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Sample records for enhances viral protein

  1. Annexin II Binds to Capsid Protein VP1 of Enterovirus 71 and Enhances Viral Infectivity ▿

    PubMed Central

    Yang, Su-Lin; Chou, Ying-Ting; Wu, Cheng-Nan; Ho, Mei-Shang

    2011-01-01

    Enterovirus type 71 (EV71) causes hand, foot, and mouth disease (HFMD), which is mostly self-limited but may be complicated with a severe to fatal neurological syndrome in some children. Understanding the molecular basis of virus-host interactions might help clarify the largely unknown neuropathogenic mechanisms of EV71. In this study, we showed that human annexin II (Anx2) protein could bind to the EV71 virion via the capsid protein VP1. Either pretreatment of EV71 with soluble recombinant Anx2 or pretreatment of host cells with an anti-Anx2 antibody could result in reduced viral attachment to the cell surface and a reduction of the subsequent virus yield in vitro. HepG2 cells, which do not express Anx2, remained permissive to EV71 infection, though the virus yield was lower than that for a cognate lineage expressing Anx2. Stable transfection of plasmids expressing Anx2 protein into HepG2 cells (HepG2-Anx2 cells) could enhance EV71 infectivity, with an increased virus yield, especially at a low infective dose, and the enhanced infectivity could be reversed by pretreating HepG2-Anx2 cells with an anti-Anx2 antibody. The Anx2-interacting domain was mapped by yeast two-hybrid analysis to VP1 amino acids 40 to 100, a region different from the known receptor binding domain on the surface of the picornavirus virion. Our data suggest that binding of EV71 to Anx2 on the cell surface can enhance viral entry and infectivity, especially at a low infective dose. PMID:21900167

  2. Flavivirus NS1 protein in infected host sera enhances viral acquisition by mosquitoes.

    PubMed

    Liu, Jianying; Liu, Yang; Nie, Kaixiao; Du, Senyan; Qiu, Jingjun; Pang, Xiaojing; Wang, Penghua; Cheng, Gong

    2016-01-01

    The arbovirus life cycle involves viral transfer between a vertebrate host and an arthropod vector, and acquisition of virus from an infected mammalian host by a vector is an essential step in this process. Here, we report that flavivirus nonstructural protein-1 (NS1), which is abundantly secreted into the serum of an infected host, plays a critical role in flavivirus acquisition by mosquitoes. The presence of dengue virus (DENV) and Japanese encephalitis virus NS1s in the blood of infected interferon-α and γ receptor-deficient mice (AG6) facilitated virus acquisition by their native mosquito vectors because the protein enabled the virus to overcome the immune barrier of the mosquito midgut. Active immunization of AG6 mice with a modified DENV NS1 reduced DENV acquisition by mosquitoes and protected mice against a lethal DENV challenge, suggesting that immunization with NS1 could reduce the number of virus-carrying mosquitoes as well as the incidence of flaviviral diseases. Our study demonstrates that flaviviruses utilize NS1 proteins produced during their vertebrate phases to enhance their acquisition by vectors, which might be a result of flavivirus evolution to adapt to multiple host environments. PMID:27562253

  3. Viruses and viral proteins

    PubMed Central

    Verdaguer, Nuria; Ferrero, Diego; Murthy, Mathur R. N.

    2014-01-01

    For more than 30 years X-ray crystallography has been by far the most powerful approach for determining the structures of viruses and viral proteins at atomic resolution. The information provided by these structures, which covers many important aspects of the viral life cycle such as cell-receptor recognition, viral entry, nucleic acid transfer and genome replication, has extensively enriched our vision of the virus world. Many of the structures available correspond to potential targets for antiviral drugs against important human pathogens. This article provides an overview of the current knowledge of different structural aspects of the above-mentioned processes. PMID:25485129

  4. Heat shock protein 70 is associated with CSFV NS5A protein and enhances viral RNA replication.

    PubMed

    Zhang, Chengcheng; Kang, Kai; Ning, Pengbo; Peng, Yangxin; Lin, Zhi; Cui, Hongjie; Cao, Zhi; Wang, Jing; Zhang, Yanming

    2015-08-01

    The non-structural 5A (NS5A) protein of classical swine fever virus (CSFV) is proven to be involved in viral replication and can also modulate cellular signaling via to its ability to interact with various cellular proteins. Here, HSP70/NS5A complex formation is confirmed by coimmunoprecipitation and GST-pulldown studies. Additionally, the N-terminal amino acids (29-240) of NS5A were identified as the interaction region through in vivo deletion analyses, and confocal microscopy showed that NS5A and HSP70 colocalized in the cytoplasm. Overexpression of HSP70 via the eukaryotic expression plasmid pDsRED N1 or lentivirus significantly promoted viral RNA synthesis. Whereas the knockdown of HSP70 by lentivirus-mediated shRNA or inhibition by quercetin markedly decreased the viral load. These data suggest that HSP70 plays a critical role in the viral life cycle, particularly during the virus RNA replication period. The investigation of HSP70 protein functions may be beneficial for developing new strategies to treat CSFV infection. PMID:25827528

  5. The viral RNA-based transfection of enhanced green fluorescent protein (EGFP) in the parasitic protozoan Trichomonas vaginalis.

    PubMed

    Li, Wei; Ding, He; Zhang, Xinxin; Cao, Lili; Li, Jianhua; Gong, Pengtao; Li, He; Zhang, Guocai; Li, Shuhong; Zhang, Xichen

    2012-03-01

    Here we have developed methods to transiently and stably transfect the human pathogenic protist Trichomonas vaginalis. The viral RNA-based transfection vector pTVV-EGFP/NEO was constructed by using enhanced green fluorescent protein gene (EGFP) and neomycin resistance gene (NEO) in tandem to replace the whole gene encoding region of T. vaginalis virus (TVV). The in vitro transcripts of linearized pTVV-EGFP/NEO were electroporated into trophozoites and the transfectants transiently expressed EGFP after 16 h postincubation. Stable expression of EGFP was persistently detected by fluorescence microscopy and by RT-PCR in transfected trophozoites under G418 selection. Our study provides a novel and valuable approach for genetic study of T. vaginalis. PMID:21861063

  6. Rice Dwarf Virus P2 Protein Hijacks Auxin Signaling by Directly Targeting the Rice OsIAA10 Protein, Enhancing Viral Infection and Disease Development.

    PubMed

    Jin, Lian; Qin, Qingqing; Wang, Yu; Pu, Yingying; Liu, Lifang; Wen, Xing; Ji, Shaoyi; Wu, Jianguo; Wei, Chunhong; Ding, Biao; Li, Yi

    2016-09-01

    The phytohormone auxin plays critical roles in regulating myriads of plant growth and developmental processes. Microbe infection can disturb auxin signaling resulting in defects in these processes, but the underlying mechanisms are poorly understood. Auxin signaling begins with perception of auxin by a transient co-receptor complex consisting of an F-box transport inhibitor response 1/auxin signaling F-box (TIR1/AFB) protein and an auxin/indole-3-acetic acid (Aux/IAA) protein. Auxin binding to the co-receptor triggers ubiquitination and 26S proteasome degradation of the Aux/IAA proteins, leading to subsequent events, including expression of auxin-responsive genes. Here we report that Rice dwarf virus (RDV), a devastating pathogen of rice, causes disease symptoms including dwarfing, increased tiller number and short crown roots in infected rice as a result of reduced sensitivity to auxin signaling. The RDV capsid protein P2 binds OsIAA10, blocking the interaction between OsIAA10 and OsTIR1 and inhibiting 26S proteasome-mediated OsIAA10 degradation. Transgenic rice plants overexpressing wild-type or a dominant-negative (degradation-resistant) mutant of OsIAA10 phenocopy RDV symptoms are more susceptible to RDV infection; however, knockdown of OsIAA10 enhances the resistance of rice to RDV infection. Our findings reveal a previously unknown mechanism of viral protein reprogramming of a key step in auxin signaling initiation that enhances viral infection and pathogenesis. PMID:27606959

  7. Susceptibility to viral infection is enhanced by stable expression of 3A or 3AB proteins from foot-and-mouth disease virus

    SciTech Connect

    Rosas, Maria F.; Vieira, Yuri A.; Postigo, Raul; Martin-Acebes, Miguel A.; Armas-Portela, Rosario; Martinez-Salas, Encarnacion; Sobrino, Francisco

    2008-10-10

    The foot-and-mouth disease virus (FMDV) 3A protein is involved in virulence and host range. A distinguishing feature of FMDV 3B among picornaviruses is that three non-identical copies are encoded in the viral RNA and required for optimal replication in cell culture. Here, we have studied the involvement of the 3AB region on viral infection using constitutive and transient expression systems. BHK-21 stably transformed clones expressed low levels of FMDV 3A or 3A(B) proteins in the cell cytoplasm. Transformed cells stably expressing these proteins did not exhibit inner cellular rearrangements detectable by electron microscope analysis. Upon FMDV infection, clones expressing either 3A alone or 3A(B) proteins showed a significant increase in the percentage of infected cells, the number of plaque forming units and the virus yield. The 3A-enhancing effect was specific for FMDV as no increase in viral multiplication was observed in transformed clones infected with another picornavirus, encephalomyocarditis virus, or the negative-strand RNA virus vesicular stomatitis virus. A potential role of 3A protein in viral RNA translation was discarded by the lack of effect on FMDV IRES-dependent translation. Increased viral susceptibility was not caused by a released factor; neither the supernatant of transformed clones nor the addition of purified 3A protein to the infection medium was responsible for this effect. Unlike stable expression, high levels of 3A or 3A(B) protein transient expression led to unspecific inhibition of viral infection. Therefore, the effect observed on viral yield, which inversely correlated with the intracellular levels of 3A protein, suggests a transacting role operating on the FMDV multiplication cycle.

  8. 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

  9. Going Viral with Fluorescent Proteins

    PubMed Central

    Costantini, Lindsey M.

    2015-01-01

    Many longstanding questions about dynamics of virus-cell interactions can be answered by combining fluorescence imaging techniques with fluorescent protein (FP) tagging strategies. Successfully creating a FP fusion with a cellular or viral protein of interest first requires selecting the appropriate FP. However, while viral architecture and cellular localization often dictate the suitability of a FP, a FP's chemical and physical properties must also be considered. Here, we discuss the challenges of and offer suggestions for identifying the optimal FPs for studying the cell biology of viruses. PMID:26202231

  10. IFITM Proteins Restrict Viral Membrane Hemifusion

    PubMed Central

    Golfetto, Ottavia; Bungart, Brittani; Li, Minghua; Ding, Shilei; He, Yuxian; Liang, Chen; Lee, James C.; Gratton, Enrico; Cohen, Fredric S.; Liu, Shan-Lu

    2013-01-01

    The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from hemifusion to full fusion, was unable to rescue the IFITM-mediated restriction on fusion. In contrast, oleic acid (OA), a lipid analog that generates negative spontaneous curvature and thereby promotes hemifusion, virtually overcame the restriction. To explore the possible effect of IFITM proteins on membrane molecular order and fluidity, we performed fluorescence labeling with Laurdan, in conjunction with two-photon laser scanning and fluorescence-lifetime imaging microscopy (FLIM). We observed that the generalized polarizations (GPs) and fluorescence lifetimes of cell membranes expressing IFITM proteins were greatly enhanced, indicating higher molecularly ordered and less fluidized membranes. Collectively, our data demonstrated that IFITM proteins suppress viral membrane fusion before the creation of hemifusion, and suggested that they may do so by reducing membrane fluidity and conferring a positive spontaneous

  11. An integrated approach for enhanced protein conjugation and capture with viral nanotemplates and hydrogel microparticle platforms via rapid bioorthogonal reactions.

    PubMed

    Jung, Sukwon; Yi, Hyunmin

    2014-07-01

    We demonstrate significantly enhanced protein conjugation and target protein capture capacity by exploiting tobacco mosaic virus (TMV) templates assembled with hydrogel microparticles. Protein conjugation results with a red fluorescent protein R-Phycoerythrin (R-PE) show significantly enhanced protein conjugation capacity of TMV-assembled particles (TMV-particles) compared to planar substrates or hydrogel microparticles. In-depth examination of protein conjugation kinetics via tetrazine (Tz)-trans-cyclooctene (TCO) cycloaddition and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction demonstrates that TMV-particles provide a less hindered environment for protein conjugation. Target protein capture results using an anti-R-PE antibody (R-Ab)-R-PE pair also show substantially improved capture capacity of R-Ab conjugated TMV-particles over R-Ab conjugated hydrogel microparticles. We further demonstrate readily controlled protein and antibody conjugation capacity by simply varying TMV concentrations, which show negligible negative impact of densely assembled TMVs on protein conjugation and capture capacity. Combined, these results illustrate a facile postfabrication protein conjugation approach with TMV templates assembled onto hydrogel microparticles for improved and controlled protein conjugation and sensing platforms. We anticipate that our approach can be readily applied to various protein sensing applications. PMID:24937661

  12. Coxsackievirus group B type 3 infection upregulates expression of monocyte chemoattractant protein 1 in cardiac myocytes, which leads to enhanced migration of mononuclear cells in viral myocarditis.

    PubMed

    Shen, Yan; Xu, Wei; Chu, Yi-Wei; Wang, Ying; Liu, Quan-Sheng; Xiong, Si-Dong

    2004-11-01

    Coxsackievirus group B type 3 (CVB3) is an important cause of viral myocarditis. The infiltration of mononuclear cells into the myocardial tissue is one of the key events in viral myocarditis. Immediately after CVB3 infects the heart, the expression of chemokine(s) by infected myocardial cells may be the first trigger for inflammatory infiltration and immune response. However, it is unknown whether CVB3 can induce the chemokine expression in cardiac myocytes. Monocyte chemoattractant protein 1 (MCP-1) is a potent chemokine that stimulates the migration of mononuclear cells. The objective of the present study was to investigate the effect of CVB3 infection on MCP-1 expression in murine cardiac myocytes and the role of MCP-1 in migration of mononuclear cells in viral myocarditis. Our results showed that the expression of MCP-1 was significantly increased in cardiac myocytes after wild-type CVB3 infection in a time- and dose-dependent manner, which resulted in enhanced migration of mononuclear cells in mice with viral myocarditis. The migration of mononuclear cells was partially abolished by antibodies specific for MCP-1 in vivo and in vitro. Administration of anti-MCP-1 antibody prevented infiltration of mononuclear cells bearing the MCP-1 receptor CCR2 in mice with viral myocarditis. Infection by UV-irradiated CVB3 induced rapid and transient expression of MCP-1 in cardiac myocytes. In conclusion, our results indicate that CVB3 infection stimulates the expression of MCP-1 in myocardial cells, which subsequently leads to migration of mononuclear cells in viral myocarditis. PMID:15507642

  13. Viral adaptation to an antiviral protein enhances the fitness level to above that of the uninhibited wild type.

    PubMed

    Cherwa, James E; Sanchez-Soria, Pablo; Wichman, Holly A; Fane, Bentley A

    2009-11-01

    Viruses often evolve resistance to antiviral agents. While resistant strains are able to replicate in the presence of the agent, they generally exhibit lower fitness than the wild-type strain in the absence of the inhibitor. In some cases, resistant strains become dependent on the antiviral agent. However, the agent rarely, if ever, elevates dependent strain fitness above the uninhibited wild-type level. This would require an adaptive mechanism to convert the antiviral agent into a beneficial growth factor. Using an inhibitory scaffolding protein that specifically blocks phiX174 capsid assembly, we demonstrate that such mechanisms are possible. To obtain the quintuple-mutant resistant strain, the wild-type virus was propagated for approximately 150 viral life cycles in the presence of increasing concentrations of the inhibitory protein. The expression of the inhibitory protein elevated the strain's fitness significantly above the uninhibited wild-type level. Thus, selecting for resistance coselected for dependency, which was characterized and found to operate on the level of capsid nucleation. To the best of our knowledge, this is the first report of a virus evolving a mechanism to productively utilize an antiviral agent to stimulate its fitness above the uninhibited wild-type level. The results of this study may be predictive of the types of resistant phenotypes that could be selected by antiviral agents that specifically target capsid assembly. PMID:19726521

  14. The Oncogenic Small Tumor Antigen of Merkel Cell Polyomavirus Is an Iron-Sulfur Cluster Protein That Enhances Viral DNA Replication

    PubMed Central

    Tsang, Sabrina H.; Wang, Ranran; Nakamaru-Ogiso, Eiko; Knight, Simon A. B.; Buck, Christopher B.

