly suppressed in the presence of 25, 50, and 100 μg/mL of Hatay propolis. We found that propolis began to inhibit HSV-1 replication after 24 h of incubation and propolis activity against HSV-2 was found to start at 48 h following incubation. The activity of propolis against both HSV-1 and HSV-2 was confirmed by a significant decrease in the number of viral copies. Conclusions We determined that Hatay propolis samples have important antiviral effects compared with acyclovir. In particular, the synergy produced by antiviral activity of propolis and acyclovir combined had a stronger effect against HSV-1 and HSV-2 than acyclovir alone. PMID:26856414

  15. Cellular DDX21 RNA helicase inhibits influenza A virus replication but is counteracted by the viral NS1 protein.

    PubMed

    Chen, Guifang; Liu, Chien-Hung; Zhou, Ligang; Krug, Robert M

    2014-04-01

    Influenza A virus RNA synthesis is catalyzed by the viral polymerase comprised of the PA, PB1, and PB2 proteins. We show that the host DDX21 RNA helicase restricts influenza A virus by binding PB1 and inhibiting polymerase assembly, resulting in reduced viral RNA and protein synthesis. Later during infection, the viral NS1 protein overcomes this restriction by binding to DDX21 and displacing PB1. DDX21 binds to a region of the NS1 N-terminal domain that also participates in other critical functions. A virus mutant whose NS1 protein is unable to bind DDX21 exhibits reduced viral protein synthesis at both late and early times of infection, a phenotype converted to wild-type upon DDX21 knockdown. As sequential interaction of PB1 and NS1 with DDX21 leads to temporal regulation of viral gene expression, influenza A virus likely uses the DDX21-NS1 interaction not only to overcome restriction, but also to regulate the viral life cycle.

  16. Blocking of Exchange Proteins Directly Activated by cAMP Leads to Reduced Replication of Middle East Respiratory Syndrome Coronavirus

    PubMed Central

    Tao, Xinrong; Mei, Feng; Agrawal, Anurodh; Peters, Clarence J.; Ksiazek, Thomas G.

    2014-01-01

    The outbreak of Middle East respiratory syndrome coronavirus (MERS-CoV) infections and diseases represents a potential threat for worldwide spread and requires development of effective therapeutic strategies. In this study, we revealed a novel positive function of an exchange protein directly activated by cyclic AMP 1 (cAMP-1; Epac-1) on MERS-CoV replication. Specifically, we have shown that Epac-specific inhibitor treatment or silencing Epac-1 gene expression rendered cells resistant to viral infection. We believe Epac-1 inhibitors deserve further study as potential therapeutic agents for MERS-CoV infection. PMID:24453361

  17. Blocking CXCL9 Decreases HIV-1 Replication and Enhances the Activity of Prophylactic Antiretrovirals in Human Cervical Tissues

    PubMed Central

    Macura, Sherrill L.; Lathrop, Melissa J.; Gui, Jiang; Doncel, Gustavo F.; Rollenhagen, Christiane

    2016-01-01

    Objectives: The interferon-gamma–induced chemokine CXCL9 is expressed in a wide range of inflammatory conditions including those affecting the female genital tract. CXCL9 promotes immune cell recruitment, activation, and proliferation. The role of CXCL9 in modulating HIV-1 infection of cervicovaginal tissues, a main portal of viral entry, however, has not been established. We report a link between CXCL9 and HIV-1 replication in human cervical tissues and propose CXCL9 as a potential target to enhance the anti–HIV-1 activity of prophylactic antiretrovirals. Design: Using ex vivo infection of human cervical tissues as a model of mucosal HIV-1 acquisition, we described the effect of CXCL9 neutralization on HIV-1 gene expression and mucosal CD4+ T-cell activation. The anti-HIV-1 activity of tenofovir, the leading mucosal pre-exposure prophylactic microbicide, alone or in combination with CXCL9 neutralization was also studied. Methods: HIV-1 replication was evaluated by p24 ELISA. HIV-1 DNA and RNA, and CD4, CCR5, and CD38 transcription were evaluated by quantitative real-time polymerase chain reaction. Frequency of activated cervical CD4+ T cells was quantified using fluorescence-activated cell sorting. Results: Antibody blocking of CXCL9 reduced HIV-1 replication by decreasing mucosal CD4+ T-cell activation. CXCL9 neutralization in combination with suboptimal concentrations of tenofovir, possibly present in the cervicovaginal tissues of women using the drug inconsistently, demonstrated an earlier and greater decrease in HIV-1 replication compared with tissues treated with tenofovir alone. Conclusions: CXCL9 neutralization reduces HIV-1 replication and may be an effective target to enhance the efficacy of prophylactic antiretrovirals. PMID:26545124

  18. Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena.

    PubMed

    Sandoval, Pamela Y; Lee, Po-Hsuen; Meng, Xiangzhou; Kapler, Geoffrey M

    2015-07-01

    The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress.

  19. Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication

    PubMed Central

    Avey, Denis; Tepper, Sarah; Li, Wenwei; Turpin, Zachary; Zhu, Fanxiu

    2015-01-01

    Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which has adapted unique mechanisms to modulate the cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed the consequences of knocking out these substrates on ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, we show data to support that ORF45 regulates the translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5’ UTR. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates

  20. A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells

    PubMed Central

    Weng, Kuo-Feng; Hung, Chuan-Tien; Hsieh, Po-Ting; Li, Mei-Ling; Chen, Guang-Wu; Kung, Yu-An; Huang, Peng-Nien; Kuo, Rei-Lin; Chen, Li-Lien; Lin, Jing-Yi; Wang, Robert Yung-Liang; Chen, Shu-Jen; Tang, Petrus; Horng, Jim-Tong; Huang, Hsing-I; Wang, Jen-Ren; Ojcius, David M.; Brewer, Gary; Shih, Shin-Ru

    2014-01-01

    The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>105 copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5′ untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES. PMID:25352551

  1. High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein.

    PubMed

    Klumpp, Klaus; Lam, Angela M; Lukacs, Christine; Vogel, Robert; Ren, Suping; Espiritu, Christine; Baydo, Ruth; Atkins, Kateri; Abendroth, Jan; Liao, Guochun; Efimov, Andrey; Hartman, George; Flores, Osvaldo A

    2015-12-01

    The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010-001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a Tm increase of more than 10 °C. NVR-010-001-E2 binds at the dimer-dimer interface of the core proteins, forms a new interaction surface promoting protein-protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010-001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein-protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties.