    2015-01-01

    ABSTRACT Merkel cell polyomavirus (MCPyV) plays an important role in Merkel cell carcinoma (MCC). MCPyV small T (sT) antigen has emerged as the key oncogenic driver in MCC carcinogenesis. It has also been shown to promote MCPyV LT-mediated replication by stabilizing LT. The importance of MCPyV sT led us to investigate sT functions and to identify potential ways to target this protein. We discovered that MCPyV sT purified from bacteria contains iron-sulfur (Fe/S) clusters. Electron paramagnetic resonance analysis showed that MCPyV sT coordinates a [2Fe-2S] and a [4Fe-4S] cluster. We also observed phenotypic conservation of Fe/S coordination in the sTs of other polyomaviruses. Since Fe/S clusters are critical cofactors in many nucleic acid processing enzymes involved in DNA unwinding and polymerization, our results suggested the hypothesis that MCPyV sT might be directly involved in viral replication. Indeed, we demonstrated that MCPyV sT enhances LT-mediated replication in a manner that is independent of its previously reported ability to stabilize LT. MCPyV sT translocates to nuclear foci containing actively replicating viral DNA, supporting a direct role for sT in promoting viral replication. Mutations of Fe/S cluster-coordinating cysteines in MCPyV sT abolish its ability to stimulate viral replication. Moreover, treatment with cidofovir, a potent antiviral agent, robustly inhibits the sT-mediated enhancement of MCPyV replication but has little effect on the basal viral replication driven by LT alone. This finding further indicates that MCPyV sT plays a direct role in stimulating viral DNA replication and introduces cidofovir as a possible drug for controlling MCPyV infection. IMPORTANCE MCPyV is associated with a highly aggressive form of skin cancer in humans. Epidemiological surveys for MCPyV seropositivity and sequencing analyses of healthy human skin suggest that MCPyV may represent a common component of the human skin microbial flora. However, much of the

  15. Low-Level Expression of the E1B 20-Kilodalton Protein by Adenovirus 14p1 Enhances Viral Immunopathogenesis.

    PubMed

    Radke, Jay R; Yong, Sherri L; Cook, James L

    2016-01-01

    Adenovirus 14p1 (Ad14p1) is an emergent variant of Ad serotype 14 (Ad14) that has caused increased severity of respiratory illnesses during globally distributed outbreaks, including cases of acute respiratory distress syndrome and death. We found that human cell infection with Ad14p1 results in markedly decreased expression of the E1B 20-kilodalton (20K) protein compared to that with infection with wild-type (wt) Ad14. This reduced Ad14p1 E1B 20K expression caused a loss-of-function phenotype of Ad-infected cell corpses that, in contrast to cells infected with wt Ad14, either failed to repress or increased NF-κB-dependent, proinflammatory cytokine responses of responder human alveolar macrophages. A small-animal model of Ad14-induced lung infection was used to test the translational relevance of these in vitro observations. Intratracheal infection of Syrian hamsters with Ad14p1 caused a marked, patchy bronchopneumonia, whereas hamster infection with wt Ad14 caused minimal peribronchial inflammation. These results suggest that this difference in E1B 20K gene expression during Ad14p1 infection and its modulating effect on the interactions between Ad14-infected cells and the host innate immune response could explain the increased immunopathogenic potential and associated increase in clinical illness in some people infected with the Ad14p1 outbreak strain.IMPORTANCE We previously reported that Ad-infected human cells exhibit E1B 19K-dependent repression of virally induced, NF-κB-dependent macrophage cytokine responses (J. R. Radke, F. Grigera, D. S. Ucker, and J. L. Cook, J Virol 88:2658-2669, 2014, http://dx.doi.org/10.1128/JVI.02372-13). The more virulent, emergent strain of Ad14, Ad14p1, causes increased cytopathology in vitro, which suggested a possible E1B 20K defect. Whether there is a linkage between these observations was unknown. We show that there is markedly reduced expression of E1B 20K in Ad14p1-infected human cells and that this causes an increased

  16. Serine 192 in the tiny RS repeat of the adenoviral L4-33K splicing enhancer protein is essential for function and reorganization of the protein to the periphery of viral replication centers

    SciTech Connect

    Oestberg, Sara; Toermaenen Persson, Heidi; Akusjaervi, Goeran

    2012-11-25

    The adenovirus L4-33K protein is a key regulator involved in the temporal shift from early to late pattern of mRNA expression from the adenovirus major late transcription unit. L4-33K is a virus-encoded alternative splicing factor, which enhances processing of 3 Prime splice sites with a weak sequence context. Here we show that L4-33K expressed from a plasmid is localized at the nuclear margin of uninfected cells. During an infection L4-33K is relocalized to the periphery of E2A-72K containing viral replication centers. We also show that serine 192 in the tiny RS repeat of the conserved carboxy-terminus of L4-33K, which is critical for the splicing enhancer function of L4-33K, is necessary for the nuclear localization and redistribution of the protein to viral replication sites. Collectively, our results show a good correlation between the activity of L4-33K as a splicing enhancer protein and its localization to the periphery of viral replication centers.

  17. Human polyoma JC virus minor capsid proteins, VP2 and VP3, enhance large T antigen binding to the origin of viral DNA replication: Evidence for their involvement in regulation of the viral DNA replication

    PubMed Central

    Saribas, A. Sami; Mun, Sarah; Johnson, Jaslyn; El-Hajmoussa, Mohammad; White, Martyn K.; Safak, Mahmut

    2014-01-01

    JC virus (JCV) lytically infects the oligodendrocytes in the central nervous system in a subset of immunocompromized patients and causes the demyelinating disease, progressive multifocal leukoencephalopathy. JCV replicates and assembles into infectious virions in the nucleus. However, understanding the molecular mechanisms of its virion biogenesis remains elusive. In this report, we have attempted to shed more light on this process by investigating molecular interactions between large T antigen (LT-Ag), Hsp70 and minor capsid proteins, VP2/VP3. We demonstrated that Hsp70 interacts with VP2/VP3 and LT-Ag; and accumulates heavily in the nucleus of the infected cells. We also showed that VP2/VP3 associates with LT-Ag through their DNA binding domains resulting in enhancement in LT-Ag DNA binding to Ori and induction in viral DNA replication. Altogether, our results suggest that VP2/VP3 and Hsp70 actively participate in JCV DNA replication and may play critical roles in coupling of viral DNA replication to virion encapsidation. PMID:24418532

  18. Characteristic amino acid changes of influenza A(H1N1)pdm09 virus PA protein enhance A(H7N9) viral polymerase activity.

    PubMed

    Liu, Jun; Huang, Feng; Zhang, Junsong; Tan, Likai; Lu, Gen; Zhang, Xu; Zhang, Hui

    2016-06-01

    Human coinfection with a novel H7N9 influenza virus and the 2009 pandemic A(H1N1) influenza virus, H1N1pdm09, has recently been reported in China. Because reassortment can occur during coinfection, it is necessary to clarify the effects of gene reassortment between these two viruses. Among the viral ribonucleoprotein complex (vRNP) genes, only the PA gene of H1N1pdm09 enhances the avian influenza viral polymerase activity. Based on a phylogenetic analysis, we show a special evolutionary feature of the H1N1pdm09 PA gene, which clustered with those of the novel H7N9 virus and related H9N2 viruses, rather than in the outgroup as the H1N1pdm09 genes do on the phylogenetic trees of other vRNP genes. Using a minigenome system of the novel H7N9 virus, we further demonstrate that replacement of its PA gene significantly enhanced its polymerase activity, whereas replacement of the other vRNP genes reduced its polymerase activity. We also show that the residues of PA evolutionarily conserved between H1N1pdm09 and the novel H7N9 virus are associated with attenuated or neutral polymerase activity. The mutations associated with the increased activity of the novel H7N9 polymerase are characteristic of the H1N1pdm09 gene, and are located almost adjacent to the surface of the PA protein. Our results suggest that the novel H7N9 virus has more effective PB1, PB2, and NP genes than H1N1pdm09, and that H1N1pdm09-like PA mutations enhance the novel H7N9 polymerase function. PMID:26980671

  19. Class III viral membrane fusion proteins

    PubMed Central

    Backovic, Marija

    2010-01-01

    SUMMARY Accumulating structural studies of viral fusion glycoproteins have revealed unanticipated structural relationships between unrelated virus families and allowed the grouping of these membrane fusogens into three distinct classes. Here we review the newly identified group of class III viral fusion proteins, whose members include fusion proteins from rhabdoviruses, herpesviruses and baculoviruses. While clearly related in structure, the class III viral fusion proteins exhibit distinct structural features in their architectures as well as in their membrane-interacting fusion loops, which are likely related to their virus-specific differences in cellular entry. Further study of the similarities and differences in the class III viral fusion glycoproteins may provide greater insights into protein:membrane interactions that are key to promoting efficient bilayer fusion during virus entry. PMID:19356922

  20. The K526R substitution in viral protein PB2 enhances the effects of E627K on influenza virus replication

    PubMed Central

    Song, Wenjun; Wang, Pui; Mok, Bobo Wing-Yee; Lau, Siu-Ying; Huang, Xiaofeng; Wu, Wai-Lan; Zheng, Min; Wen, Xi; Yang, Shigui; Chen, Yu; Li, Lanjuan; Yuen, Kwok-Yung; Chen, Honglin

    2014-01-01

    Host-adaptive strategies, such as the E627K substitution in the PB2 protein, are critical for replication of avian influenza A viruses in mammalian hosts. Here we show that mutation PB2-K526R is present in some human H7N9 influenza isolates, in nearly 80% of H5N1 human isolates from Indonesia and, in conjunction with E627K, in almost all seasonal H3N2 viruses since 1970. Polymerase complexes containing PB2-526R derived from H7N9, H5N1 or H3N2 viruses exhibit increased polymerase activity. PB2-526R also enhances viral transcription and replication in cells. In comparison with viruses carrying 627K, H7N9 viruses carrying both 526R and 627K replicate more efficiently in mammalian (but not avian) cells and in mouse lung tissues, and cause greater body weight loss and mortality in infected mice. PB2-K526R interacts with nuclear export protein and our results suggest that it contributes to enhance replication for certain influenza virus subtypes, particularly in combination with 627K. PMID:25409547

  1. A Teleost Bactericidal Permeability-Increasing Protein Kills Gram-Negative Bacteria, Modulates Innate Immune Response, and Enhances Resistance against Bacterial and Viral Infection.

    PubMed

    Sun, Yuan-Yuan; Sun, Li

    2016-01-01

    Bactericidal/permeability-increasing protein (BPI) is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria. In teleost, the function of BPI is unknown. In the present work, we studied the function of tongue sole (Cynoglossus semilaevis) BPI, CsBPI. We found that CsBPI was produced extracellularly by peripheral blood leukocytes (PBL). Recombinant CsBPI (rCsBPI) was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria. Binding to bacteria led to bacterial death through membrane permeabilization and structural destruction, and the bound bacteria were more readily taken up by PBL. In vivo, rCsBPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity. Furthermore, rCsBPI enhanced the resistance of tongue sole against bacterial as well as viral infection. These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense. PMID:27105425

  2. A Teleost Bactericidal Permeability-Increasing Protein Kills Gram-Negative Bacteria, Modulates Innate Immune Response, and Enhances Resistance against Bacterial and Viral Infection

    PubMed Central

    Sun, Yuan-yuan; Sun, Li

    2016-01-01

    Bactericidal/permeability-increasing protein (BPI) is an important factor of innate immunity that in mammals is known to take part in the clearance of invading Gram-negative bacteria. In teleost, the function of BPI is unknown. In the present work, we studied the function of tongue sole (Cynoglossus semilaevis) BPI, CsBPI. We found that CsBPI was produced extracellularly by peripheral blood leukocytes (PBL). Recombinant CsBPI (rCsBPI) was able to bind to a number of Gram-negative bacteria but not Gram-positive bacteria. Binding to bacteria led to bacterial death through membrane permeabilization and structural destruction, and the bound bacteria were more readily taken up by PBL. In vivo, rCsBPI augmented the expression of a wide arrange of genes involved in antibacterial and antiviral immunity. Furthermore, rCsBPI enhanced the resistance of tongue sole against bacterial as well as viral infection. These results indicate for the first time that a teleost BPI possesses immunoregulatory effect and plays a significant role in antibacterial and antiviral defense. PMID:27105425

  3. O-GlcNAc modification of the coat protein of the potyvirus Plum pox virus enhances viral infection

    PubMed Central

    de Jesús Pérez, José; Udeshi, Namrata D.; Shabanowitz, Jeffrey; Ciordia, Sergio; Juárez, Silvia; Scott, Cheryl L.; Olszewski, Neil E.; Hunt, Donald F.; García, Juan Antonio

    2015-01-01

    O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum pox virus (PPV) capsid protein (CP). Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was out-competed by wild-type virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type virus. PMID:23639873

  4. Trapping mammalian protein complexes in viral particles

    PubMed Central

    Eyckerman, Sven; Titeca, Kevin; Van Quickelberghe, Emmy; Cloots, Eva; Verhee, Annick; Samyn, Noortje; De Ceuninck, Leentje; Timmerman, Evy; De Sutter, Delphine; Lievens, Sam; Van Calenbergh, Serge; Gevaert, Kris; Tavernier, Jan

    2016-01-01

    Cell lysis is an inevitable step in classical mass spectrometry–based strategies to analyse protein complexes. Complementary lysis conditions, in situ cross-linking strategies and proximal labelling techniques are currently used to reduce lysis effects on the protein complex. We have developed Virotrap, a viral particle sorting approach that obviates the need for cell homogenization and preserves the protein complexes during purification. By fusing a bait protein to the HIV-1 GAG protein, we show that interaction partners become trapped within virus-like particles (VLPs) that bud from mammalian cells. Using an efficient VLP enrichment protocol, Virotrap allows the detection of known binary interactions and MS-based identification of novel protein partners as well. In addition, we show the identification of stimulus-dependent interactions and demonstrate trapping of protein partners for small molecules. Virotrap constitutes an elegant complementary approach to the arsenal of methods to study protein complexes. PMID:27122307

  5. Hsp90 Inhibitors Are Efficacious against Kaposi Sarcoma by Enhancing the Degradation of the Essential Viral Gene LANA, of the Viral Co-Receptor EphA2 as well as Other Client Proteins

    PubMed Central

    Chen, Wuguo; Sin, Sang-Hoon; Wen, Kwun Wah; Damania, Blossom; Dittmer, Dirk P.

    2012-01-01

    Heat-shock protein 90 (Hsp90) inhibitors exhibit activity against human cancers. We evaluated a series of new, oral bioavailable, chemically diverse Hsp90 inhibitors (PU-H71, AUY922, BIIB021, NVP-BEP800) against Kaposi sarcoma (KS). All Hsp90 inhibitors exhibited nanomolar EC50 in culture and AUY922 reduced tumor burden in a xenograft model of KS. KS is associated with KS-associated herpesvirus (KSHV). We identified the viral latency associated nuclear antigen (LANA) as a novel client protein of Hsp90 and demonstrate that the Hsp90 inhibitors diminish the level of LANA through proteasomal degradation. These Hsp90 inhibitors also downregulated EphA2 and ephrin-B2 protein levels. LANA is essential for viral maintenance and EphA2 has recently been shown to facilitate KSHV infection; which in turn feeds latent persistence. Further, both molecules are required for KS tumor formation and both were downregulated in response to Hsp90 inhibitors. This provides a rationale for clinical testing of Hsp90 inhibitors in KSHV-associated cancers and in the eradication of latent KSHV reservoirs. PMID:23209418

  6. TDP-43 regulates endogenous retrovirus-K viral protein accumulation.

    PubMed

    Manghera, Mamneet; Ferguson-Parry, Jennifer; Douville, Renée N

    2016-10-01

    The concomitant expression of neuronal TAR DNA binding protein 43 (TDP-43) and human endogenous retrovirus-K (ERVK) is a hallmark of ALS. Since the involvement of TDP-43 in retrovirus replication remains controversial, we sought to evaluate whether TDP-43 exerts an effect on ERVK expression. In this study, TDP-43 bound the ERVK promoter in the context of inflammation or proteasome inhibition, with no effect on ERVK transcription. However, over-expression of ALS-associated aggregating forms of TDP-43, but not wild-type TDP-43, significantly enhanced ERVK viral protein accumulation. Human astrocytes and neurons further demonstrated cell-type specific differences in their ability to express and clear ERVK proteins during inflammation and proteasome inhibition. Astrocytes, but not neurons, were able to clear excess ERVK proteins through stress granule formation and autophagy. In vitro findings were validated in autopsy motor cortex tissue from patients with ALS and neuro-normal controls. We further confirmed marked enhancement of ERVK in cortical neurons of patients with ALS. Despite evidence of enhanced stress granule and autophagic response in ALS cortical neurons, these cells failed to clear excess ERVK protein accumulation. This highlights how multiple cellular pathways, in conjunction with disease-associated mutations, can converge to modulate the expression and clearance of viral gene products from genomic elements such as ERVK. In ALS, ERVK protein aggregation is a novel aspect of TDP-43 misregulation contributing towards the pathology of this neurodegenerative disease. PMID:27370226

  7. Viral and host proteins involved in picornavirus life cycle.

    PubMed

    Lin, Jing-Yi; Chen, Tzu-Chun; Weng, Kuo-Feng; Chang, Shih-Cheng; Chen, Li-Lien; Shih, Shin-Ru

    2009-01-01

    Picornaviruses cause several diseases, not only in humans but also in various animal hosts. For instance, human enteroviruses can cause hand-foot-and-mouth disease, herpangina, myocarditis, acute flaccid paralysis, acute hemorrhagic conjunctivitis, severe neurological complications, including brainstem encephalitis, meningitis and poliomyelitis, and even death. The interaction between the virus and the host is important for viral replication, virulence and pathogenicity. This article reviews studies of the functions of viral and host factors that are involved in the life cycle of picornavirus. The interactions of viral capsid proteins with host cell receptors is discussed first, and the mechanisms by which the viral and host cell factors are involved in viral replication, viral translation and the switch from translation to RNA replication are then addressed. Understanding how cellular proteins interact with viral RNA or viral proteins, as well as the roles of each in viral infection, will provide insights for the design of novel antiviral agents based on these interactions. PMID:19925687

  8. Curvature Sensing by a Viral Scission Protein.

    PubMed

    Martyna, Agnieszka; Gómez-Llobregat, Jordi; Lindén, Martin; Rossman, Jeremy S

    2016-06-28

    Membrane scission is the final step in all budding processes wherein a membrane neck is sufficiently constricted so as to allow for fission and the release of the budded particle. For influenza viruses, membrane scission is mediated by an amphipathic helix (AH) domain in the viral M2 protein. While it is known that the M2AH alters membrane curvature, it is not known how the protein is localized to the center neck of budding virions where it would be able to cause membrane scission. Here, we use molecular dynamics simulations on buckled lipid bilayers to show that the M2AH senses membrane curvature and preferentially localizes to regions of high membrane curvature, comparable to that seen at the center neck of budding influenza viruses. These results were then validated using in vitro binding assays to show that the M2AH senses membrane curvature by detecting lipid packing defects in the membrane. Our results show that the M2AH senses membrane curvature and suggest that the AH domain may localize the protein at the viral neck where it can then mediate membrane scission and the release of budding viruses. PMID:27299375

  9. Superresolution imaging of viral protein trafficking

    PubMed Central

    Salka, Kyle; Bhuvanendran, Shivaprasad; Yang, David

    2015-01-01

    The endoplasmic reticulum (ER) membrane is closely apposed to the outer mitochondrial membrane (OMM), which facilitates communication between these organelles. These contacts, known as mitochondria-associated membranes (MAM), facilitate calcium signaling, lipid transfer, as well as antiviral and stress responses. How cellular proteins traffic to the MAM, are distributed therein, and interact with ER and mitochondrial proteins are subject of great interest. The human cytomegalovirus UL37 exon 1 protein or viral mitochondria-localized inhibitor of apoptosis (vMIA) is crucial for viral growth. Upon synthesis at the ER, vMIA traffics to the MAM and OMM, where it reprograms the organization and function of these compartments. vMIA significantly changes the abundance of cellular proteins at the MAM and OMM, including proteins that regulate calcium homeostasis and cell death. Through the use of superresolution imaging, we have shown that vMIA is distributed at the OMM in nanometer scale clusters. This is similar to the clusters reported for the mitochondrial calcium channel, VDAC, as well as electron transport chain, translocase of the OMM complex, and mitochondrial inner membrane organizing system components. Thus, aside from addressing how vMIA targets the MAM and regulates survival of infected cells, biochemical studies and superresolution imaging of vMIA offer insights into the formation, organization, and functioning of MAM. Here, we discuss these insights into trafficking, function, and organization of vMIA at the MAM and OMM and discuss how the use of superresolution imaging is contributing to the study of the formation and trafficking of viruses. PMID:25724304

  10. The Association of Receptor of Activated Protein Kinase C 1(RACK1) with Infectious Bursal Disease Virus Viral Protein VP5 and Voltage-dependent Anion Channel 2 (VDAC2) Inhibits Apoptosis and Enhances Viral Replication*

    PubMed Central

    Lin, Wencheng; Zhang, Zhiqiang; Xu, Zhichao; Wang, Bin; Li, Xiaoqi; Cao, Hong; Wang, Yongqiang; Zheng, Shijun J.

    2015-01-01

    Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Our previous report indicates that IBDV VP5 induces apoptosis via interaction with voltage-dependent anion channel 2 (VDAC2). However, the underlying molecular mechanism is still unclear. We report here that receptor of activated protein kinase C 1 (RACK1) interacts with both VDAC2 and VP5 and that they could form a complex. We found that overexpression of RACK1 inhibited IBDV-induced apoptosis in DF-1 cells and that knockdown of RACK1 by small interfering RNA induced apoptosis associated with activation of caspases 9 and 3 and suppressed IBDV growth. These results indicate that RACK1 plays an antiapoptotic role during IBDV infection via interaction with VDAC2 and VP5, suggesting that VP5 sequesters RACK1 and VDAC2 in the apoptosis-inducing process. PMID:25583988

  11. X-Linked Inhibitor of Apoptosis Protein-Mediated Attenuation of Apoptosis, Using a Novel Cardiac-Enhanced Adeno-Associated Viral Vector

    PubMed Central

    Piacentino III, Valentino; Milano, Carmelo A.; Bolanos, Michael; Schroder, Jacob; Messina, Emily; Cockrell, Adam S.; Jones, Edward; Krol, Ava; Bursac, Nenad; Mao, Lan; Devi, Gayathri R.; Samulski, R. Jude

    2012-01-01

    Abstract Successful amelioration of cardiac dysfunction and heart failure through gene therapy approaches will require a transgene effective at attenuating myocardial injury, and subsequent remodeling, using an efficient and safe delivery vehicle. Our laboratory has established a well-curated, high-quality repository of human myocardial tissues that we use as a discovery engine to identify putative therapeutic transgene targets, as well as to better understand the molecular basis of human heart failure. By using this rare resource we were able to examine age- and sex-matched left ventricular samples from (1) end-stage failing human hearts and (2) nonfailing human hearts and were able to identify the X-linked inhibitor of apoptosis protein (XIAP) as a novel target for treating cardiac dysfunction. We demonstrate that XIAP is diminished in failing human hearts, indicating that this potent inhibitor of apoptosis may be central in protecting the human heart from cellular injury culminating in heart failure. Efforts to ameliorate heart failure through delivery of XIAP compelled the design of a novel adeno-associated viral (AAV) vector, termed SASTG, that achieves highly efficient transduction in mouse heart and in cultured neonatal rat cardiomyocytes. Increased XIAP expression achieved with the SASTG vector inhibits caspase-3/7 activity in neonatal cardiomyocytes after induction of apoptosis through three common cardiac stresses: protein kinase C-γ inhibition, hypoxia, or β-adrenergic receptor agonist. These studies demonstrate the potential benefit of XIAP to correct heart failure after highly efficient delivery to the heart with the rationally designed SASTG AAV vector. PMID:22339372

  12. Enhanced enteroviral infectivity via viral protease-mediated cleavage of Grb2-associated binder 1.

    PubMed

    Deng, Haoyu; Fung, Gabriel; Shi, Junyan; Xu, Suowen; Wang, Chen; Yin, Meimei; Hou, Jun; Zhang, Jingchun; Jin, Zheng-Gen; Luo, Honglin

    2015-11-01

    Coxsackievirus B3 (CVB3), an important human causative pathogen for viral myocarditis, pancreatitis, and meningitis, has evolved different strategies to manipulate the host signaling machinery to ensure successful viral infection. We previously revealed a crucial role for the ERK1/2 signaling pathway in regulating viral infectivity. However, the detail mechanism remains largely unknown. Grb2-associated binder 1 (GAB1) is an important docking protein responsible for intracellular signaling assembly and transduction. In this study, we demonstrated that GAB1 was proteolytically cleaved after CVB3 infection at G175 and G436 by virus-encoded protease 2A(pro), independent of caspase activation. Knockdown of GAB1 resulted in a significant reduction of viral protein expression and virus titers. Moreover, we showed that virus-induced cleavage of GAB1 is beneficial to viral growth as the N-terminal proteolytic product of GAB1 (GAB1-N1-174) further enhances ERK1/2 activation and promotes viral replication. Our results collectively suggest that CVB3 targets host GAB1 to generate a GAB1-N1-174 fragment that enhances viral infectivity, at least in part, via activation of the ERK pathway. The findings in this study suggest a novel mechanism that CVB3 employs to subvert the host signaling and facilitate consequent viral replication. PMID:26183772

  13. POTENCY RANKING OF CHEMICALS BASED ON ENHANCEMENT OF VIRAL TRANSFORMATION

    EPA Science Inventory

    Treating primary hamster embryo cells with various classes of chemical carcinogens and mutagens leads to enhancement of transformation by simian adenovirus SA7. It appears that carcinogenic chemicals render the individual cells more sensitive to viral transformation, thus increas...

  14. Structures and Mechanisms of Viral Membrane Fusion Proteins

    PubMed Central

    White, Judith M.; Delos, Sue E.; Brecher, Matthew; Schornberg, Kathryn

    2009-01-01

    Recent work has identified three distinct classes of viral membrane fusion proteins based on structural criteria. In addition, there are at least four distinct mechanisms by which viral fusion proteins can be triggered to undergo fusion-inducing conformational changes. Viral fusion proteins also contain different types of fusion peptides and vary in their reliance on accessory proteins. These differing features combine to yield a rich diversity of fusion proteins. Yet despite this staggering diversity, all characterized viral fusion proteins convert from a fusion-competent state (dimers or trimers, depending on the class) to a membrane-embedded homotrimeric prehairpin, and then to a trimer-of-hairpins that brings the fusion peptide, attached to the target membrane, and the transmembrane domain, attached to the viral membrane, into close proximity thereby facilitating the union of viral and target membranes. During these conformational conversions, the fusion proteins induce membranes to progress through stages of close apposition, hemifusion, and then the formation of small, and finally large, fusion pores. Clearly, highly divergent proteins have converged on the same overall strategy to mediate fusion, an essential step in the life cycle of every enveloped virus. PMID:18568847

  15. Engineering Biomaterial Systems to Enhance Viral Vector Gene Delivery

    PubMed Central

    Jang, Jae-Hyung; Schaffer, David V; Shea, Lonnie D

    2011-01-01

    Integrating viral gene delivery with engineered biomaterials is a promising strategy to overcome a number of challenges associated with virus-mediated gene delivery, including inefficient delivery to specific cell types, limited tropism, spread of vectors to distant sites, and immune responses. Viral vectors can be combined with biomaterials either through encapsulation within the material or immobilization onto a material surface. Subsequent biomaterial-based delivery can increase the vector's residence time within the target site, thereby potentially providing localized delivery, enhancing transduction, and extending the duration of gene expression. Alternatively, physical or chemical modification of viral vectors with biomaterials can be employed to modulate the tropism of viruses or reduce inflammatory and immune responses, both of which may benefit transduction. This review describes strategies to promote viral gene delivery technologies using biomaterials, potentially providing opportunities for numerous applications of gene therapy to inherited or acquired disorders, infectious disease, and regenerative medicine. PMID:21629221

  16. Illuminating structural proteins in viral "dark matter" with metaproteomics.

    PubMed

    Brum, Jennifer R; Ignacio-Espinoza, J Cesar; Kim, Eun-Hae; Trubl, Gareth; Jones, Robert M; Roux, Simon; VerBerkmoes, Nathan C; Rich, Virginia I; Sullivan, Matthew B

    2016-03-01

    Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional "viral dark matter." Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional dark matter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore, four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world's oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Together, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter. PMID:26884177

  17. Illuminating structural proteins in viral "dark matter" with metaproteomics

    DOE PAGESBeta

    Brum, Jennifer R.; Ignacio-Espinoza, J. Cesar; Kim, Eun -Hae; Trubl, Gareth; Jones, Robert M.; Roux, Simon; Verberkmoes, Nathan C.; Rich, Virginia I.; Sullivan, Matthew B.

    2016-02-16

    Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional "viral dark matter." Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional darkmatter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore,more » four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world's oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Altogether, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter.« less

  18. HMGB1 Protein Binds to Influenza Virus Nucleoprotein and Promotes Viral Replication

    PubMed Central

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

    2012-01-01

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

  19. The Protein Kinase Double-Stranded RNA-Dependent (PKR) Enhances Protection against Disease Cause by a Non-Viral Pathogen

    PubMed Central

    White, Christine L.; Patel, Krupen; Lamb, Bruce; Sen, Ganes C.; Subauste, Carlos S.

    2013-01-01

    PKR is well characterized for its function in antiviral immunity. Using Toxoplasma gondii, we examined if PKR promotes resistance to disease caused by a non-viral pathogen. PKR−/− mice infected with T. gondii exhibited higher parasite load and worsened histopathology in the eye and brain compared to wild-type controls. Susceptibility to toxoplasmosis was not due to defective expression of IFN-γ, TNF-α, NOS2 or IL-6 in the retina and brain, differences in IL-10 expression in these organs or to impaired induction of T. gondii-reactive T cells. While macrophages/microglia with defective PKR signaling exhibited unimpaired anti-T. gondii activity in response to IFN-γ/TNF-α, these cells were unable to kill the parasite in response to CD40 stimulation. The TRAF6 binding site of CD40, but not the TRAF2,3 binding sites, was required for PKR phosphorylation in response to CD40 ligation in macrophages. TRAF6 co-immunoprecipitated with PKR upon CD40 ligation. TRAF6-PKR interaction appeared to be indirect, since TRAF6 co-immunoprecipitated with TRAF2 and TRAF2 co-immunoprecipitated with PKR, and deficiency of TRAF2 inhibited TRAF6-PKR co-immunoprecipitation as well as PKR phosphorylation induced by CD40 ligation. PKR was required for stimulation of autophagy, accumulation the autophagy molecule LC3 around the parasite, vacuole-lysosomal fusion and killing of T. gondii in CD40-activated macrophages and microglia. Thus, our findings identified PKR as a mediator of anti-microbial activity and promoter of protection against disease caused by a non-viral pathogen, revealed that PKR is activated by CD40 via TRAF6 and TRAF2, and positioned PKR as a link between CD40-TRAF signaling and stimulation of the autophagy pathway. PMID:23990781

  20. New insights into an X-traordinary viral protein

    PubMed Central

    Schaller, Torsten; Bauby, Hélène; Hué, Stéphane; Malim, Michael H.; Goujon, Caroline

    2014-01-01

    Vpx is a protein encoded by members of the HIV-2/SIVsmm and SIVrcm/SIVmnd-2 lineages of primate lentiviruses, and is packaged into viral particles. Vpx plays a critical role during the early steps of the viral life cycle and has been shown to counteract SAMHD1, a restriction factor in myeloid and resting T cells. However, it is becoming evident that Vpx is a multifunctional protein in that SAMHD1 antagonism is likely not its sole role. This review summarizes the current knowledge on this X-traordinary protein. PMID:24782834

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

    PubMed Central

    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. PMID:25938226

  2. Anti-Viral Antibody Profiling by High Density Protein Arrays

    PubMed Central

    Bian, Xiaofang; Wiktor, Peter; Kahn, Peter; Brunner, Al; Khela, Amritpal; Karthikeyan, Kailash; Barker, Kristi; Yu, Xiaobo; Magee, Mitch; Wasserfall, Clive H.; Gibson, David; Rooney, Madeleine E; Qiu, Ji; LaBaer, Joshua

    2015-01-01

    Viral infections elicit anti-viral antibodies and have been associated with various chronic diseases. Detection of these antibodies can facilitate diagnosis, treatment of infection and understanding of the mechanisms of virus associated diseases. In this work, we assayed anti-viral antibodies using a novel high density-nucleic acid programmable protein array (HD-NAPPA) platform. Individual viral proteins were expressed in situ directly from plasmids encoding proteins in an array of microscopic reaction chambers. Quality of protein display and serum response was assured by comparing intra- and inter- array correlation within or between printing batches with average correlation coefficients of 0.91 and 0.96, respectively. HD-NAPPA showed higher signal to background (S/B) ratio compared with standard NAPPA on planar glass slides and ELISA. Antibody responses to 761 antigens from 25 different viruses were profiled among patients with juvenile idiopathic arthritis (JIA) and type 1 diabetes (T1D). Common as well as unique antibody reactivity patterns were detected between patients and healthy controls. We believe HD-viral-NAPPA will enable the study of host-pathogen interactions at unprecedented dimensions and elucidate the role of pathogen infections in disease development. PMID:25758251

  3. Discovery of host-viral protein complexes during infection

    PubMed Central

    Rowles, Daniell L.; Terhune, Scott S.; Cristea, Ileana M.