  2. The combination of tacrolimus and entecavir improves the remission of HBV-associated glomerulonephritis without enhancing viral replication

    PubMed Central

    Wang, Lifen; Ye, Zhiming; Liang, Huaban; Zhang, Bin; Xu, Lixia; Feng, Zhonglin; Liu, Shuangxin; Shi, Wei

    2016-01-01

    Background: Tacrolimus inhibits hepatitis B virus entry into hepatocytes through targeting the HBV receptor, sodium taurocholate cotransporting polypeptide. This study was performed to evaluate the efficacy and safety of Tacrolimus combined with entecavir antiviral therapy for HBV-associated glomerulonephritis patients with biopsy-proven membranous nephropathy. Method: A cohort of 42 patients was enrolled in this retrospective study. Twenty-three patients received Tacrolimus (0.05 mg/kg/day) in combination entecavir over 24 weeks, whereas the other 19 patients only received entecavir monotherapy. Results: The probability of proteinuria remission in the Tacrolimus+entecavir group was 69 and 87% after 12 and 24 weeks, whereas was only 26 and 42%, respectively, in the entecavir group. The mean time to partial or complete remission was 18.6 weeks in the Tacrolimus+entecavir group and 34.3 weeks in the entecavir group (P<0.001). A decrease in the HBV DNA titer was observed in all patients with active HBV replication. None of the HBV carriers in the Tacrolimus+entecavir group showed evidence of HBV reactivation. The serum creatinine and alanine aminotransferase levels remained stable in both groups. The Tacrolimus target trough concentration was 5-10 ng/mL. Conclusion: Tacrolimus combined with entecavir rapidly and effectively induced remission of HBV-GN in Chinese adults. Furthermore, Tacrolimus may have a synergistic antiviral effect with entecavir. PMID:27186284

  3. High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein

    PubMed Central

    Klumpp, Klaus; Lam, Angela M.; Lukacs, Christine; Vogel, Robert; Ren, Suping; Espiritu, Christine; Baydo, Ruth; Atkins, Kateri; Abendroth, Jan; Liao, Guochun; Efimov, Andrey; Hartman, George; Flores, Osvaldo A.

    2015-01-01

    The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010–001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a Tm increase of more than 10 °C. NVR-010–001-E2 binds at the dimer–dimer interface of the core proteins, forms a new interaction surface promoting protein–protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010–001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein–protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties. PMID:26598693

  4. High-resolution crystal structure of a hepatitis B virus replication inhibitor bound to the viral core protein.

    PubMed

    Klumpp, Klaus; Lam, Angela M; Lukacs, Christine; Vogel, Robert; Ren, Suping; Espiritu, Christine; Baydo, Ruth; Atkins, Kateri; Abendroth, Jan; Liao, Guochun; Efimov, Andrey; Hartman, George; Flores, Osvaldo A

    2015-12-01

    The hepatitis B virus (HBV) core protein is essential for HBV replication and an important target for antiviral drug discovery. We report the first, to our knowledge, high-resolution crystal structure of an antiviral compound bound to the HBV core protein. The compound NVR-010-001-E2 can induce assembly of the HBV core wild-type and Y132A mutant proteins and thermostabilize the proteins with a Tm increase of more than 10 °C. NVR-010-001-E2 binds at the dimer-dimer interface of the core proteins, forms a new interaction surface promoting protein-protein interaction, induces protein assembly, and increases stability. The impact of naturally occurring core protein mutations on antiviral activity correlates with NVR-010-001-E2 binding interactions determined by crystallography. The crystal structure provides understanding of a drug efficacy mechanism related to the induction and stabilization of protein-protein interactions and enables structure-guided design to improve antiviral potency and drug-like properties. PMID:26598693

  5. Dual effect of nitric oxide on SARS-CoV replication: Viral RNA production and palmitoylation of the S protein are affected

    SciTech Connect

    Akerstroem, Sara; Gunalan, Vithiagaran; Keng, Choong Tat; Tan, Yee-Joo; Mirazimi, Ali

    2009-12-05

    Nitric oxide is an important molecule playing a key role in a broad range of biological process such as neurotransmission, vasodilatation and immune responses. While the anti-microbiological properties of nitric oxide-derived reactive nitrogen intermediates (RNI) such as peroxynitrite, are known, the mechanism of these effects are as yet poorly studied. Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) belongs to the family Coronaviridae, was first identified during 2002-2003. Mortality in SARS patients ranges from between 6 to 55%. We have previously shown that nitric oxide inhibits the replication cycle of SARS-CoV in vitro by an unknown mechanism. In this study, we have further investigated the mechanism of the inhibition process of nitric oxide against SARS-CoV. We found that peroxynitrite, an intermediate product of nitric oxide in solution formed by the reaction of NO with superoxide, has no effect on the replication cycle of SARS-CoV, suggesting that the inhibition is either directly effected by NO or a derivative other than peroxynitrite. Most interestingly, we found that NO inhibits the replication of SARS-CoV by two distinct mechanisms. Firstly, NO or its derivatives cause a reduction in the palmitoylation of nascently expressed spike (S) protein which affects the fusion between the S protein and its cognate receptor, angiotensin converting enzyme 2. Secondly, NO or its derivatives cause a reduction in viral RNA production in the early steps of viral replication, and this could possibly be due to an effect on one or both of the cysteine proteases encoded in Orf1a of SARS-CoV.

  6. Continuous expression and replication of the hepatitis delta virus genome in Hep G2 hepatoblastoma cells transfected with cloned viral DNA.

    PubMed

    Chen, P J; Kuo, M Y; Chen, M L; Tu, S J; Chiu, M N; Wu, H L; Hsu, H C; Chen, D S

    1990-07-01

    To establish stable cell clones allowing continuous replication of hepatitis delta virus (HDV), Hep G2, a hepatoblastoma cell line containing no hepatitis B virus (HBV) DNA sequences, was transfected with a recombinant plasmid containing a tandem trimer of HDV cDNA (driven by the simian virus 40 late promoter) and a neomycin-resistance gene. After selection with the neomycin analogue G418, at least two of the resistant clones were shown to have intact delta antigen by specific immunoblotting, and the delta antigen was located in the cell nucleus by immunofluorescence. Transfected cloned viral DNAs were found to be integrated into cell chromosomes. Replication of the HDV genome was demonstrated by the presence of not only genomic and antigenomic HDV RNAs but also HDV RNAs in multimeric and circular forms. In addition, a 0.8-kilobase antigenomic RNA containing a poly(A) tail and encoding the delta-antigen open reading frame was documented. Continuous replication and transcription of the HDV genome was thus achieved in these transfected cell lines. The results confirmed that replication of HDV was unassisted by HBV. Stable passage of such cell lines strongly suggests that HDV lacks direct cytopathicity in hepatocytes. These clones should be useful in studying the details of the HDV life cycle and the relationship between HDV and its helper virus, HBV.