    2014-01-01

    Summary Viruses have co-evolved with their hosts, developing effective approaches for hijacking and manipulating host cellular processes. Therefore, for their efficient replication and spread, viruses depend on dynamic and temporally-regulated interactions with host proteins. The rapid identification of host proteins targeted by viral proteins during infection provides significant insights into mechanisms of viral protein function. The resulting discoveries often lead to unique and innovative hypotheses on viral protein function. Here, we describe a robust method for identifying virus-host protein interactions and protein complexes, which we have successfully utilized to characterize spatial-temporal protein interactions during infections with either DNA or RNA viruses, including human cytomegalovirus (HCMV), herpes simplex virus type 1 (HSV-1), pseudorabies virus (PRV), human immunodeficiency virus (HIV-1), Sindbis, and West Nile virus (WNV). This approach involves cryogenic cell lysis, rapid immunoaffinity purification targeting a virus or host protein, followed by identification of associated proteins using mass spectrometry. Like most proteomic approaches, this methodology has evolved over the past few years and continues to evolve. We are presenting here the updated approaches for each step, and discuss alternative strategies allowing for the protocol to be optimized for different biological systems. PMID:23996249

  4. Manipulation of Plant Host Susceptibility: An Emerging Role for Viral Movement Proteins?

    PubMed Central

    Amari, Khalid; Vazquez, Franck; Heinlein, Manfred

    2012-01-01

    Viruses encode viral suppressors of RNA silencing (VSRs) to counteract RNA silencing, a major antiviral defense response in plants. Recent studies indicate a role of virus-derived siRNAs in manipulating the expression of specific host genes and that certain plant viral movement proteins (MPs) can act as viral enhancers of RNA silencing (VERs) by stimulating the spread of silencing between cells. This suggests that viruses have evolved complex responses capable to efficiently hijack the host RNA silencing machinery to their own advantage. We draw here a dynamic model of the interaction of plant viruses with the silencing machinery during invasion of the host. The model proposes that cells at the spreading front of infection, where infection starts from zero and the VSR levels are supposedly low, represent potential sites for viral manipulation of host gene expression by using virus- and host-derived small RNAs. Viral MPs may facilitate the spread of silencing to produce a wave of small RNA-mediated gene expression changes ahead of the infection to increase host susceptibility. When experimentally ascertained, this hypothetical model will call for re-defining viral movement and the function of viral MPs. PMID:22639637

  5. Nucleotides in the polyomavirus enhancer that control viral transcription and DNA replication.

    PubMed Central

    Tang, W J; Berger, S L; Triezenberg, S J; Folk, W R

    1987-01-01

    The polyomavirus enhancer is required in cis for high-level expression of the viral early region and for replication of the viral genome. We introduced multiple mutations in the enhancer which reduced transcription and DNA replication. Polyomaviruses with these mutant enhancers formed very small plaques in whole mouse embryo cells. Revertants of the viral mutants were isolated and characterized. Reversion occurred by any of the following events: restoration of guanosines at nucleotide (nt) 5134 and nt 5140 within the adenovirus 5 E1A enhancer core AGGAAGTGACT; acquisition of an A----G mutation at nt 5258, which is the same mutation that enables polyomavirus to grow in embryonal carcinoma F9 cells; duplication of mutated sequences between nt 5146 and 5292 (including sequences homologous with immunoglobulin G, simian virus 40, and bovine papillomavirus enhancer elements). Reversion restored both the replicative and transcriptional functions of the viruses. Revertants that acquired the F9 mutation at nt 5258 grew at least 20-fold better than the original mutant in whole mouse embryo cells, but replicated only marginally better than the original mutant in 3T6 cells. Viruses with a reversion of the mutation at nt 5140 replicated equally well in both types of cells. Since individual nucleotides in the polyomavirus enhancer simultaneously altered DNA replication and transcription in specific cell types, it is likely that these processes rely upon a common element, such as an enhancer-binding protein. Images PMID:3037332

  6. Theoretical studies of viral capsid proteins.

    PubMed

    Phelps, D K; Speelman, B; Post, C B

    2000-04-01

    Recent results in structural biology and increases in computer power have prompted initial theoretical studies on capsids of nonenveloped icosahedral viruses. The macromolecular assembly of 60 to 180 protein copies into a protein shell results in a structure of considerable size for molecular dynamics simulations. Nonetheless, progress has been made in examining these capsid assemblies from molecular dynamics calculations and kinetic models. The goals of these studies are to understand capsid function and structural properties, including quarternary structural stability, effects of antiviral compounds that bind the capsid and the self-assembly process. The insight that can be gained from the detailed information provided by simulations is demonstrated in studies of human rhinovirus; an entropic basis for the antiviral activity of hydrophobic compounds, predicted from calculated compressibility values, has been corroborated by experimental measurements on poliovirus. PMID:10753813

  7. Geminivirus C3 Protein: Replication Enhancement and Protein Interactions

    PubMed Central

    Settlage, Sharon B.; See, Renee G.; Hanley-Bowdoin, Linda

    2005-01-01

    Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants. PMID:16014949

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

    PubMed Central

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

    2015-01-01

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

  9. Viral and host proteins that modulate filovirus budding

    PubMed Central

    Liu, Yuliang; Harty, Ronald N

    2010-01-01

    The filoviruses, Ebola and Marburg, utilize a multifaceted mechanism for assembly and budding of infectious virions from mammalian cells. Growing evidence not only demonstrates the importance of multiple viral proteins for efficient assembly and budding, but also the exploitation of various host proteins/pathways by the virus during this late stage of filovirus replication, including endocytic compartments, vacuolar protein sorting pathways, ubiquitination machinery, lipid rafts and cytoskeletal components. Continued elucidation of these complex and orchestrated virus-host interactions will provide a fundamental understanding of the molecular mechanisms of filovirus assembly/budding and ultimately lead to the development of novel viral- and/or host-oriented therapeutics to inhibit filovirus egress and spread. This article will focus on the most recent studies on host interactions and modulation of filovirus budding and summarize the key findings from these investigations. PMID:20730024

  10. HIV1-viral protein R (Vpr) mutations: associated phenotypes and relevance for clinical pathologies.

    PubMed

    Soares, Rui; Rocha, Graça; Meliço-Silvestre, António; Gonçalves, Teresa

    2016-09-01

    Over the last 30 years, research into HIV has advanced the knowledge of virus genetics and the development of efficient therapeutic strategies. HIV-1 viral protein R (Vpr) is a specialized and multifunctional protein that plays important roles at multiple stages of the HIV-1 viral life cycle. This protein interacts with a number of cellular and viral proteins and with multiple activities including nuclear transport of the pre-integration complex (PIC) to the nucleus, transcriptional activation, cell cycle arrest at G2/M transition phase and induction of cell death via apoptosis. Specifically, Vpr has been shown to control many host cell functions through a variety of biological processes and by interaction with several cellular pathways. The different functions of Vpr may enhance viral replication and impair the immune system in HIV-1 infected patients. Importantly, functional defects induced by mutations in the Vpr protein correlate with slow disease progression of HIV-infected patients. Vpr is also associated with other concomitant pathologies developed by these patients, which may lead it to be considered as a potential novel therapeutic target. This review will focus on HIV-1 Vpr, mainly on the importance of its structural mutations on the progression of HIV infection, associated phenotypes and relevance for clinical pathologies. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27264019

  11. 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

  12. Uncovering Viral Protein-Protein Interactions and their Role in Arenavirus Life Cycle

    PubMed Central

    Loureiro, Maria Eugenia; D’Antuono, Alejandra; Levingston Macleod, Jesica M.; López, Nora

    2012-01-01

    The Arenaviridae family includes widely distributed pathogens that cause severe hemorrhagic fever in humans. Replication and packaging of their single-stranded RNA genome involve RNA recognition by viral proteins and a number of key protein-protein interactions. Viral RNA synthesis is directed by the virus-encoded RNA dependent-RNA polymerase (L protein) and requires viral RNA encapsidation by the Nucleoprotein. In addition to the role that the interaction between L and the Nucleoprotein may have in the replication process, polymerase activity appears to be modulated by the association between L and the small multifunctional Z protein. Z is also a structural component of the virions that plays an essential role in viral morphogenesis. Indeed, interaction of the Z protein with the Nucleoprotein is critical for genome packaging. Furthermore, current evidence suggests that binding between Z and the viral envelope glycoprotein complex is required for virion infectivity, and that Z homo-oligomerization is an essential step for particle assembly and budding. Efforts to understand the molecular basis of arenavirus life cycle have revealed important details on these viral protein-protein interactions that will be reviewed in this article. PMID:23170177

  13. Uncovering viral protein-protein interactions and their role in arenavirus life cycle.

    PubMed

    Loureiro, Maria Eugenia; D'Antuono, Alejandra; Levingston Macleod, Jesica M; López, Nora

    2012-09-01

    The Arenaviridae family includes widely distributed pathogens that cause severe hemorrhagic fever in humans. Replication and packaging of their single-stranded RNA genome involve RNA recognition by viral proteins and a number of key protein-protein interactions. Viral RNA synthesis is directed by the virus-encoded RNA dependent-RNA polymerase (L protein) and requires viral RNA encapsidation by the Nucleoprotein. In addition to the role that the interaction between L and the Nucleoprotein may have in the replication process, polymerase activity appears to be modulated by the association between L and the small multifunctional Z protein. Z is also a structural component of the virions that plays an essential role in viral morphogenesis. Indeed, interaction of the Z protein with the Nucleoprotein is critical for genome packaging. Furthermore, current evidence suggests that binding between Z and the viral envelope glycoprotein complex is required for virion infectivity, and that Z homo-oligomerization is an essential step for particle assembly and budding. Efforts to understand the molecular basis of arenavirus life cycle have revealed important details on these viral protein-protein interactions that will be reviewed in this article. PMID:23170177

  14. Serum amyloid A protein in acute viral infections.

    PubMed Central

    Miwata, H; Yamada, T; Okada, M; Kudo, T; Kimura, H; Morishima, T

    1993-01-01

    Concentrations of serum amyloid A protein (SAA) were measured in 254 children with viral diseases, including measles, varicella, rubella, mumps, echo-30 meningitis, chronic hepatitis B and C, and in eight with Kawasaki disease. Latex agglutination nephelometric immunoassay was used for assaying SAA. In 191 out of 195 patients (98%), SAA concentrations became markedly raised in the acute phase of the viral disease: measles (97%), varicella (100%), mumps (95%), and echo-30 meningitis (99%) with mean titres of 82.4, 80.5, 60.2, 75.2, and 101.1 micrograms/ml respectively. This increase in SAA was followed by a rapid return to normal concentrations (< 5 micrograms/ml) during convalescence. Remarkably higher concentrations of SAA (mean 1630 micrograms/ml) were detected in the acute phase of patients with Kawasaki disease, but in most of the children with chronic hepatitis B or C, the titres of SAA remained normal. There was no close correlation between SAA and serum concentrations for alpha 1-acid glycoprotein, beta 2-microglobulin, transferrin, and IgG. There was a clear correlation between SAA and C reactive protein concentrations, although SAA showed a greater incremental change than C reactive protein in the acute phase. In the acute phase of these viral diseases, 56% of the patients had raised SAA concentrations (> or = 5 micrograms/ml) with normal C reactive protein concentrations (< 5 micrograms/ml). These results indicate that SAA could be useful as an inflammatory marker in children with acute viral infections. PMID:8481043

  15. Controlled Assembly of Viral Surface Proteins into Biological Nanoparticles

    NASA Astrophysics Data System (ADS)

    Nakatani-Webster, Eri

    In recent years, therapeutic use of engineered particles on the 1-1,000 nm scale has gained popularity; these nanoparticles have been developed for use in drug delivery, gene therapy, vaccine preparation, and diagnostics. Often, viral proteins are utilized in the design of such species, and outlined here are completed studies on the in vitro assembly of nanoparticles derived from two very different viral systems. The incorporation of the human immunodeficiency virus (HIV) envelope glycoprotein precursor gp160 into phospholipid bilayer nanodiscs is discussed as a potential platform for vaccine design; efforts were successful, however yield currently limits the practical application of this approach. The utility of bacteriophage lambda procapsids and virus-like particles in therapeutic nanoparticle design is also outlined, as are efforts toward the structural and thermodynamic characterization of a urea-triggered capsid maturation event. It is demonstrated that lambda virus-like particles can be assembled from purified capsid and scaffolding proteins, and that these particles undergo urea-triggered maturation and in vitro decoration protein addition similar to that seen in lambda procapsids. The studies on lambda provided materials for the further development of nanoparticles potentially useful in a clinical setting, as well as shedding light on critical viral assembly and maturation events as they may take place in vivo.

  16. Cementing proteins provide extra mechanical stabilization to viral cages

    NASA Astrophysics Data System (ADS)

    Hernando-Pérez, M.; Lambert, S.; Nakatani-Webster, E.; Catalano, C. E.; de Pablo, P. J.

    2014-07-01

    The study of virus shell stability is key not only for gaining insights into viral biological cycles but also for using viral capsids in materials science. The strength of viral particles depends profoundly on their structural changes occurring during maturation, whose final step often requires the specific binding of ‘decoration’ proteins (such as gpD in bacteriophage lambda) to the viral shell. Here we characterize the mechanical stability of gpD-free and gpD-decorated bacteriophage lambda capsids. The incorporation of gpD into the lambda shell imparts a major mechanical reinforcement that resists punctual deformations. We further interrogate lambda particle stability with molecular fatigue experiments that resemble the sub-lethal Brownian collisions of virus shells with macromolecules in crowded environments. Decorated particles are especially robust against collisions of a few kBT (where kB is the Boltzmann’s constant and T is the temperature ~300 K), which approximate those anticipated from molecular insults in the environment.

  17. Cementing proteins provide extra mechanical stabilization to viral cages.

    PubMed

    Hernando-Pérez, M; Lambert, S; Nakatani-Webster, E; Catalano, C E; de Pablo, P J

    2014-01-01

    The study of virus shell stability is key not only for gaining insights into viral biological cycles but also for using viral capsids in materials science. The strength of viral particles depends profoundly on their structural changes occurring during maturation, whose final step often requires the specific binding of 'decoration' proteins (such as gpD in bacteriophage lambda) to the viral shell. Here we characterize the mechanical stability of gpD-free and gpD-decorated bacteriophage lambda capsids. The incorporation of gpD into the lambda shell imparts a major mechanical reinforcement that resists punctual deformations. We further interrogate lambda particle stability with molecular fatigue experiments that resemble the sub-lethal Brownian collisions of virus shells with macromolecules in crowded environments. Decorated particles are especially robust against collisions of a few kBT (where kB is the Boltzmann's constant and T is the temperature ~300 K), which approximate those anticipated from molecular insults in the environment. PMID:25072871

  18. Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins.

    PubMed

    Kudryashova, Elena; Koneru, Pratibha C; Kvaratskhelia, Mamuka; Strömstedt, Adam A; Lu, Wuyuan; Kudryashov, Dmitri S

    2016-01-01

    Human defensins are innate immune defense peptides with a remarkably broad repertoire of anti-pathogen activities. In addition to modulating immune response, inflammation, and angiogenesis, disintegrating bacterial membranes, and inactivating bacterial toxins, defensins are known to intercept various viruses at different stages of their life cycles, while remaining relatively benign towards human cells and proteins. Recently we have found that human defensins inactivate proteinaceous bacterial toxins by taking advantage of their low thermodynamic stability and acting as natural "anti-chaperones", i.e. destabilizing the native conformation of the toxins. In the present study we tested various proteins produced by several viruses (HIV-1, PFV, and TEV) and found them to be susceptible to destabilizing effects of human α-defensins HNP-1 and HD-5 and the synthetic θ-defensin RC-101, but not β-defensins hBD-1 and hBD-2 or structurally related plant-derived peptides. Defensin-induced unfolding promoted exposure of hydrophobic groups otherwise confined to the core of the viral proteins. This resulted in precipitation, an enhanced susceptibility to proteolytic cleavage, and a loss of viral protein activities. We propose, that defensins recognize and target a common and essential physico-chemical property shared by many bacterial toxins and viral proteins - the intrinsically low thermodynamic protein stability. PMID:27581352

  19. Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins

    PubMed Central

    Kudryashova, Elena; Koneru, Pratibha C.; Kvaratskhelia, Mamuka; Strömstedt, Adam A.; Lu, Wuyuan; Kudryashov, Dmitri S.

    2016-01-01

    Human defensins are innate immune defense peptides with a remarkably broad repertoire of anti-pathogen activities. In addition to modulating immune response, inflammation, and angiogenesis, disintegrating bacterial membranes, and inactivating bacterial toxins, defensins are known to intercept various viruses at different stages of their life cycles, while remaining relatively benign towards human cells and proteins. Recently we have found that human defensins inactivate proteinaceous bacterial toxins by taking advantage of their low thermodynamic stability and acting as natural “anti-chaperones”, i.e. destabilizing the native conformation of the toxins. In the present study we tested various proteins produced by several viruses (HIV-1, PFV, and TEV) and found them to be susceptible to destabilizing effects of human α-defensins HNP-1 and HD-5 and the synthetic θ-defensin RC-101, but not β-defensins hBD-1 and hBD-2 or structurally related plant-derived peptides. Defensin-induced unfolding promoted exposure of hydrophobic groups otherwise confined to the core of the viral proteins. This resulted in precipitation, an enhanced susceptibility to proteolytic cleavage, and a loss of viral protein activities. We propose, that defensins recognize and target a common and essential physico-chemical property shared by many bacterial toxins and viral proteins – the intrinsically low thermodynamic protein stability. PMID:27581352

  20. Rift Valley Fever Virus Nonstructural Protein NSs Promotes Viral RNA Replication and Transcription in a Minigenome System

    PubMed Central

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

    2005-01-01

    Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-α/β) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-α/β production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis. PMID:15827175

  1. Transactivation of programmed ribosomal frameshifting by a viral protein.

    PubMed

    Li, Yanhua; Treffers, Emmely E; Napthine, Sawsan; Tas, Ali; Zhu, Longchao; Sun, Zhi; Bell, Susanne; Mark, Brian L; van Veelen, Peter A; van Hemert, Martijn J; Firth, Andrew E; Brierley, Ian; Snijder, Eric J; Fang, Ying

    2014-05-27

    Programmed -1 ribosomal frameshifting (-1 PRF) is a widely used translational mechanism facilitating the expression of two polypeptides from a single mRNA. Commonly, the ribosome interacts with an mRNA secondary structure that promotes -1 frameshifting on a homopolymeric slippery sequence. Recently, we described an unusual -2 frameshifting (-2 PRF) signal directing efficient expression of a transframe protein [nonstructural protein 2TF (nsp2TF)] of porcine reproductive and respiratory syndrome virus (PRRSV) from an alternative reading frame overlapping the viral replicase gene. Unusually, this arterivirus PRF signal lacks an obvious stimulatory RNA secondary structure, but as confirmed here, can also direct the occurrence of -1 PRF, yielding a third, truncated nsp2 variant named "nsp2N." Remarkably, we now show that both -2 and -1 PRF are transactivated by a protein factor, specifically a PRRSV replicase subunit (nsp1β). Embedded in nsp1β's papain-like autoproteinase domain, we identified a highly conserved, putative RNA-binding motif that is critical for PRF transactivation. The minimal RNA sequence required for PRF was mapped within a 34-nt region that includes the slippery sequence and a downstream conserved CCCANCUCC motif. Interaction of nsp1β with the PRF signal was demonstrated in pull-down assays. These studies demonstrate for the first time, to our knowledge, that a protein can function as a transactivator of ribosomal frameshifting. The newly identified frameshifting determinants provide potential antiviral targets for arterivirus disease control and prevention. Moreover, protein-induced transactivation of frameshifting may be a widely used mechanism, potentially including previously undiscovered viral strategies to regulate viral gene expression and/or modulate host cell translation upon infection. PMID:24825891

  2. Chemokine binding proteins: An immunomodulatory strategy going viral.

    PubMed

    González-Motos, Víctor; Kropp, Kai A; Viejo-Borbolla, Abel

    2016-08-01

    Chemokines are chemotactic cytokines whose main function is to direct cell migration. The chemokine network is highly complex and its deregulation is linked to several diseases including immunopathology, cancer and chronic pain. Chemokines also play essential roles in the antiviral immune response. Viruses have therefore developed several counter strategies to modulate chemokine activity. One of these is the expression of type I transmembrane or secreted proteins with the ability to bind chemokines and modulate their activity. These proteins, termed viral chemokine binding proteins (vCKBP), do not share sequence homology with host proteins and are immunomodulatory in vivo. In this review we describe the discovery and characterization of vCKBP, explain their role in the context of infection in vivo and discuss relevant novel findings. PMID:26987612

  3. Cholesterol-binding viral proteins in virus entry and morphogenesis.

    PubMed

    Schroeder, Cornelia

    2010-01-01

    Up to now less than a handful of viral cholesterol-binding proteins have been characterized, in HIV, influenza virus and Semliki Forest virus. These are proteins with roles in virus entry or morphogenesis. In the case of the HIV fusion protein gp41 cholesterol binding is attributed to a cholesterol recognition consensus (CRAC) motif in a flexible domain of the ectodomain preceding the trans-membrane segment. This specific CRAC sequence mediates gp41 binding to a cholesterol affinity column. Mutations in this motif arrest virus fusion at the hemifusion stage and modify the ability of the isolated CRAC peptide to induce segregation of cholesterol in artificial membranes.Influenza A virus M2 protein co-purifies with cholesterol. Its proton translocation activity, responsible for virus uncoating, is not cholesterol-dependent, and the transmembrane channel appears too short for integral raft insertion. Cholesterol binding may be mediated by CRAC motifs in the flexible post-TM domain, which harbours three determinants of binding to membrane rafts. Mutation of the CRAC motif of the WSN strain attenuates virulence for mice. Its affinity to the raft-non-raft interface is predicted to target M2 protein to the periphery of lipid raft microdomains, the sites of virus assembly. Its influence on the morphology of budding virus implicates M2 as factor in virus fission at the raft boundary. Moreover, M2 is an essential factor in sorting the segmented genome into virus particles, indicating that M2 also has a role in priming the outgrowth of virus buds.SFV E1 protein is the first viral type-II fusion protein demonstrated to directly bind cholesterol when the fusion peptide loop locks into the target membrane. Cholesterol binding is modulated by another, proximal loop, which is also important during virus budding and as a host range determinant, as shown by mutational studies. PMID:20213541

  4. Role of the SV40 enhancer in the early to late shift in viral transcription.

    PubMed Central

    Kelly, J J; Wildeman, A G

    1991-01-01

    Simian virus 40 large tumor antigen is a multifunctional protein, with two of its roles being the promotion of viral DNA replication and replication-independent activation of viral transcription. Replication leads to a shift in transcription from the early-early to the late and late-early cap sites, through mechanisms poorly understood. The viral transcription enhancer contains sequences important for both early and late transcription, and we therefore have carried out experiments to evaluate its role in these events. We find that the ability of replication to lead to a shift diminishes when early-early transcription is made increasingly stronger by multimerizing the enhancer, and suggest that replication might lead to the shift by interfering with the ability of the enhancer to direct initiation to those sites. The natural situation in the virus of having two copies of this element might represent a compromise between maximizing both T antigen expression early in infection and late gene expression after replication begins. We also show that replication-independent transcription activation by T antigen is bidirectional and involves at least in part elements to which the factor TEF-1 binds. Images PMID:1662364

  5. The Viral Restriction Factor Tetherin Prevents Leucine-rich Pentatricopeptide Repeat-containing Protein (LRPPRC) from Association with Beclin 1 and B-cell CLL/lymphoma 2 (Bcl-2) and Enhances Autophagy and Mitophagy*

    PubMed Central

    Zou, Jing; Li, Wenjiao; Misra, Anisha; Yue, Fei; Song, Kun; Chen, Qi; Guo, Guanghua; Yi, Jinglin; Kimata, Jason T.; Liu, Leyuan

    2015-01-01

    Tetherin has been characterized as a key factor that restricts viral particles such as HIV and hepatitis C virus on plasma membranes, acts as a ligand of the immunoglobulin-like transcript 7 (ILT7) receptor in tumor cells, and suppresses antiviral innate immune responses mediated by human plasmacytoid dendritic cells. However, the normal cellular function of Tetherin without viral infection is unknown. Here we show that Tetherin not only serves as a substrate of autophagy but itself regulates the initiation of autophagy. Tetherin interacts with the autophagy/mitophagy suppressor LRPPRC and prevents LRPPRC from forming a ternary complex with Beclin 1 and Bcl-2 so that Beclin 1 is released to bind with PI3KCIII (class III PI3K) to activate the initiation of autophagy. Suppression of Tetherin leads to impairment of autophagy, whereas overexpression of Tetherin causes activation of autophagy. Under mitophagic stress, Tetherin is concentrated on mitochondria engulfed in autophagosomes. Tetherin plays a general role in the degradation of autophagosomes containing not only the symbiotic mitochondria but also, possibly, the infected virus. Therefore, Tetherin may enhance autophagy and mitophagy to suppress tumorigenesis, enhance innate immune responses, or prevent T cell apoptosis or pyroptosis. PMID:25631043

  6. 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

  7. 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

  8. Human cytomegalovirus RL13 protein interacts with host NUDT14 protein affecting viral DNA replication.

    PubMed

    Wang, Guili; Ren, Gaowei; Cui, Xin; Lu, Zhitao; Ma, Yanping; Qi, Ying; Huang, Yujing; Liu, Zhongyang; Sun, Zhengrong; Ruan, Qiang

    2016-03-01

    The interaction between the host and human cytomegalovirus (HCMV) is important in determining the outcome of a viral infection. The HCMV RL13 gene product exerts independent, inhibitory effects on viral growth in fibroblasts and epithelial cells. At present, there are few reports on the interactions between the HCMV RL13 protein and human host proteins. The present study provided direct evidence for the specific interaction between HCMV RL13 and host nucleoside diphosphate linked moiety X (nudix)‑type motif 14 (NUDT14), a UDP‑glucose pyrophosphatase, using two‑hybrid screening, an in vitro glutathione S‑transferase pull‑down assay, and co‑immunoprecipitation in human embryonic kidney HEK293 cells. Additionally, the RL13 protein was shown to co‑localize with the NUDT14 protein in the HEK293 cell membrane and cytoplasm, demonstrated using fluorescence confocal microscopy. Decreasing the expression level of NUDT14 via NUDT14‑specific small interfering RNAs increased the number of viral DNA copies in the HCMV‑infected cells. However, the overexpression of NUDT14 in a stably expressing cell line did not affect viral DNA levels significantly in the HCMV infected cells. Based on the known functions of NUDT14, the results of the present study suggested that the interaction between the RL13 protein and NUDT14 protein may be involved in HCMV DNA replication, and that NUDT14 may offer potential in the modulation of viral infection. PMID:26781650

  9. 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

  10. Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression.

    PubMed

    Kennedy, Edward M; Bogerd, Hal P; Kornepati, Anand V R; Kang, Dong; Ghoshal, Delta; Marshall, Joy B; Poling, Brigid C; Tsai, Kevin; Gokhale, Nandan S; Horner, Stacy M; Cullen, Bryan R

    2016-05-11

    Covalent addition of a methyl group to adenosine N(6) (m(6)A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m(6)A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m(6)A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m(6)A sequencing techniques to precisely map several m(6)A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3' untranslated region (3' UTR). Viral 3' UTR m(6)A sites or analogous cellular m(6)A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m(6)A "reader" proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m(6)A editing and the resultant recruitment of YTHDF proteins as major positive regulators of HIV-1 mRNA expression. PMID:27117054

  11. Roles of Serine and Threonine Residues of Mumps Virus P Protein in Viral Transcription and Replication

    PubMed Central

    Pickar, Adrian; Xu, Pei; Elson, Andrew; Li, Zhuo; Zengel, James

    2014-01-01

    ABSTRACT Mumps virus (MuV), a paramyxovirus containing a negative-sense nonsegmented RNA genome, is a human pathogen that causes an acute infection with symptoms ranging from parotitis to mild meningitis and severe encephalitis. Vaccination against mumps virus has been effective in reducing mumps cases. However, recently large outbreaks have occurred in vaccinated populations. There is no anti-MuV drug. Understanding replication of MuV may lead to novel antiviral strategies. MuV RNA-dependent RNA polymerase minimally consists of the phosphoprotein (P) and the large protein (L). The P protein is heavily phosphorylated. To investigate the roles of serine (S) and threonine (T) residues of P in viral RNA transcription and replication, P was subjected to mass spectrometry and mutational analysis. P, a 392-amino acid residue protein, has 64 S and T residues. We have found that mutating nine S/T residues significantly reduced and mutating residue T at 101 to A (T101A) significantly enhanced activity in a minigenome system. A recombinant virus containing the P-T101A mutation (rMuV-P-T101A) was recovered and analyzed. rMuV-P-T101A grew to higher titers and had increased protein expression at early time points. Together, these results suggest that phosphorylation of MuV-P-T101 plays a negative role in viral RNA synthesis. This is the first time that the P protein of a paramyxovirus has been systematically analyzed for S/T residues that are critical for viral RNA synthesis. IMPORTANCE Mumps virus (MuV) is a reemerging paramyxovirus that caused large outbreaks in the United States, where vaccination coverage is very high. There is no anti-MuV drug. In this work, we have systematically analyzed roles of Ser/Thr residues of MuV P in viral RNA synthesis. We have identified S/T residues of P critical for MuV RNA synthesis and phosphorylation sites that are important for viral RNA synthesis. This work leads to a better understanding of viral RNA synthesis as well as to potential

  12. WSV399, a viral tegument protein, interacts with the shrimp protein PmVRP15 to facilitate viral trafficking and assembly.