  7. Natural Single-Nucleotide Variations in the HIV-1 Genomic SA1prox Region Can Alter Viral Replication Ability by Regulating Vif Expression Levels

    PubMed Central

    Nomaguchi, Masako; Doi, Naoya; Sakai, Yosuke; Ode, Hirotaka; Iwatani, Yasumasa; Matsumoto, Yui; Miyazaki, Yasuyuki; Masuda, Takao

    2016-01-01

    ABSTRACT We previously found that natural single-nucleotide variations located within a proximal region of splicing acceptor 1 (SA1prox) in the HIV-1 genome could alter the viral replication potential and mRNA expression pattern, especially the vif mRNA level. Here, we studied the virological and molecular basis of nucleotide sequence variations in SA1prox for alterations of viral replication ability. Consistent with our previous findings, variant clones indeed expressed Vif at different levels and grew distinctively in cells with various APOBEC3G expression levels. Similar effects were observed for natural variations found in HIV-2 SA1prox, suggesting the importance of the SA1prox sequence. To define nucleotides critical for the regulation of HIV-1 Vif expression, effects of natural SA1prox variations newly found in the HIV Sequence Compendium database on vif mRNA/Vif protein levels were examined. Seven out of nine variations were found to produce Vif at lower, higher, or more excessive levels than wild-type NL4-3. Combination experiments of variations giving distinct Vif levels suggested that the variations mutually affected vif transcript production. While low and high producers of Vif grew in an APOBEC3G-dependent manner, excessive expressers always showed an impeded growth phenotype due to defects in single-cycle infectivity and/or virion production levels. The phenotype of excessive expressers was not due primarily to inadequate expression of Tat or Rev, although SA1prox variations altered the overall HIV-1 mRNA expression pattern. Collectively, our results demonstrate that HIV SA1prox regulates Vif expression levels and suggest a relationship between SA1prox and viral adaptation/evolution given that variations occurred naturally. IMPORTANCE While human cells possess restriction factors to inhibit HIV-1 replication, HIV-1 encodes antagonists to overcome these barriers. Conflicts between host restriction factors and viral counterparts are critical driving

  8. Inhibition of viral replication reduces regulatory T cells and enhances the antiviral immune response in chronic hepatitis B

    SciTech Connect

    Stoop, Jeroen N. . E-mail: j.n.stoop@erasmusmc.nl; Molen, Renate G. van der . E-mail: r.vandermolen@erasmusmc.nl; Kuipers, Ernst J. . E-mail: e.j.kuipers@erasmusmc.nl; Kusters, Johannes G. . E-mail: j.g.kusters@erasmusmc.nl; Janssen, Harry L.A. . E-mail: h.janssen@erasmusmc.nl

    2007-04-25

    Regulatory T cells (Treg) play a key role in the impaired immune response that is typical for a chronic Hepatitis B virus (HBV) infection. To gain more insight in the mechanism that is responsible for this impaired immune response, the effect of viral load reduction resulting from treatment with the nucleotide analogue adefovir dipivoxil on the percentages of Treg and HBV-specific T-cell responses was analyzed. Peripheral blood mononuclear cells (PBMC) of 12 patients were collected at baseline and during treatment. In parallel to the decline in viral load, we found a decline in circulating Treg, combined with an increase in HBV core antigen-specific IFN-{gamma} production and proliferation. The production of IL10 did not decrease during therapy. In conclusion, adefovir induced viral load reduction results in a decline of circulating Treg together with a partial recovery of the immune response.

  9. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    PubMed

    Neuvonen, Maarit; Ahola, Tero

    2009-01-01

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  10. Genome-wide screening using RNAi (RNA interference) to study host factors in viral replication and pathogenesis

    PubMed Central

    Houzet, Laurent; Jeang, Kuan-Teh

    2012-01-01

    With the recent development of siRNA and shRNA expression libraries, RNAi technology has been extensively employed to identify genes involved in diverse cellular processes, such as signal transduction, cell cycle, cancer biology and host-pathogen interactions. In the field of viral infection, this approach has already identified hundreds of new genes not previously known to be important for various virus lifecycles. In this brief review, we focus on recent studies performed using genome-wide RNAi-based screens in mammalian cells for the identification of essential host factors for viral infection and pathogenesis. PMID:21727185

  11. Role of BK virus infection in end-stage renal disease patients waiting for kidney transplantation--viral replication dynamics from pre- to post-transplant.

    PubMed

    Mitterhofer, Anna Paola; Tinti, Francesca; Pietropaolo, Valeria; Umbro, Ilaria; Anzivino, Elena; Bellizzi, Anna; Zavatto, Assunta; Poli, Luca; Berloco, Pasquale Bartolomeo; Taliani, Gloria

    2014-03-01

    We report the prevalence of BK virus (BKV) infection before renal transplantation and the dynamics of BKV viremia from pre- to post-transplantation. We assessed 60 kidney transplanted patients from a single cohort in Italy, treated with identical immunosuppressive therapy, for BK viremia at pre-transplantation, 12 h, and three and six months post-transplantation. Polymerase chain reaction showed that the prevalence of plasma BKV replication--considered a marker of infection--was 20% in pre-transplant patients. All pre-transplant-positive patients remained positive post-transplant, whereas the majority of pre-transplant-negative patients remained negative. Viremia dynamics classification revealed three clusters of patients: Cluster A++, pre-transplant-positive patients (20%) who tested positive at least once post-transplant; Cluster B-+, pre-transplant-negative patients (28%) who tested positive at least once post-transplant; and Cluster C- -, pre-transplant-negative patients (52%) who remained negative throughout. These clusters presented significant differences related to the prevalence of substantially positive patients with high plasma viral load (>10(3) copies/mL) in cluster A, but not in donors' or grafts' characteristics. We suggest that pre-transplant viral status should be considered as an additional risk factor for post-transplant BKV replication. Therefore, pre-transplant BKV infection screening in kidney transplant patients should be performed for improving planning of personalized immunosuppressant schemes and specific post-transplant surveillance.

  12. Interactome analysis of the EV71 5' untranslated region in differentiated neuronal cells SH-SY5Y and regulatory role of FBP3 in viral replication.

    PubMed

    Huang, Hsing-I; Chang, Ying-Ying; Lin, Jhao-Yin; Kuo, Rei-Lin; Liu, Hao-Ping; Shih, Shin-Ru; Wu, Chih-Ching

    2016-09-01

    Enterovirus 71 (EV71), a single-stranded RNA virus, is one of the most serious neurotropic pathogens in the Asia-Pacific region. Through interactions with host proteins, the 5' untranslated region (5'UTR) of EV71 is important for viral replication. To gain a protein profile that interact with the EV71 5'UTR in neuronal cells, we performed a biotinylated RNA-protein pull-down assay in conjunction with LC-MS/MS analysis. A total of 109 proteins were detected and subjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analyses. These proteins were found to be highly correlated with biological processes including RNA processing/splicing, epidermal cell differentiation, and protein folding. A protein-protein interaction network was constructed using the STRING online database to illustrate the interactions of those proteins that are mainly involved in RNA processing/splicing or protein folding. Moreover, we confirmed that the far-upstream element binding protein 3 (FBP3) was able to bind to the EV71 5'UTR. The redistribution of FBP3 in subcellular compartments was observed after EV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibited viral replication. Our results reveal various host proteins that potentially interact with the EV71 5'UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells. PMID:27291656

  13. Interactome analysis of the EV71 5' untranslated region in differentiated neuronal cells SH-SY5Y and regulatory role of FBP3 in viral replication.

    PubMed

    Huang, Hsing-I; Chang, Ying-Ying; Lin, Jhao-Yin; Kuo, Rei-Lin; Liu, Hao-Ping; Shih, Shin-Ru; Wu, Chih-Ching

    2016-09-01

    Enterovirus 71 (EV71), a single-stranded RNA virus, is one of the most serious neurotropic pathogens in the Asia-Pacific region. Through interactions with host proteins, the 5' untranslated region (5'UTR) of EV71 is important for viral replication. To gain a protein profile that interact with the EV71 5'UTR in neuronal cells, we performed a biotinylated RNA-protein pull-down assay in conjunction with LC-MS/MS analysis. A total of 109 proteins were detected and subjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analyses. These proteins were found to be highly correlated with biological processes including RNA processing/splicing, epidermal cell differentiation, and protein folding. A protein-protein interaction network was constructed using the STRING online database to illustrate the interactions of those proteins that are mainly involved in RNA processing/splicing or protein folding. Moreover, we confirmed that the far-upstream element binding protein 3 (FBP3) was able to bind to the EV71 5'UTR. The redistribution of FBP3 in subcellular compartments was observed after EV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibited viral replication. Our results reveal various host proteins that potentially interact with the EV71 5'UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells.

  14. Recursion-based depletion of human immunodeficiency virus-specific naive CD4(+) T cells may facilitate persistent viral replication and chronic viraemia leading to acquired immunodeficiency syndrome.

    PubMed

    Tsukamoto, Tetsuo; Yamamoto, Hiroyuki; Okada, Seiji; Matano, Tetsuro

    2016-09-01

    Although antiretroviral therapy has made human immunodeficiency virus (HIV) infection a controllable disease, it is still unclear how viral replication persists in untreated patients and causes CD4(+) T-cell depletion leading to acquired immunodeficiency syndrome (AIDS) in several years. Theorists tried to explain it with the diversity threshold theory in which accumulated mutations in the HIV genome make the virus so diverse that the immune system will no longer be able to recognize all the variants and fail to control the viraemia. Although the theory could apply to a number of cases, macaque AIDS models using simian immunodeficiency virus (SIV) have shown that failed viral control at the set point is not always associated with T-cell escape mutations. Moreover, even monkeys without a protective major histocompatibility complex (MHC) allele can contain replication of a super infected SIV following immunization with a live-attenuated SIV vaccine, while those animals are not capable of fighting primary SIV infection. Here we propose a recursion-based virus-specific naive CD4(+) T-cell depletion hypothesis through thinking on what may happen in individuals experiencing primary immunodeficiency virus infection. This could explain the mechanism for impairment of virus-specific immune response in the course of HIV infection.

  15. Stability in controlling viral replication identifies long-term nonprogressors as a distinct subgroup among human immunodeficiency virus type 1-infected persons.

    PubMed Central

    Vesanen, M; Stevens, C E; Taylor, P E; Rubinstein, P; Saksela, K

    1996-01-01

    Long-term nonprogressors (LTNPs) of human immunodeficiency virus type 1 (HIV-1) infection are characterized by low levels of HIV-1 replication and viral load. However, it has not been established whether they differ in this regard from progressors from the very early stage of infection. By studying peripheral blood mononuclear cell (PBMC) specimens from a longitudinally monitored cohort of HIV-1-infected men, we found that HIV-1 proviral copy numbers and HIV-1 mRNA expression levels as low or lower than those seen in seven carefully selected LTNPs were commonly observed in specimens collected soon after seroconversion from 28 subjects who became infected while under observation. However, only the LTNPs were able to stably maintain such an efficient viral control over time. Because of the instability of the early control of HIV-1 replication, the predictive value of HIV-1 mRNA expression in PBMCs at postseroconversion was found to be limited but significantly increased during the first year of infection. Besides their diagnostic implications, these data support the idea that LTNPs may be a pathophysiologically distinct subgroup among persons infected with HIV-1. PMID:8971039

  16. Recursion-based depletion of human immunodeficiency virus-specific naive CD4(+) T cells may facilitate persistent viral replication and chronic viraemia leading to acquired immunodeficiency syndrome.

    PubMed

    Tsukamoto, Tetsuo; Yamamoto, Hiroyuki; Okada, Seiji; Matano, Tetsuro

    2016-09-01

    Although antiretroviral therapy has made human immunodeficiency virus (HIV) infection a controllable disease, it is still unclear how viral replication persists in untreated patients and causes CD4(+) T-cell depletion leading to acquired immunodeficiency syndrome (AIDS) in several years. Theorists tried to explain it with the diversity threshold theory in which accumulated mutations in the HIV genome make the virus so diverse that the immune system will no longer be able to recognize all the variants and fail to control the viraemia. Although the theory could apply to a number of cases, macaque AIDS models using simian immunodeficiency virus (SIV) have shown that failed viral control at the set point is not always associated with T-cell escape mutations. Moreover, even monkeys without a protective major histocompatibility complex (MHC) allele can contain replication of a super infected SIV following immunization with a live-attenuated SIV vaccine, while those animals are not capable of fighting primary SIV infection. Here we propose a recursion-based virus-specific naive CD4(+) T-cell depletion hypothesis through thinking on what may happen in individuals experiencing primary immunodeficiency virus infection. This could explain the mechanism for impairment of virus-specific immune response in the course of HIV infection. PMID:27515208

  17. Cellular minichromosome maintenance complex component 5 (MCM5) is incorporated into HIV-1 virions and modulates viral replication in the newly infected cells

    PubMed Central

    Santos, Steven; Obukhov, Yuri; Nekhai, Sergei; Pushkarsky, Tatiana; Brichacek, Beda; Bukrinsky, Michael; Iordanskiy, Sergey

    2016-01-01

    The post-entry events of HIV-1 infection occur within reverse transcription complexes derived from the viral cores entering the target cell. HIV-1 cores contain host proteins incorporated from virus-producing cells. In this report, we show that MCM5, a subunit of the hexameric minichromosome maintenance (MCM) DNA helicase complex, associates with Gag polyprotein and is incorporated into HIV-1 virions. The progeny virions depleted of MCM5 demonstrated reduced reverse transcription in newly infected cells, but integration and subsequent replication steps were not affected. Interestingly, increased packaging of MCM5 into the virions also led to reduced reverse transcription, but here viral replication was impaired. Our data suggest that incorporation of physiological amounts of MCM5 promotes aberrant reverse transcription, leading to partial incapacitation of cDNA, whereas increased MCM5 abundance leads to reduced reverse transcription and infection. Therefore, MCM5 has the properties of an inhibitory factor that interferes with production of an integration-competent cDNA product. PMID:27414250

  18. Activation of a dormant replication origin is essential for Haloferax mediterranei lacking the primary origins

    PubMed Central

    Yang, Haibo; Wu, Zhenfang; Liu, Jingfang; Liu, Xiaoqing; Wang, Lei; Cai, Shuangfeng; Xiang, Hua

    2015-01-01

    The use of multiple origins for chromosome replication has been demonstrated in archaea. Similar to the dormant origins in eukaryotes, some potential origins in archaea appear to be inactive during genome replication. We have comprehensively explored the origin utilization in Haloferax mediterranei. Here we report three active chromosomal origins by genome-wide replication profiling, and demonstrate that when these three origins are deleted, a dormant origin becomes activated. Notably, this dormant origin cannot be further deleted when the other origins are already absent and vice versa. Interestingly, a potential origin that appears to stay dormant in its native host H. volcanii lacking the main active origins becomes activated and competent for replication of the entire chromosome when integrated into the chromosome of origin-deleted H. mediterranei. These results indicate that origin-dependent replication is strictly required for H. mediterranei and that dormant replication origins in archaea can be activated if needed. PMID:26374389

  19. Replicative fitness of transmitted HIV-1 drives acute immune activation, proviral load in memory CD4+ T cells, and disease progression.