    PubMed

    Jaree, Phattarunda; Senapin, Saengchan; Hirono, Ikuo; Lo, Chu-Fang; Tassanakajon, Anchalee; Somboonwiwat, Kunlaya

    2016-06-01

    Viral responsive protein 15 (PmVRP15) has been identified as a highly up-regulated gene in the hemocyte of white spot syndrome virus (WSSV)-infected shrimp Penaeus monodon. However, the function of PmVRP15 in host-viral interaction was still unclear. To elucidate PmVRP15 function, the interacting partner of PmVRP15 from WSSV was screened by yeast two-hybrid assay and then confirmed by co-immunoprecipitation (Co-IP). Only WSV399 protein was identified as a PmVRP15 binding protein; however, the function of WSV399 has not been characterized. Localization of WSV399 on the WSSV virion was revealed by immunoblotting analysis (in vitro) and immunoelectron microscopy (in vivo). The results showed that WSV399 is a structural protein of the WSSV virion and is particularly located on the tegument. Gene silencing of wsv399 in WSSV-infected shrimp reduced the percentage of cumulative mortality by 74%, although the expression level of a viral replication marker gene, vp28, was not changed suggesting that WSV399 might not involved in viral replication but viral assembly. Because it has already been known that tegument proteins function in capsid transport during viral trafficking and assembly, interaction between PmVRP15 on hemocyte nuclear membrane and the WSV399 viral tegument protein suggests that PmVRP15 might be required for trafficking and assembly of WSSV during infection. PMID:26828390

  13. Rodent models of HAND and drug abuse: exogenous administration of viral protein(s) and cocaine.

    PubMed

    Yao, Honghong; Buch, Shilpa

    2012-06-01

    Humans and chimpanzees are the natural hosts for HIV. Non-human primate models of SIV/SHIV infection in rhesus, cynomologus and pigtail macaques have been used extensively as excellent model systems for pathogenesis and vaccine studies. However, owing to the variability of disease progression in infected macaques, a phenomenon identical to humans, coupled with their prohibitive costs, there exists a critical need for the development of small-animal models in which to study the untoward effects of HIV-1 infection. Owing to the fact that rodents are not the natural permissive hosts for lentiviral infection, development of small animal models for studying virus infection has used strategies that circumvent the steps of viral entry and infection. Such strategies involve overexpression of toxic viral proteins, SCID mice engrafted with the human PBLs or macrophages, and EcoHIV chimeric virus wherein the gp120 of HIV-1 was replaced with the gp80 of the ecotropic murine leukemia virus. Additional strategy that is often used by investigators to study the toxic effect of viral proteins involves direct stereotactic injection of the viral protein(s) into specific brain regions. The present report is a compilation of the applications of direct administration of Tat into the striatum to mimic the effects of the viral neurotoxin in the CNS. Added advantage of this model is that it is also amenable to repeated intraperitoneal cocaine injections, thereby allowing the study of the additive/synergistic effects of both the viral protein and cocaine. Such a model system recapitulates aspects of HAND in the context of drug abuse. PMID:22447295

  14. Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein

    PubMed Central

    King, Cason R.; Cohen, Michael J.; Fonseca, Gregory J.; Dirk, Brennan S.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2016-01-01

    The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A’s role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs. PMID:27137912

  15. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein

    PubMed Central

    Bugli, Francesca; Caprettini, Valeria; Cacaci, Margherita; Martini, Cecilia; Paroni Sterbini, Francesco; Torelli, Riccardo; Della Longa, Stefano; Papi, Massimiliano; Palmieri, Valentina; Giardina, Bruno; Posteraro, Brunella; Sanguinetti, Maurizio; Arcovito, Alessandro

    2014-01-01

    In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here – providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures – opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes. PMID:24936129

  16. Synthesis and characterization of different immunogenic viral nanoconstructs from rotavirus VP6 inner capsid protein.

    PubMed

    Bugli, Francesca; Caprettini, Valeria; Cacaci, Margherita; Martini, Cecilia; Paroni Sterbini, Francesco; Torelli, Riccardo; Della Longa, Stefano; Papi, Massimiliano; Palmieri, Valentina; Giardina, Bruno; Posteraro, Brunella; Sanguinetti, Maurizio; Arcovito, Alessandro

    2014-01-01

    In order to deliver low-cost viral capsomeres from a large amount of soluble viral VP6 protein from human rotavirus, we developed and optimized a biotechnological platform in Escherichia coli. Specifically, three different expression protocols were compared, differing in their genetic constructs, ie, a simple native histidine-tagged VP6 sequence, VP6 fused to thioredoxin, and VP6 obtained with the newly described small ubiquitin-like modifier (SUMO) fusion system. Our results demonstrate that the histidine-tagged protein does not escape the accumulation in the inclusion bodies, and that SUMO is largely superior to the thioredoxin-fusion tag in enhancing the expression and solubility of VP6 protein. Moreover, the VP6 protein produced according to the SUMO fusion tag displays well-known assembly properties, as observed in both transmission electron microscopy and atomic force microscopy images, giving rise to either VP6 trimers, 60 nm spherical virus-like particles, or nanotubes a few microns long. This different quaternary organization of VP6 shows a higher level of immunogenicity for the elongated structures with respect to the spheres or the protein trimers. Therefore, the expression and purification strategy presented here - providing a large amount of the viral capsid protein in the native form with relatively simple, rapid, and economical procedures - opens a new route toward large-scale production of a more efficient antigenic compound to be used as a vaccination tool or as an adjuvant, and also represents a top-quality biomaterial to be further modified for biotechnological purposes. PMID:24936129

  17. Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy.

    PubMed

    Ou, Horng D; Deerinck, Thomas J; Bushong, Eric; Ellisman, Mark H; O'Shea, Clodagh C

    2015-11-15

    Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host's cellular environment, their natural in situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940's and subsequent application to cells in the 1950's. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and

  18. Endolysosomal trafficking of viral G protein-coupled receptor functions in innate immunity and control of viral oncogenesis.

    PubMed

    Dong, Xiaonan; Cheng, Adam; Zou, Zhongju; Yang, Yih-Sheng; Sumpter, Rhea M; Huang, Chou-Long; Bhagat, Govind; Virgin, Herbert W; Lira, Sergio A; Levine, Beth

    2016-03-15

    The ubiquitin-proteasome system degrades viral oncoproteins and other microbial virulence factors; however, the role of endolysosomal degradation pathways in these processes is unclear. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, and a constitutively active viral G protein-coupled receptor (vGPCR) contributes to the pathogenesis of KSHV-induced tumors. We report that a recently discovered autophagy-related protein, Beclin 2, interacts with KSHV GPCR, facilitates its endolysosomal degradation, and inhibits vGPCR-driven oncogenic signaling. Furthermore, monoallelic loss of Becn2 in mice accelerates the progression of vGPCR-induced lesions that resemble human Kaposi's sarcoma. Taken together, these findings indicate that Beclin 2 is a host antiviral molecule that protects against the pathogenic effects of KSHV GPCR by facilitating its endolysosomal degradation. More broadly, our data suggest a role for host endolysosomal trafficking pathways in regulating viral pathogenesis and oncogenic signaling. PMID:26929373

  19. Nuclear Protein Sam68 Interacts with the Enterovirus 71 Internal Ribosome Entry Site and Positively Regulates Viral Protein Translation

    PubMed Central

    Zhang, Hua; Song, Lei; Cong, Haolong

    2015-01-01

    ABSTRACT Enterovirus 71 (EV71) recruits various cellular factors to assist in the replication and translation of its genome. Identification of the host factors involved in the EV71 life cycle not only will enable a better understanding of the infection mechanism but also has the potential to be of use in the development of antiviral therapeutics. In this study, we demonstrated that the cellular factor 68-kDa Src-associated protein in mitosis (Sam68) acts as an internal ribosome entry site (IRES) trans-acting factor (ITAF) that binds specifically to the EV71 5′ untranslated region (5′UTR). Interaction sites in both the viral IRES (stem-loops IV and V) and the heterogeneous nuclear ribonucleoprotein K homology (KH) domain of Sam68 protein were further mapped using an electrophoretic mobility shift assay (EMSA) and biotin RNA pulldown assay. More importantly, dual-luciferase (firefly) reporter analysis suggested that overexpression of Sam68 positively regulated IRES-dependent translation of virus proteins. In contrast, both IRES activity and viral protein translation significantly decreased in Sam68 knockdown cells compared with the negative-control cells treated with short hairpin RNA (shRNA). However, downregulation of Sam68 did not have a significant inhibitory effect on the accumulation of the EV71 genome. Moreover, Sam68 was redistributed from the nucleus to the cytoplasm and interacts with cellular factors, such as poly(rC)-binding protein 2 (PCBP2) and poly(A)-binding protein (PABP), during EV71 infection. The cytoplasmic relocalization of Sam68 in EV71-infected cells may be involved in the enhancement of EV71 IRES-mediated translation. Since Sam68 is known to be a RNA-binding protein, these results provide direct evidence that Sam68 is a novel ITAF that interacts with EV71 IRES and positively regulates viral protein translation. IMPORTANCE The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection

  20. HUMORAL ANTIBODY RESPONSE TO INDIVIDUAL VIRAL PROTEINS AFTER MURINE CYTOMEGALOVIRUS INFECTION

    EPA Science Inventory

    The purpose of this study was to identify viral proteins that played an important role in the humoral immune response to murine cytomegalovirus (MCMV). Viral proteins were separated from a purified virus preparation on polyacrylamide gels, were blotted onto nitrocellulose strips,...

  1. ReVOLT: radiation-enhanced viral oncolytic therapy

    SciTech Connect

    Advani, Sunil J.; Mezhir, James J.; Roizman, Bernard; Weichselbaum, Ralph R. . E-mail: rrw@rover.uchicago.edu

    2006-11-01

    Viral oncolytic therapy has been pursued with renewed interest as the molecular basis of carcinogenesis and viral replication has been elucidated. Genetically engineered, attenuated viruses have been rationally constructed to achieve a therapeutic index in tumor cells compared with surrounding normal tissue. Many of these attenuated mutant viruses have entered clinical trials. Here we review the preclinical literature demonstrating the interaction of oncolytic viruses with ionizing radiation and provides a basis for future clinical trials.

  2. A core viral protein binds host nucleosomes to sequester immune danger signals.

    PubMed

    Avgousti, Daphne C; Herrmann, Christin; Kulej, Katarzyna; Pancholi, Neha J; Sekulic, Nikolina; Petrescu, Joana; Molden, Rosalynn C; Blumenthal, Daniel; Paris, Andrew J; Reyes, Emigdio D; Ostapchuk, Philomena; Hearing, Patrick; Seeholzer, Steven H; Worthen, G Scott; Black, Ben E; Garcia, Benjamin A; Weitzman, Matthew D

    2016-07-01

    Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important part in innate immune responses. Viral-encoded core basic proteins compact viral genomes, but their impact on host chromatin structure and function remains unexplored. Adenoviruses encode a highly basic protein called protein VII that resembles cellular histones. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles, it is unknown whether protein VII affects cellular chromatin. Here we show that protein VII alters cellular chromatin, leading us to hypothesize that this has an impact on antiviral responses during adenovirus infection in human cells. We find that protein VII forms complexes with nucleosomes and limits DNA accessibility. We identified post-translational modifications on protein VII that are responsible for chromatin localization. Furthermore, proteomic analysis demonstrated that protein VII is sufficient to alter the protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in the chromatin of members of the high-mobility-group protein B family (HMGB1, HMGB2 and HMGB3). HMGB1 is actively released in response to inflammatory stimuli and functions as a danger signal to activate immune responses. We showed that protein VII can directly bind HMGB1 in vitro and further demonstrated that protein VII expression in mouse lungs is sufficient to decrease inflammation-induced HMGB1 content and neutrophil recruitment in the bronchoalveolar lavage fluid. Together, our in vitro and in vivo results show that protein VII sequesters HMGB1 and can prevent its release. This study uncovers a viral strategy in which nucleosome binding is exploited to control extracellular immune signaling. PMID:27362237

  3. Viral load and clinical disease enhancement associated with a lentivirus cytotoxic T lymphocyte vaccine regimen

    PubMed Central

    Mealey, Robert H.; Leib, Steven R.; Littke, Matt H.; Wagner, Bettina; Horohov, David W.; McGuire, Travis C.

    2009-01-01

    Effective DNA-based vaccines against lentiviruses will likely induce CTL against conserved viral proteins. Equine infectious anemia virus (EIAV) infects horses worldwide, and serves as a useful model for lentiviral immune control. Although attenuated live EIAV vaccines have induced protective immune responses, DNA-based vaccines have not. In particular, DNA-based vaccines have had limited success in inducing CTL responses against intracellular pathogens in the horse. We hypothesized that priming with a codon-optimized plasmid encoding EIAV Gag p15/p26 with co-administration of a plasmid encoding an equine IL-2/IgG fusion protein as a molecular adjuvant, followed by boosting with a vaccinia vector expressing Gag p15/p26, would induce protective Gag-specific CTL responses. Although the regimen induced Gag-specific CTL in four of seven vaccinated horses, CTL were not detected until after the vaccinia boost, and protective effects were not observed in EIAV challenged vaccinates. Unexpectedly, vaccinates had significantly higher viral loads and more severe clinical disease, associated with the presence of vaccine-induced CTL. It was concluded that 1.) further optimization of the timing and route of DNA immunization was needed for efficient CTL priming in vivo, 2.) co-administration of the IL-2/IgG plasmid did not enhance CTL priming by the Gag p15/p26 plasmid, 3.) vaccinia vectors are useful for lentivirus-specific CTL induction in the horse, 4.) Gag-specific CTL alone are either insufficient or a more robust Gag-specific CTL response is needed to limit EIAV viremia and clinical disease, and 5.) CTL-inducing vaccines lacking envelope immunogens can result in lentiviral disease enhancement. Although the mechanisms for enhancement associated with this vaccine regimen remain to be elucidated, these results have important implications for development of lentivirus T cell vaccines. PMID:19368787

  4. 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

  5. Exploitation of Lipid Components by Viral and Host Proteins for Hepatitis C Virus Infection

    PubMed Central

    Moriishi, Kohji; Matsuura, Yashiharu

    2012-01-01

    Hepatitis C virus (HCV), which is a major causative agent of blood-borne hepatitis, has chronically infected about 170 million individuals worldwide and leads to chronic infection, resulting in development of steatosis, cirrhosis, and eventually hepatocellular carcinoma. Hepatocellular carcinoma associated with HCV infection is not only caused by chronic inflammation, but also by the biological activity of HCV proteins. HCV core protein is known as a main component of the viral nucleocapsid. It cooperates with host factors and possesses biological activity causing lipid alteration, oxidative stress, and progression of cell growth, while other viral proteins also interact with host proteins including molecular chaperones, membrane-anchoring proteins, and enzymes associated with lipid metabolism to maintain the efficiency of viral replication and production. HCV core protein is localized on the surface of lipid droplets in infected cells. However, the role of lipid droplets in HCV infection has not yet been elucidated. Several groups recently reported that other viral proteins also support viral infection by regulation of lipid droplets and core localization in infected cells. Furthermore, lipid components are required for modification of host factors and the intracellular membrane to maintain or up-regulate viral replication. In this review, we summarize the current status of knowledge regarding the exploitation of lipid components by viral and host proteins in HCV infection. PMID:22347882

  6. Roles of Phosphorylation of the Nucleocapsid Protein of Mumps Virus in Regulating Viral RNA Transcription and Replication

    PubMed Central

    Zengel, James; Pickar, Adrian; Xu, Pei; Lin, Alita

    2015-01-01

    ABSTRACT Mumps virus (MuV) is a paramyxovirus with a negative-sense nonsegmented RNA genome. The viral RNA genome is encapsidated by the nucleocapsid protein (NP) to form the ribonucleoprotein (RNP), which serves as a template for transcription and replication. In this study, we investigated the roles of phosphorylation sites of NP in MuV RNA synthesis. Using radioactive labeling, we first demonstrated that NP was phosphorylated in MuV-infected cells. Using both liquid chromatography-mass spectrometry (LC-MS) and in silico modeling, we identified nine putative phosphorylated residues within NP. We mutated these nine residues to alanine. Mutation of the serine residue at position 439 to alanine (S439A) was found to reduce the phosphorylation of NP in transfected cells by over 90%. The effects of these mutations on the MuV minigenome system were examined. The S439A mutant was found to have higher activity, four mutants had lower activity, and four mutants had similar activity compared to wild-type NP. MuV containing the S439A mutation had 90% reduced phosphorylation of NP and enhanced viral RNA synthesis and viral protein expression at early time points after infection, indicating that S439 is the major phosphorylation site of NP and its phosphorylation plays an important role in downregulating viral RNA synthesis. IMPORTANCE Mumps virus (MuV), a paramyxovirus, is an important human pathogen that is reemerging in human populations. Nucleocapsid protein (NP) of MuV is essential for viral RNA synthesis. We have identified the major phosphorylation site of NP. We have found that phosphorylation of NP plays a critical role in regulating viral RNA synthesis. The work will lead to a better understanding of viral RNA synthesis and possible novel targets for antiviral drug development. PMID:25948749

  7. Membrane Requirements for Uridylylation of the Poliovirus VPg Protein and Viral RNA Synthesis In Vitro

    PubMed Central

    Fogg, Mark H.; Teterina, Natalya L.; Ehrenfeld, Ellie

    2003-01-01

    Efficient translation of poliovirus (PV) RNA in uninfected HeLa cell extracts generates all of the viral proteins required to carry out viral RNA replication and encapsidation and to produce infectious virus in vitro. In infected cells, viral RNA replication occurs in ribonucleoprotein complexes associated with clusters of vesicles that are formed from preexisting intracellular organelles, which serve as a scaffold for the viral RNA replication complex. In this study, we have examined the role of membranes in viral RNA replication in vitro. Electron microscopic and biochemical examination of extracts actively engaged in viral RNA replication failed to reveal a significant increase in vesicular membrane structures or the protective aggregation of vesicles observed in PV-infected cells. Viral, nonstructural replication proteins, however, bind to heterogeneous membrane fragments in the extract. Treatment of the extracts with nonionic detergents, a membrane-altering inhibitor of fatty acid synthesis (cerulenin), or an inhibitor of intracellular membrane trafficking (brefeldin A) prevents the formation of active replication complexes in vitro, under conditions in which polyprotein synthesis and processing occur normally. Under all three of these conditions, synthesis of uridylylated VPg to form the primer for initiation of viral RNA synthesis, as well as subsequent viral RNA replication, was inhibited. Thus, although organized membranous structures morphologically similar to the vesicles observed in infected cells do not appear to form in vitro, intact membranes are required for viral RNA synthesis, including the first step of forming the uridylylated VPg primer for RNA chain elongation. PMID:14557626

  8. [The use of a dried protein mixture in treating patients with viral hepatitis].