    PubMed

    Claiborne, Daniel T; Prince, Jessica L; Scully, Eileen; Macharia, Gladys; Micci, Luca; Lawson, Benton; Kopycinski, Jakub; Deymier, Martin J; Vanderford, Thomas H; Nganou-Makamdop, Krystelle; Ende, Zachary; Brooks, Kelsie; Tang, Jianming; Yu, Tianwei; Lakhi, Shabir; Kilembe, William; Silvestri, Guido; Douek, Daniel; Goepfert, Paul A; Price, Matthew A; Allen, Susan A; Paiardini, Mirko; Altfeld, Marcus; Gilmour, Jill; Hunter, Eric

    2015-03-24

    HIV-1 infection is characterized by varying degrees of chronic immune activation and disruption of T-cell homeostasis, which impact the rate of disease progression. A deeper understanding of the factors that influence HIV-1-induced immunopathology and subsequent CD4(+) T-cell decline is critical to strategies aimed at controlling or eliminating the virus. In an analysis of 127 acutely infected Zambians, we demonstrate a dramatic and early impact of viral replicative capacity (vRC) on HIV-1 immunopathogenesis that is independent of viral load (VL). Individuals infected with high-RC viruses exhibit a distinct inflammatory cytokine profile as well as significantly elevated T-cell activation, proliferation, and CD8(+) T-cell exhaustion, during the earliest months of infection. Moreover, the vRC of the transmitted virus is positively correlated with the magnitude of viral burden in naive and central memory CD4(+) T-cell populations, raising the possibility that transmitted viral phenotypes may influence the size of the initial latent viral reservoir. Taken together, these findings support an unprecedented role for the replicative fitness of the founder virus, independent of host protective genes and VL, in influencing multiple facets of HIV-1-related immunopathology, and that a greater focus on this parameter could provide novel approaches to clinical interventions.

  20. The effect of IL-2 expression by recombinant Newcastle disease virus on host immune response, viral replication and pathogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Interleukin 2 (IL-2) is a soluble cytokine that stimulates the cell-mediated immune response. Virus constructs, such as recombinant vaccinia virus, expressing chicken IL-2 have been shown to improve viral clearance by natural killer cells in mice. We have inserted the open-reading frame of the chi...

  1. Polygonum cuspidatum and Its Active Components Inhibit Replication of the Influenza Virus through Toll-Like Receptor 9-Induced Interferon Beta Expression

    PubMed Central

    Lin, Chao-jen; Lin, Hui-Ju; Chen, Ter-Hsin; Hsu, Yu-An; Liu, Chin-San; Hwang, Guang-Yuh; Wan, Lei

    2015-01-01

    Influenza virus infection is a global public health issue. The effectiveness of antiviral therapies for influenza has been limited by the emergence of drug-resistant viral strains. Therefore, there is an urgent need to identify novel antiviral therapies. Here we tested the effects of 300 traditional Chinese medicines on the replication of various influenza virus strains in a lung cell line, A549, using an influenza-specific luciferase reporter assay. Of the traditional medicines tested, Polygonum cuspidatum (PC) and its active components, resveratrol and emodin, were found to attenuate influenza viral replication in A549 cells. Furthermore, they preferentially inhibited the replication of influenza A virus, including clinical strains isolated in 2009 and 2011 in Taiwan and the laboratory strain A/WSN/33 (H1N1). In addition to inhibiting the expression of hemagglutinin and neuraminidase, PC, emodin, and resveratrol also increased the expression of interferon beta (IFN-β) through Toll-like receptor 9 (TLR9). Moreover, the anti-viral activity of IFN-β or resveratrol was reduced when the A549 cells were treated with neutralizing anti-IFN-β antibodies or a TLR9 inhibitor, suggesting that IFN-β likely acts synergistically with resveratrol to inhibit H1N1 replication. This potential antiviral mechanism, involving direct inhibition of virus replication and simultaneous activation of the host immune response, has not been previously described for a single antiviral molecule. In conclusion, our data support the use of PC, resveratrol or emodin for inhibiting influenza virus replication directly and via TLR-9–induced IFN-β production. PMID:25658356

  2. Polygonum cuspidatum and its active components inhibit replication of the influenza virus through toll-like receptor 9-induced interferon beta expression.

    PubMed

    Lin, Chao-Jen; Lin, Hui-Ju; Chen, Ter-Hsin; Hsu, Yu-An; Liu, Chin-San; Hwang, Guang-Yuh; Wan, Lei

    2015-01-01

    Influenza virus infection is a global public health issue. The effectiveness of antiviral therapies for influenza has been limited by the emergence of drug-resistant viral strains. Therefore, there is an urgent need to identify novel antiviral therapies. Here we tested the effects of 300 traditional Chinese medicines on the replication of various influenza virus strains in a lung cell line, A549, using an influenza-specific luciferase reporter assay. Of the traditional medicines tested, Polygonum cuspidatum (PC) and its active components, resveratrol and emodin, were found to attenuate influenza viral replication in A549 cells. Furthermore, they preferentially inhibited the replication of influenza A virus, including clinical strains isolated in 2009 and 2011 in Taiwan and the laboratory strain A/WSN/33 (H1N1). In addition to inhibiting the expression of hemagglutinin and neuraminidase, PC, emodin, and resveratrol also increased the expression of interferon beta (IFN-β) through Toll-like receptor 9 (TLR9). Moreover, the anti-viral activity of IFN-β or resveratrol was reduced when the A549 cells were treated with neutralizing anti-IFN-β antibodies or a TLR9 inhibitor, suggesting that IFN-β likely acts synergistically with resveratrol to inhibit H1N1 replication. This potential antiviral mechanism, involving direct inhibition of virus replication and simultaneous activation of the host immune response, has not been previously described for a single antiviral molecule. In conclusion, our data support the use of PC, resveratrol or emodin for inhibiting influenza virus replication directly and via TLR-9-induced IFN-β production. PMID:25658356

  3. miR-122 Stimulates Hepatitis C Virus RNA Synthesis by Altering the Balance of Viral RNAs Engaged in Replication Versus Translation

    PubMed Central

    Masaki, Takahiro; Arend, Kyle C.; Li, You; Yamane, Daisuke; McGivern, David R.; Kato, Takanobu; Wakita, Takaji; Moorman, Nathaniel J.; Lemon, Stanley M.