    PubMed

    Dunaevskiĭ, G A; Karpenko, P A; Denisiako, E I; Tsimbal, A E; Iurkovskaia, N B

    1989-07-01

    The authors studied the effect of a dry protein mixture on the clinical course and protein and pigmentary metabolism, functional tests of the liver in 223 patients with viral hepatitis. It was found that this mixture favours reduction of the icteric period, more rapid restoration of the liver function and improves prognosis. The dry protein mixture is recommended to be included in dietotherapy of patients with viral hepatitis. PMID:2800480

  9. The use of a viral 2A sequence for the simultaneous over-expression of both the vgf gene and enhanced green fluorescent protein (eGFP) in vitro and in vivo

    PubMed Central

    Lewis, Jo E.; Brameld, John M.; Hill, Phil; Barrett, Perry; Ebling, Francis J.P.; Jethwa, Preeti H.

    2015-01-01

    Introduction The viral 2A sequence has become an attractive alternative to the traditional internal ribosomal entry site (IRES) for simultaneous over-expression of two genes and in combination with recombinant adeno-associated viruses (rAAV) has been used to manipulate gene expression in vitro. New method To develop a rAAV construct in combination with the viral 2A sequence to allow long-term over-expression of the vgf gene and fluorescent marker gene for tracking of the transfected neurones in vivo. Results Transient transfection of the AAV plasmid containing the vgf gene, viral 2A sequence and eGFP into SH-SY5Y cells resulted in eGFP fluorescence comparable to a commercially available reporter construct. This increase in fluorescent cells was accompanied by an increase in VGF mRNA expression. Infusion of the rAAV vector containing the vgf gene, viral 2A sequence and eGFP resulted in eGFP fluorescence in the hypothalamus of both mice and Siberian hamsters, 32 weeks post infusion. In situ hybridisation confirmed that the location of VGF mRNA expression in the hypothalamus corresponded to the eGFP pattern of fluorescence. Comparison with old method The viral 2A sequence is much smaller than the traditional IRES and therefore allowed over-expression of the vgf gene with fluorescent tracking without compromising viral capacity. Conclusion The use of the viral 2A sequence in the AAV plasmid allowed the simultaneous expression of both genes in vitro. When used in combination with rAAV it resulted in long-term over-expression of both genes at equivalent locations in the hypothalamus of both Siberian hamsters and mice, without any adverse effects. PMID:26300182

  10. Poly(A)-binding protein interacts with the nucleocapsid protein of porcine reproductive and respiratory syndrome virus and participates in viral replication.

    PubMed

    Wang, Xiaoye; Bai, Juan; Zhang, Lili; Wang, Xianwei; Li, Yufeng; Jiang, Ping

    2012-12-01

    Interactions between host factors and the viral protein play important roles in host adaptation and regulation of virus replication. Poly(A)-binding protein (PABP), a host cellular protein that enhances translational efficiency by circularizing mRNAs, was identified by yeast two-hybrid screening as a cellular partner for PRRSV nucleocapsid (N) protein in porcine alveolar macrophages. The specific interaction of PRRSV N protein with PABP was confirmed in infected cells by co-immunoprecipitation and in vitro by GST pull-down assay. We showed by confocal microscopy that the PABP co-localized with the PRRSV N protein. Using a series of deletion mutants, the interactive domain of N protein with PABP was mapped to a region of amino acids 52-69. For PABP, C-terminal half, which interestingly interacts other translation regulators, was determined to be the domain interactive with N protein. Short hairpin RNA (shRNA)-mediated silencing of PABP in cells resulted in significantly reduced PRRSV RNA synthesis, viral encoded protein expression and viral titer. Overall, the results presented here point toward an important role for PABP in regulating PRRSV replication. PMID:22985629

  11. HSV Usurps Eukaryotic Initiation Factor 3 Subunit M for Viral Protein Translation: Novel Prevention Target

    PubMed Central

    Cheshenko, Natalia; Trepanier, Janie B.; Segarra, Theodore J.; Fuller, A. Oveta; Herold, Betsy C.

    2010-01-01

    Prevention of genital herpes is a global health priority. B5, a recently identified ubiquitous human protein, was proposed as a candidate HSV entry receptor. The current studies explored its role in HSV infection. Viral plaque formation was reduced by ∼90% in human cells transfected with small interfering RNA targeting B5 or nectin-1, an established entry receptor. However, the mechanisms were distinct. Silencing of nectin-1 prevented intracellular delivery of viral capsids, nuclear transport of a viral tegument protein, and release of calcium stores required for entry. In contrast, B5 silencing had no effect on these markers of entry, but inhibited viral protein translation. Specifically, viral immediate early genes, ICP0 and ICP4, were transcribed, polyadenylated and transported from the nucleus to the cytoplasm, but the viral transcripts did not associate with ribosomes or polysomes in B5-silenced cells. In contrast, immediate early gene viral transcripts were detected in polysome fractions isolated from control cells. These findings are consistent with sequencing studies demonstrating that B5 is eukaryotic initiation factor 3 subunit m (eIF3m). Although B5 silencing altered the polysome profile of cells, silencing had little effect on cellular RNA or protein expression and was not cytotoxic, suggesting that this subunit is not essential for host cellular protein synthesis. Together these results demonstrate that B5 plays a major role in the initiation of HSV protein translation and could provide a novel target for strategies to prevent primary and recurrent herpetic disease. PMID:20676407

  12. A Trio of Viral Proteins Tunes Aphid-Plant Interactions in Arabidopsis thaliana

    PubMed Central

    Du, Zhiyou; Murphy, Alex M.; Anggoro, Damar Tri; Tungadi, Trisna; Luang-In, Vijitra; Lewsey, Mathew G.; Rossiter, John T.; Powell, Glen; Smith, Alison G.; Carr, John P.

    2013-01-01

    Background Virus-induced deterrence to aphid feeding is believed to promote plant virus transmission by encouraging migration of virus-bearing insects away from infected plants. We investigated the effects of infection by an aphid-transmitted virus, cucumber mosaic virus (CMV), on the interaction of Arabidopsis thaliana, one of the natural hosts for CMV, with Myzus persicae (common names: ‘peach-potato aphid’, ‘green peach aphid’). Methodology/Principal Findings Infection of Arabidopsis (ecotype Col-0) with CMV strain Fny (Fny-CMV) induced biosynthesis of the aphid feeding-deterrent 4-methoxy-indol-3-yl-methylglucosinolate (4MI3M). 4MI3M inhibited phloem ingestion by aphids and consequently discouraged aphid settling. The CMV 2b protein is a suppressor of antiviral RNA silencing, which has previously been implicated in altering plant-aphid interactions. Its presence in infected hosts enhances the accumulation of CMV and the other four viral proteins. Another viral gene product, the 2a protein (an RNA-dependent RNA polymerase), triggers defensive signaling, leading to increased 4MI3M accumulation. The 2b protein can inhibit ARGONAUTE1 (AGO1), a host factor that both positively-regulates 4MI3M biosynthesis and negatively-regulates accumulation of substance(s) toxic to aphids. However, the 1a replicase protein moderated 2b-mediated inhibition of AGO1, ensuring that aphids were deterred from feeding but not poisoned. The LS strain of CMV did not induce feeding deterrence in Arabidopsis ecotype Col-0. Conclusions/Significance Inhibition of AGO1 by the 2b protein could act as a booby trap since this will trigger antibiosis against aphids. However, for Fny-CMV the interplay of three viral proteins (1a, 2a and 2b) appears to balance the need of the virus to inhibit antiviral silencing, while inducing a mild resistance (antixenosis) that is thought to promote transmission. The strain-specific effects of CMV on Arabidopsis-aphid interactions, and differences between

  13. Multivalent display of proteins on viral nanoparticles using molecular recognition and chemical ligation strategies.

    PubMed

    Venter, P Arno; Dirksen, Anouk; Thomas, Diane; Manchester, Marianne; Dawson, Philip E; Schneemann, Anette

    2011-06-13

    Multivalent display of heterologous proteins on viral nanoparticles forms a basis for numerous applications in nanotechnology, including vaccine development, targeted therapeutic delivery, and tissue-specific bioimaging. In many instances, precise placement of proteins is required for optimal functioning of the supramolecular assemblies, but orientation- and site-specific coupling of proteins to viral scaffolds remains a significant technical challenge. We have developed two strategies that allow for controlled attachment of a variety of proteins on viral particles using covalent and noncovalent principles. In one strategy, an interaction between domain 4 of anthrax protective antigen and its receptor was used to display multiple copies of a target protein on virus-like particles. In the other, expressed protein ligation and aniline-catalyzed oximation was used to display covalently a model protein. The latter strategy, in particular, yielded nanoparticles that induced potent immune responses to the coupled protein, suggesting potential applications in vaccine development. PMID:21545187

  14. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    NASA Astrophysics Data System (ADS)

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-06-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development.

  15. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness.

    PubMed

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  16. Codon optimization of the adenoviral fiber negatively impacts structural protein expression and viral fitness

    PubMed Central

    Villanueva, Eneko; Martí-Solano, Maria; Fillat, Cristina

    2016-01-01

    Codon usage adaptation of lytic viruses to their hosts is determinant for viral fitness. In this work, we analyzed the codon usage of adenoviral proteins by principal component analysis and assessed their codon adaptation to the host. We observed a general clustering of adenoviral proteins according to their function. However, there was a significant variation in the codon preference between the host-interacting fiber protein and the rest of structural late phase proteins, with a non-optimal codon usage of the fiber. To understand the impact of codon bias in the fiber, we optimized the Adenovirus-5 fiber to the codon usage of the hexon structural protein. The optimized fiber displayed increased expression in a non-viral context. However, infection with adenoviruses containing the optimized fiber resulted in decreased expression of the fiber and of wild-type structural proteins. Consequently, this led to a drastic reduction in viral release. The insertion of an exogenous optimized protein as a late gene in the adenovirus with the optimized fiber further interfered with viral fitness. These results highlight the importance of balancing codon usage in viral proteins to adequately exploit cellular resources for efficient infection and open new opportunities to regulate viral fitness for virotherapy and vaccine development. PMID:27278133

  17. Generation of Recombinant Viral Hemorrhagic Septicemia Virus (rVHSV) Expressing Two Foreign Proteins and Effect of Lengthened Viral Genome on Viral Growth and In Vivo Virulence.

    PubMed

    Kim, Min Sun; Lee, Su Jin; Kim, Dong Soo; Kim, Ki Hong

    2016-04-01

    In this study, a new recombinant VHSV (rVHSV-Arfp-Bgfp) was generated by insertion of a red fluorescent protein (RFP) gene between N and P genes, a green fluorescent protein (GFP) gene between P and M genes of VHSV genome, the expression of each heterologous gene in infected cells, and effects of the lengthened recombinant VHSV's genome on the replication ability and in vivo virulence to olive flounder (Paralichthys olivaceus) fingerlings were compared with previously generated rVHSVs (rVHSV-wild, rVHSV-Arfp, and rVHSV-Brfp). The expression of RFP and GFP in cells infected with rVHSV-Arfp-Bgfp was verified through fluorescent microscopy and FACS analysis. In the viral growth analysis, rVHSV-Arfp and rVHSV-Brfp showed significantly lower viral titers than rVHSV-wild, and the replication of rVHSV-Arfp-Bgfp was significantly decreased compared to that of even rVHSV-Arfp or rVHSV-Brfp. These results suggest that the genome length is a critical factor for the determination of rVHSVs replication efficiency. In the in vivo virulence experiment, the cumulative mortalities of olive flounder fingerlings infected with each rVHSV were inversely proportional to the length of the viral genome, suggesting that decreased viral growth rate due to the lengthened viral genome is accompanied with the decrease of in vivo virulence of rVHSVs. Recombinant viruses expressing multiple foreign antigens can be used for the development of combined vaccines. However, as the present rVHSV-Arfp-Bgfp still possesses an ability to kill hosts (although very weakened), researches on the producing more attenuated viruses or propagation-deficient replicon particles are needed to solve safety-related problems. PMID:26921191

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

    PubMed

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

    2016-07-29

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

  19. Platelet Factor 4 Inhibits and Enhances HIV-1 Infection in a Concentration-Dependent Manner by Modulating Viral Attachment.

    PubMed

    Parker, Zahra F; Rux, Ann H; Riblett, Amber M; Lee, Fang-Hua; Rauova, Lubica; Cines, Douglas B; Poncz, Mortimer; Sachais, Bruce S; Doms, Robert W

    2016-07-01

    Platelet factor 4 (PF4) has been recently shown to inhibit infection by a broad range of human immunodeficiency virus type 1 (HIV-1) isolates in vitro. We found that the inhibitory effects of PF4 are limited to a defined concentration range where PF4 exists largely in a monomeric state. Under these conditions, PF4 bound the HIV-1 envelope protein and inhibited HIV-1 attachment to the cell surface. However, as concentrations increased to the point where PF4 exists largely in tetrameric or higher-order forms, viral infection in vitro was enhanced. Enhancement could be inhibited by mutations in PF4 that shift the oligomeric equilibrium toward the monomeric state, or by using soluble glycosaminoglycans (GAGs) to which tetrameric PF4 avidly binds. We conclude that at physiologically relevant concentrations, oligomeric PF4 enhances infection by HIV-1 by interacting with the viral envelope protein as well as cell surface GAGs, enhancing virus attachment to the cell surface. This effect was not specific to HIV-1, as enhancement was seen with some but not all other viruses tested. The biphasic effects of PF4 on HIV-1 infection suggest that native PF4 will not be a useful antiviral agent and that PF4 could contribute to the hematologic abnormalities commonly seen in HIV-infected individuals by enhancing virus infection in the bone marrow. PMID:26847431

  20. Heat shock protein-90-beta facilitates enterovirus 71 viral particles assembly

    SciTech Connect

    Wang, Robert Y.L.; Kuo, Rei-Lin; Ma, Wei-Chieh; Huang, Hsing-I; Yu, Jau-Song; Yen, Sih-Min; Huang, Chi-Ruei; Shih, Shin-Ru

    2013-09-01

    Molecular chaperones are reported to be crucial for virus propagation, but are not yet addressed in Human Enterovirus 71 (EV71). Here we describe the specific association of heat shock protein-90-beta (Hsp90β), but not alpha form (Hsp90α), with EV71 viral particles by the co-purification with virions using sucrose density gradient ultracentrifugation, and by the colocalization with viral particles, as assessed by immunogold electron microscopy. The reduction of the Hsp90β protein using RNA interference decreased the correct assembly of viral particles, without affecting EV71 replication levels. Tracking ectopically expressed Hsp90β protein associated with EV71 virions revealed that Hsp90β protein was transmitted to new host cells through its direct association with infectious viral particles. Our findings suggest a new antiviral strategy in which extracellular Hsp90β protein is targeted to decrease the infectivity of EV71 and other enteroviruses, without affecting the broader functions of this constitutively expressed molecular chaperone. - Highlights: • Hsp90β is associated with EV71 virion and is secreted with the release virus. • Hsp90β effects on the correct assembly of viral particles. • Viral titer of cultured medium was reduced in the presence of geldanamycin. • Viral titer was also reduced when Hsp90β was suppressed by siRNA treatment. • The extracellular Hsp90β was also observed in other RNA viruses-infected cells.