    2015-01-01

    SUMMARY The liver-specific microRNA, miR-122, stabilizes hepatitis C virus (HCV) RNA genomes by recruiting host argonaute 2 (AGO2) to the 5′ end and preventing decay mediated by exonuclease Xrn1. However, HCV replication requires miR-122 in Xrn1-depleted cells, indicating additional function s. We show that miR-122 enhances HCV RNA levels by altering the fraction of HCV genomes available for RNA synthesis. Exogenous miR-122 increases viral RNA and protein levels in Xrn1-depleted cells, with enhanced RNA synthesis occurring before heightened protein synthesis. Inhibiting protein translation blocks miR-122-mediated increases in RNA synthesis, but independently enhances RNA synthesis by releasing ribosomes from viral genomes. Additionally, miR-122 reduces the fraction of viral genomes engaged in protein translation. Depleting AGO2 or PCBP2, which binds HCV RNA in competition with miR-122 and promotes translation, eliminates miR-122 stimulation of RNA synthesis. Thus, by displacing PCBP2, miR-122 reduces HCV genomes engaged in translation while increasing the fraction available for RNA synthesis. PMID:25662750

  4. Small interfering RNAs targeting peste des petits ruminants virus M mRNA increase virus-mediated fusogenicity and inhibit viral replication in vitro.

    PubMed

    Liu, Fuxiao; Wu, Xiaodong; Zou, Yanli; Li, Lin; Liu, Shan; Chi, Tianying; Wang, Zhiliang

    2015-11-01

    Peste des petits ruminants (PPR), caused by peste des petits ruminants virus (PPRV), is an acute or subacute, highly contagious and economically important disease of small ruminants. The PPRV is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) protein possesses an intrinsic ability to bind to lipid membranes, and plays a crucial role in viral assembly and further budding. In this study, three different small interfering RNAs (siRNA) were designed on the basis of translated region for PPRV Nigeria 75/1M mRNA, and were subsequently synthesized for their transfection into Vero-SLAM cells, followed by infection with PPRVs. The results showed that two out of three siRNAs robustly induced cell-to-cell fusion as early as 36h post-infection with PPRVs, effectively suppressed expression of the M protein by interference for the M mRNA, and eventually inhibited viral replication in vitro. These findings led us to speculate that siRNA-mediated knockdown of the M protein would alter its interaction with viral glycoproteins, thus exacerbating intercellular fusion but hampering virus release. PMID:26318517

  5. The Replicative Consequences of Papillomavirus E2 Protein Binding to the Origin Replication Factor ORC2

    PubMed Central

    DeSmet, Marsha; Kanginakudru, Sriramana; Rietz, Anne; Wu, Wai-Hong; Roden, Richard

    2016-01-01

    The origin recognition complex (ORC) coordinates a series of events that lead to initiation of DNA strand duplication. As a nuclear double stranded DNA plasmid, the papillomavirus (PV) genome resembles a mini-chromosome in infected cells. To initiate its replication, the viral E2 protein binds to and recruits the E1 DNA helicase at the viral origin. PV genome replication program exhibits three stages: initial amplification from a single genome upon infection to a few copies per cell, a cell cycle linked maintenance phase, and a differentiation dependent late stage where the genome is amplified to thousands of copies. Involvement of ORC or other pre-replication complex (pre-RC) factors has not been described. We report that human PV (HPV) and bovine PV (BPV-1) E2 proteins bind to ORC2, however, ORC2 was not detected at the viral origin. Depletion of ORC2 enhanced PV replication in a transient replication model and in keratinocytes stably maintaining viral episomes, while there was no effect on copy number in a cell line with integrated HPV genomes. Consistent with this, occupancy of E1 and E2 at the viral origin increased following ORC2 silencing. These data imply that ORC2 is not necessary for activation of the PV origin by E1 and E2 but instead suppresses E2 replicative function. Furthermore, we observed that over-expression of HPV E2 decreased ORC2 occupation at two known mammalian origins of replication, suggesting that E2 restricts pre-ORC assembly that could otherwise compete for host replication complexes necessary for viral genome amplification. We infer that the ORC2 complex with E2 restricts viral replication in the maintenance phase of the viral replication program and that elevated levels of E2 that occur during the differentiation dependent amplification stage subvert ORC loading and hence DNA synthesis at cellular origins. PMID:27701460

  6. Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication

    PubMed Central

    Chang, FuJung; Riera, Alberto; Evrin, Cecile; Sun, Jingchuan; Li, Huilin; Speck, Christian; Weinreich, Michael

    2015-01-01

    To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant ‘Cdc6-E224Q’ promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo. DOI: http://dx.doi.org/10.7554/eLife.05795.001 PMID:26305410

  7. Cdc6 ATPase activity disengages Cdc6 from the pre-replicative complex to promote DNA replication.

    PubMed

    Chang, FuJung; Riera, Alberto; Evrin, Cecile; Sun, Jingchuan; Li, Huilin; Speck, Christian; Weinreich, Michael

    2015-08-25

    To initiate DNA replication, cells first load an MCM helicase double hexamer at origins in a reaction requiring ORC, Cdc6, and Cdt1, also called pre-replicative complex (pre-RC) assembly. The essential mechanistic role of Cdc6 ATP hydrolysis in this reaction is still incompletely understood. Here, we show that although Cdc6 ATP hydrolysis is essential to initiate DNA replication, it is not essential for MCM loading. Using purified proteins, an ATPase-defective Cdc6 mutant 'Cdc6-E224Q' promoted MCM loading on DNA. Cdc6-E224Q also promoted MCM binding at origins in vivo but cells remained blocked in G1-phase. If after loading MCM, Cdc6-E224Q was degraded, cells entered an apparently normal S-phase and replicated DNA, a phenotype seen with two additional Cdc6 ATPase-defective mutants. Cdc6 ATP hydrolysis is therefore required for Cdc6 disengagement from the pre-RC after helicase loading to advance subsequent steps in helicase activation in vivo.

  8. Control of DNA replication in a transformed lymphoid cell line: coexistence of activator and inhibitor activities.

    PubMed

    Coffman, F D; Fresa, K L; Oglesby, I; Cohen, S

    1991-12-01

    Proliferating lymphocytes contain an intracellular factor, ADR (activator of DNA replication), which can initiate DNA synthesis in isolated quiescent nuclei. Resting lymphocytes lack ADR activity and contain an intracellular inhibitory factor that suppresses DNA synthesis in normal but not transformed nuclei. In this study we describe a MOLT-4 subline that produces both the activator and inhibitory activities which can be separated by ammonium sulfate fractionation. The inhibitor is heat stable and inhibits ADR-mediated DNA replication in a dose-dependent manner. It does not inhibit DNA polymerase alpha activity. The inhibitor must be present at the initiation of DNA replication to be effective, as it loses most of its effectiveness if it is added after replication has begun. The presence of inhibitory activity in proliferating MOLT-4 cells, taken with the previous observation that inhibitor derived from normal resting cells does not affect DNA synthesis by MOLT-4 nuclei, suggests that failure of a down-regulating signal may play an important role in proliferative disorder. PMID:1934078

  9. Natural Killer cell-dependent and non-dependent anti-viral activity of 2-Cys Peroxiredoxin against HIV

    PubMed Central

    Asmal, Mohammed; Letvin, Norman L.; Geiben-Lynn, Ralf

    2013-01-01

    2-cys peroxiredoxins (Prx), a group of anti-oxidative enzyme proteins, act directly on virally-infected cells to inhibit HIV-1 replication, and indirectly through destruction of HIV infected cells by stimulation of Natural Killer (NK) cell-mediated immune responses. We assayed for antibody-dependent NK cell mediated viral inhibition (ADCVI) using plasma from SIV-infected rhesus macaques. We found that Prx-1 strongly increased ADCVI in a dose-dependent manner, suggesting augmentation of NK cell killing. We also investigated the effect of Prx-1 on NK cell-independent HIV-1 and HIV-2 inhibition. We found that primary HIV isolates were potently inhibited at nM concentrations, regardless of viral clade, receptor usage or anti-retroviral drug resistance. During NK cell independent inhibition, we found that Prx-1 reversed the HIV-1 induced gene expression of Heat shock protein 90 kDa alpha (cystolic), class A member 2, (HSP90), a protein of the stress pathway. Prx-1 highly activated Cyclin-dependent kinase inhibitor 2B (CDKN2B), a gene of the TGF-β pathway, and Baculoviral IAP repeat-containing 2 (Birc-2), an anti-apoptotic gene of the NF-κB pathway. We identified gene-expression networks highly dependent on the NFκB and ERK1/2 pathways. Our findings demonstrate that Prx-1 inhibits HIV replication through NK cell-dependent and NK cell-independent mechanisms. PMID:24244928

  10. Well begun is half done: Rubella virus perturbs autophagy signaling, thereby facilitating the construction of viral replication compartments.

    PubMed

    Orosz, László; Megyeri, Klára

    2016-04-01

    The rubella virus is the causative agent of postnatal German measles and the congenital rubella syndrome. The majority of the rubella virus replication complexes originate from the endomembrane system. The rubella virus perturbs the signaling pathways regulating the formation of autophagic membranes in the infected cells, including the Ras/Raf/MEK/ERK and PI3K/Akt pathways. It is widely accepted that these pathways inhibit autophagy. In contrast, the class III PI3K enzymes are essential for autophagy initiation. By manipulating the Ras/Raf/MEK/ERK, class I PI3K/Akt and class III PI3K axes of signal transduction, the rubella virus may differentially regulate the autophagic cascade, with consequent stimulation of the initiation and strong suppression of the later phases. Dysregulation of autophagy by this virus can have a significant impact on the construction of replication compartments by regulating membrane trafficking. We hypothesize that the rubella virus perturbs the autophagic process in order to prevent the degradation of the virus progeny, and to ensure its replication by hijacking omegasomes for the construction of the replication complexes. The virus is therefore able to utilize an antiviral mechanism to its own advantage. Therapeutic modalities targeting the autophagic process may help to ameliorate the serious consequences of the congenital rubella syndrome. PMID:26968901

  11. Functional Centromeres Determine the Activation Time of Pericentric Origins of DNA Replication in Saccharomyces cerevisiae

    PubMed Central

    Pohl, Thomas J.; Brewer, Bonita J.; Raghuraman, M. K.

    2012-01-01

    The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation. PMID:22589733

  12. Functional centromeres determine the activation time of pericentric origins of DNA replication in Saccharomyces cerevisiae.

    PubMed

    Pohl, Thomas J; Brewer, Bonita J; Raghuraman, M K

    2012-01-01

    The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.

  13. Flaviviral NS4b, chameleon and jack‐in‐the‐box roles in viral replication and pathogenesis, and a molecular target for antiviral intervention

    PubMed Central

    Zmurko, Joanna; Dallmeier, Kai

    2015-01-01

    Summary Dengue virus and other flaviviruses such as the yellow fever, West Nile, and Japanese encephalitis viruses are emerging vector‐borne human pathogens that affect annually more than 100 million individuals and that may cause debilitating and potentially fatal hemorrhagic and encephalitic diseases. Currently, there are no specific antiviral drugs for the treatment of flavivirus‐associated disease. A better understanding of the flavivirus–host interactions during the different events of the flaviviral life cycle may be essential when developing novel antiviral strategies. The flaviviral non‐structural protein 4b (NS4b) appears to play an important role in flaviviral replication by facilitating the formation of the viral replication complexes and in counteracting innate immune responses such as the following: (i) type I IFN signaling; (ii) RNA interference; (iii) formation of stress granules; and (iv) the unfolded protein response. Intriguingly, NS4b has recently been shown to constitute an excellent target for the selective inhibition of flavivirus replication. We here review the current knowledge on NS4b. © 2015 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd. PMID:25828437

  14. Persistence of viral genes in a variant of MDBK cell after productive replication of a mutant of influenza virus A/WSN.

    PubMed

    Urabe, M; Tanaka, T; Odagiri, T; Tashiro, M; Tobita, K

    1993-01-01

    The MDBK-R cell line is a variant of the MDBK cell line, which was derived by three consecutive high multiplicity superinfections of MDBK cells with AWBY-140 virus, a mutant of influenza virus A/WSN (H 1N 1). MDBK-R cells are permissive for productive replication of AWBY-140, but resist lysis by the virus and grew normally without producing infectious virus after replication of the mutant occurred there. By polymerase chain reaction (PCR), we demonstrated nucleotide sequences specific to all the 8 genes of AWBY-140 in MDBK-R cells which had been infected with the mutant at a high multiplicity and subsequently received 25 passages. This suggests that the genes of influenza virus mutant persisted in the dividing host cells for a long time after productive infection, when none of the cells was producing virus. We were also able to amplify the M gene related sequence of the mutant from both poly(A)+ and poly(A)- fractions of the RNA extracted from the cells at 27th passage level by PCR, which suggests that the persisting genes were replicated and transcribed, but we failed to demonstrate any viral protein in the cells by Western blotting.

  15. Roles of stress-activated protein kinases in the replication of Singapore grouper iridovirus and regulation of the inflammatory responses in grouper cells.

    PubMed

    Huang, Xiaohong; Huang, Youhua; OuYang, Zhengliang; Cai, Jia; Yan, Yang; Qin, Qiwei

    2011-06-01

    Stress-activated protein kinases (SAPKs), including p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun N-terminal kinase (JNK), are usually activated in response to different environmental stimuli, including virus infection. In the present study, the roles of SAPKs during Singapore grouper iridovirus (SGIV) infection were investigated in fish cells. The results showed that increased phosphorylation of JNK1/2 and p38 MAPK occurred during active replication of SGIV in grouper cell cultures. Moreover, downstream effectors (c-Jun, MAPK-activated protein kinase 2, p53, activator protein 1, Myc and nuclear factor of activated T cells) were activated after SGIV infection, suggesting that SGIV replication activated the JNK and p38 MAPK signalling pathways. Notably, using specific inhibitors, it was found that viral gene transcripts, protein expression and viral titres were not affected by inhibition of p38 MAPK but were suppressed significantly by inhibiting JNK1/2 activation. In addition, transcription of grouper immune genes including interferon regulatory factor 1, interleukin-8 and tumour necrosis factor alpha (TNF-α) were regulated by JNK, whilst only TNF-α was regulated by p38 MAPK. It is proposed that the JNK pathway is important for SGIV replication and modulates the inflammatory responses during virus infection.

  16. bta-miR-29b attenuates apoptosis by directly targeting caspase-7 and NAIF1 and suppresses bovine viral diarrhea virus replication in MDBK cells.

    PubMed

    Fu, Qiang; Shi, Huijun; Shi, Mengting; Meng, Luping; Zhang, Hui; Ren, Yan; Guo, Fei; Jia, Bin; Wang, Pengyan; Ni, Wei; Chen, Chuangfu

    2014-07-01

    MicroRNAs (miRNAs) are small, endogenous, noncoding RNA molecules that serve as powerful regulators of multiple cellular processes, including apoptosis, differentiation, growth, and proliferation. Bovine viral diarrhea virus (BVDV) contributes significantly to health-related economic losses in the beef and dairy industries. Although BVDV-induced apoptosis correlates with increased intracellular viral RNA accumulation and with bta-miR-29b (miR-29b) expression upregulation in Madin-Darby bovine kidney (MDBK) cells infected with BVDV strain NADL, the role of miR-29b in regulating BVDV-infection-related apoptosis remains unexplored. Here, we report that miR-29b serves as a new miRNA regulating apoptosis. We showed that miR-29b target sequences were present in the 3' untranslated regions of 2 key apoptosis regulators mRNAs, cysteine aspartases-7 (caspase-7) and nuclear apoptosis-inducing factor 1 (NAIF1). Indeed, upon miRNA overexpression, both mRNA and protein levels of caspase-7 and NAIF1 were decreased. We further found that miR-29b attenuated apoptosis by directly regulating intracellular levels of caspase-7 and NAIF1. Moreover, apoptosis blockage by miR-29b was rescued upon co-infection of MDBK cells with lentiviruses expressing caspase-7 and NAIF1. Importantly, miR-29b decreased BVDV NADL envelope glycoprotein E1 mRNA levels and suppressed viral replication. These studies advance our understanding of the mechanisms of miRNAs in mediating the cells combating viral infections.

  17. Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid

    PubMed Central

    Peng, Ke; Muranyi, Walter; Glass, Bärbel; Laketa, Vibor; Yant, Stephen R; Tsai, Luong; Cihlar, Tomas; Müller, Barbara; Kräusslich, Hans-Georg

    2014-01-01

    The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules. DOI: http://dx.doi.org/10.7554/eLife.04114.001 PMID:25517934

  18. Two homologous host proteins interact with potato virus X RNAs and CPs and affect viral replication and movement

    PubMed Central

    Choi, Hoseong; Cho, Won Kyong; Kim, Kook-Hyung

    2016-01-01

    Because viruses encode only a small number of proteins, all steps of virus infection rely on specific interactions between viruses and hosts. We previously screened several Nicotiana benthamiana (Nb) proteins that interact with the stem-loop 1 (SL1) RNA structure located at the 5′ end of the potato virus X (PVX) genome. In this study, we characterized two of these proteins (NbCPIP2a and NbCPIP2b), which are homologous and are induced upon PVX infection. Electrophoretic mobility shift assay confirmed that both proteins bind to either SL1(+) or SL1(−) RNAs of PVX. The two proteins also interact with the PVX capsid protein (CP) in planta. Overexpression of NbCPIP2a positively regulated systemic movement of PVX in N. benthamiana, whereas NbCPIP2b overexpression did not affect systemic movement of PVX. Transient overexpression and silencing experiments demonstrated that NbCPIP2a and NbCPIP2b are positive regulators of PVX replication and that the effect on replication was greater for NbCPIP2a than for NbCPIP2b. Although these two host proteins are associated with plasma membranes, PVX infection did not affect their subcellular localization. Taken together, these results indicate that NbCPIP2a and NbCPIP2b specifically bind to PVX SL1 RNAs as well as to CP and enhance PVX replication and movement. PMID:27353522

  19. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    SciTech Connect

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory . E-mail: vchinchar@microbio.umsmed.edu

    2007-02-20

    Frog virus 3 (FV3) is a large DNA virus that encodes {approx} 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-II{alpha}). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-II{alpha} triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.

  20. Depth-related gradients of viral activity in Lake Pavin.

    PubMed

    Colombet, J; Sime-Ngando, T; Cauchie, H M; Fonty, G; Hoffmann, L; Demeure, G

    2006-06-01

    High-resolution vertical sampling and determination of viral and prokaryotic parameters in a deep volcanic lake shows that in the absence of thermal stratification but within light, oxygen, and chlorophyll gradients, host availability empirically is prevalent over the physical and chemical environments and favors lytic over lysogenic "viral life cycles."

  1. SUMO and KSHV Replication

    PubMed Central

    Chang, Pei-Ching; Kung, Hsing-Jien

    2014-01-01

    Small Ubiquitin-related MOdifier (SUMO) modification was initially identified as a reversible post-translational modification that affects the regulation of diverse cellular processes, including signal transduction, protein trafficking, chromosome segregation, and DNA repair. Increasing evidence suggests that the SUMO system also plays an important role in regulating chromatin organization and transcription. It is thus not surprising that double-stranded DNA viruses, such as Kaposi’s sarcoma-associated herpesvirus (KSHV), have exploited SUMO modification as a means of modulating viral chromatin remodeling during the latent-lytic switch. In addition, SUMO regulation allows the disassembly and assembly of promyelocytic leukemia protein-nuclear bodies (PML-NBs), an intrinsic antiviral host defense, during the viral replication cycle. Overcoming PML-NB-mediated cellular intrinsic immunity is essential to allow the initial transcription and replication of the herpesvirus genome after de novo infection. As a consequence, KSHV has evolved a way as to produce multiple SUMO regulatory viral proteins to modulate the cellular SUMO environment in a dynamic way during its life cycle. Remarkably, KSHV encodes one gene product (K-bZIP) with SUMO-ligase activities and one gene product (K-Rta) that exhibits SUMO-targeting ubiquitin ligase (STUbL) activity. In addition, at least two viral products are sumoylated that have functional importance. Furthermore, sumoylation can be modulated by other viral gene products, such as the viral protein kinase Orf36. Interference with the sumoylation of specific viral targets repr