  1. Osmolyte-Mediated Encapsulation of Proteins inside MS2 Viral Capsids

    PubMed Central

    Glasgow, Jeff E.; Capehart, Stacy L.; Francis, Matthew B.; Tullman-Ercek, Danielle

    2012-01-01

    The encapsulation of enzymes in nanometer-sized compartments has the potential to enhance and control enzymatic activity, both in vivo and in vitro. Despite this potential, there are little quantitative data on the effect of encapsulation in a well-defined compartment under varying conditions. To gain more insight into these effects, we have characterized two improved methods for the encapsulation of heterologous molecules inside bacteriophage MS2 viral capsids. First, attaching DNA oligomers to a molecule of interest and incubating it with MS2 coat protein dimers yielded reassembled capsids that packaged the tagged molecules. The addition of a protein stabilizing osmolyte, trimethylamine-N-oxide (TMAO), significantly increased the yields of reassembly. Second, we found that expressed proteins with genetically encoded negatively charged peptide tags could also induce capsid reassembly, resulting in high yields of reassembled capsids containing the protein. This second method was used to encapsulate alkaline phosphatase tagged with a 16 amino acid peptide. The purified encapsulated enzyme was found to have the same Km value and a slightly lower kcat value than the free enzyme, indicating that this method of encapsulation had a minimal effect on enzyme kinetics. This method provides a practical and potentially scalable way of studying the complex effects of encapsulating enzymes in protein-based compartments. PMID:22953696

  2. Role of Accessory Proteins of HTLV-1 in Viral Replication, T Cell Activation, and Cellular Gene Expression

    PubMed Central

    Michael, Bindhu; Nair, Amithraj; Lairmore, Michael D.

    2010-01-01

    Human T-cell lymphotropic virus type 1 (HTLV-1), causes adult T cell leukemia/lymphoma (ATLL), and initiates a variety of immune mediated disorders. The viral genome encodes common structural and enzymatic proteins characteristic of all retroviruses and utilizes alternative splicing and alternate codon usage to make several regulatory and accessory proteins encoded in the pX region (pX ORF I to IV). Recent studies indicate that the accessory proteins p12I, p27I, p13II, and p30II, encoded by pX ORF I and II, contribute to viral replication and the ability of the virus to maintain typical in vivo expression levels. Proviral clones that are mutated in either pX ORF I or II, while fully competent in cell culture, are severely limited in their replicative capacity in a rabbit model. These HTLV-1 accessory proteins are critical for establishment of viral infectivity, enhance T- lymphocyte activation and potentially alter gene transcription and mitochondrial function. HTLV-1 pX ORF I expression is critical to the viral infectivity in resting primary lymphocytes suggesting a role for the calcineurin-binding protein p12I in lymphocyte activation. The endoplasmic reticulum and cis-Golgi localizing p12I activates NFAT, a key T cell transcription factor, through calcium-mediated signaling pathways and may lower the threshold of lymphocyte activation via the JAK/STAT pathway. In contrast p30II localizes to the nucleus and represses viral promoter activity, but may regulate cellular gene expression through p300/CBP or related co-activators of transcription. The mitochondrial localizing p13II induces morphologic changes in the organelle and may influence energy metabolism infected cells. Future studies of the molecular details HTLV-1 “accessory” proteins interactions will provide important new directions for investigations of HTLV-1 and related viruses associated with lymphoproliferative diseases. Thus, the accessory proteins of HTLV-1, once thought to be dispensable for

  3. Endolysosomal trafficking of viral G protein-coupled receptor functions in innate immunity and control of viral oncogenesis

    PubMed Central

    Dong, Xiaonan; Cheng, Adam; Zou, Zhongju; Yang, Yih-Sheng; Sumpter, Rhea M.; Huang, Chou-Long; Bhagat, Govind; Virgin, Herbert W.; Lira, Sergio A.; Levine, Beth

    2016-01-01

    The ubiquitin-proteasome system degrades viral oncoproteins and other microbial virulence factors; however, the role of endolysosomal degradation pathways in these processes is unclear. Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma, and a constitutively active viral G protein-coupled receptor (vGPCR) contributes to the pathogenesis of KSHV-induced tumors. We report that a recently discovered autophagy-related protein, Beclin 2, interacts with KSHV GPCR, facilitates its endolysosomal degradation, and inhibits vGPCR-driven oncogenic signaling. Furthermore, monoallelic loss of Becn2 in mice accelerates the progression of vGPCR-induced lesions that resemble human Kaposi’s sarcoma. Taken together, these findings indicate that Beclin 2 is a host antiviral molecule that protects against the pathogenic effects of KSHV GPCR by facilitating its endolysosomal degradation. More broadly, our data suggest a role for host endolysosomal trafficking pathways in regulating viral pathogenesis and oncogenic signaling. PMID:26929373

  4. Maturation of the viral core enhances the fusion of HIV-1 particles with primary human T cells and monocyte-derived macrophages

    SciTech Connect

    Jiang Jiyang; Aiken, Christopher . E-mail: chris.aiken@vanderbilt.edu

    2006-03-15

    HIV-1 infection requires fusion of viral and cellular membranes in a reaction catalyzed by the viral envelope proteins gp120 and gp41. We recently reported that efficient HIV-1 particle fusion with target cells is linked to maturation of the viral core by an activity of the gp41 cytoplasmic domain. Here, we show that maturation enhances the fusion of a variety of recombinant viruses bearing primary and laboratory-adapted Env proteins with primary human CD4{sup +} T cells. Overall, HIV-1 fusion was more dependent on maturation for viruses bearing X4-tropic envelope proteins than for R5-tropic viruses. Fusion of HIV-1 with monocyte-derived macrophages was also dependent on particle maturation. We conclude that the ability to couple fusion to particle maturation is a common feature of HIV-1 Env proteins and may play an important role during HIV-1 replication in vivo.

  5. Computational analysis reveal inhibitory action of nimbin against dengue viral envelope protein.

    PubMed

    Lavanya, P; Ramaiah, Sudha; Anbarasu, Anand

    2015-12-01

    Dengue has emerged to be global health problem worldwide. Hence there is an immediate need to adopt new strategies in the development of effective anti-dengue drugs. Extracts from the leaves of Azadirachta indica has been traditionally used in folk medicine for viral infections. In the present study we report the anti-viral potency of nimbin, the active compound from the neem leaf extract against the envelope protein of dengue virus. Progression of viral entry into the host cell is facilitated by the envelope protein of dengue virus, suggesting; it as an effective anti-viral target. Nimbin is found to be effective against the envelope protein of all four types of dengue virus (dengue 1-4), which is evident from our in silico analysis. Our findings suggest the clinical importance of nimbin, which can serve as effective lead compound for further analysis. PMID:26645034

  6. 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

  7. Formation of a stable complex between the human immunodeficiency virus integrase protein and viral DNA.

    PubMed Central

    Vink, C; Lutzke, R A; Plasterk, R H

    1994-01-01

    The integrase (IN) protein of the human immunodeficiency virus (HIV) mediates two distinct reactions: (i) specific removal of two nucleotides from the 3' ends of the viral DNA and (ii) integration of the viral DNA into target DNA. Although IN discriminates between specific (viral) DNA and nonspecific DNA in physical in vitro assays, a sequence-specific DNA-binding domain could not be identified in the protein. A nonspecific DNA-binding domain, however, was found at the C terminus of the protein. We examined the DNA-binding characteristics of HIV-1 IN, and found that a stable complex of IN and viral DNA is formed in the presence of Mn2+. The IN-viral DNA complex is resistant to challenge by an excess of competitor DNA. Stable binding of IN to the viral DNA requires that the protein contains an intact N-terminal domain and active site (in the central region of the protein), in addition to the C-terminal DNA-binding domain. Images PMID:7937134

  8. Modulation of cellular and viral promoters by mutant human p53 proteins found in tumor cells.

    PubMed Central

    Deb, S; Jackson, C T; Subler, M A; Martin, D W

    1992-01-01

    Wild-type p53 has recently been shown to repress transcription from several cellular and viral promoters. Since p53 mutations are the most frequently reported genetic defects in human cancers, it becomes important to study the effects of mutations of p53 on promoter functions. We, therefore, have studied the effects of wild-type and mutant human p53 on the human proliferating-cell nuclear antigen (PCNA) promoter and on several viral promoters, including the herpes simplex virus type 1 UL9 promoter, the human cytomegalovirus major immediate-early promoter-enhancer, and the long terminal repeat promoters of Rous sarcoma virus and human T-cell lymphotropic virus type I. HeLa cells were cotransfected with a wild-type or mutant p53 expression vector and a plasmid containing a chloramphenicol acetyltransferase reporter gene under viral (or cellular) promoter control. As expected, expression of the wild-type p53 inhibited promoter function. Expression of a p53 with a mutation at any one of the four amino acid positions 175, 248, 273, or 281, however, correlated with a significant increase of the PCNA promoter activity (2- to 11-fold). The viral promoters were also activated, although to a somewhat lesser extent. We also showed that activation by a mutant p53 requires a minimal promoter containing a lone TATA box. A more significant increase (25-fold) in activation occurs when the promoter contains a binding site for the activating transcription factor or cyclic AMP response element-binding protein. Using Saos-2 cells that do not express p53, we showed that activation by a mutant p53 was a direct enhancement. The mutant forms of p53 used in this study are found in various cancer cells. The activation of PCNA by mutant p53s may indicate a way to increase cell proliferation by the mutant p53s. Thus, our data indicate a possible functional role for the mutants of p53 found in cancer cells in activating several important loci, including PCNA. Images PMID:1356162

  9. Andes Virus Nucleocapsid Protein Interrupts Protein Kinase R Dimerization To Counteract Host Interference in Viral Protein Synthesis

    PubMed Central

    Wang, Zekun

    2014-01-01

    ABSTRACT Pathogenic hantaviruses delay the type I interferon response during early stages of viral infection. However, the robust interferon response and induction of interferon-stimulated genes observed during later stages of hantavirus infection fail to combat the virus replication in infected cells. Protein kinase R (PKR), a classical interferon-stimulated gene product, phosphorylates the eukaryotic translation initiation factor eIF2α and causes translational shutdown to create roadblocks for the synthesis of viral proteins. The PKR-induced translational shutdown helps host cells to establish an antiviral state to interrupt virus replication. However, hantavirus-infected cells do not undergo translational shutdown and fail to establish an antiviral state during the course of viral infection. In this study, we showed for the first time that Andes virus infection induced PKR overexpression. However, the overexpressed PKR was not active due to a significant inhibition of autophosphorylation. Further studies revealed that Andes virus nucleocapsid protein inhibited PKR dimerization, a critical step required for PKR autophosphorylation to attain activity. The studies reported here establish a hantavirus nucleocapsid protein as a new PKR inhibitor. These studies provide mechanistic insights into hantavirus resistance to the host interferon response and solve the puzzle of the lack of translational shutdown observed in hantavirus-infected cells. The sensitivity of hantavirus replication to PKR has likely imposed a selective evolutionary pressure on hantaviruses to evade the PKR antiviral response for survival. We envision that evasion of the PKR antiviral response by NP has likely helped hantaviruses to exist during evolution and to survive in infected hosts with a multifaceted antiviral defense. IMPORTANCE Protein kinase R (PKR), a versatile antiviral host factor, shuts down the translation machinery upon activation in virus-infected cells to create hurdles for the

  10. Illuminating structural proteins in viral “dark matter” with metaproteomics

    PubMed Central

    Brum, Jennifer R.; Ignacio-Espinoza, J. Cesar; Kim, Eun-Hae; Trubl, Gareth; Jones, Robert M.; Roux, Simon; VerBerkmoes, Nathan C.; Rich, Virginia I.; Sullivan, Matthew B.

    2016-01-01

    Viruses are ecologically important, yet environmental virology is limited by dominance of unannotated genomic sequences representing taxonomic and functional “viral dark matter.” Although recent analytical advances are rapidly improving taxonomic annotations, identifying functional dark matter remains problematic. Here, we apply paired metaproteomics and dsDNA-targeted metagenomics to identify 1,875 virion-associated proteins from the ocean. Over one-half of these proteins were newly functionally annotated and represent abundant and widespread viral metagenome-derived protein clusters (PCs). One primarily unannotated PC dominated the dataset, but structural modeling and genomic context identified this PC as a previously unidentified capsid protein from multiple uncultivated tailed virus families. Furthermore, four of the five most abundant PCs in the metaproteome represent capsid proteins containing the HK97-like protein fold previously found in many viruses that infect all three domains of life. The dominance of these proteins within our dataset, as well as their global distribution throughout the world’s oceans and seas, supports prior hypotheses that this HK97-like protein fold is the most abundant biological structure on Earth. Together, these culture-independent analyses improve virion-associated protein annotations, facilitate the investigation of proteins within natural viral communities, and offer a high-throughput means of illuminating functional viral dark matter. PMID:26884177

  11. MSLVP: prediction of multiple subcellular localization of viral proteins using a support vector machine.

    PubMed

    Thakur, Anamika; Rajput, Akanksha; Kumar, Manoj

    2016-07-19

    Knowledge of the subcellular location (SCL) of viral proteins in the host cell is important for understanding their function in depth. Therefore, we have developed "MSLVP", a two-tier prediction algorithm for predicting multiple SCLs of viral proteins. For this study, data sets of comprehensive viral proteins with experimentally validated SCL annotation were collected from UniProt. Non-redundant (90%) data sets of 3480 viral proteins that belonged to single (2715), double (391) and multiple (374) sites were employed. Additionally, 1687 (30% sequence identity) viral proteins were categorised into single (1366), double (167) and multiple (154) sites. Single, double and multiple locations further comprised of eight, four and six categories, respectively. Viral protein locations include the nucleus, cytoplasm, endoplasmic reticulum, extracellular, single-pass membrane, multi-pass membrane, capsid, remaining others and combinations thereof. Support vector machine based models were developed using sequence features like amino acid composition, dipeptide composition, physicochemical properties and their hybrids. We have employed "one-versus-one" as well as "one-versus-other" strategies for multiclass classification. The performance of "one-versus-one" is better than the "one-versus-other" approach during 10-fold cross-validation. For the 90% data set, we achieved an accuracy, a Matthew's correlation coefficient (MCC) and a receiver operating characteristic (ROC) of 99.99%, 1.00, 1.00; 100.00%, 1.00, 1.00 and 99.90%; 1.00, 1.00 for single, double and multiple locations, respectively. Similar results were achieved for a 30% sequence identity data set. Predictive models for each SCL performed equally well on the independent dataset. The MSLVP web server () can predict subcellular locations i.e. single (8; including single and multi-pass membrane), double (4) and multiple (6). This would be helpful for elucidating the functional annotation of viral proteins and potential drug

  12. Polyethylene glycol enhanced protein refolding.

    PubMed

    Cleland, J L; Builder, S E; Swartz, J R; Winkler, M; Chang, J Y; Wang, D I

    1992-09-01

    Previous studies on the refolding of recombinant bovine carbonic anhydrase B (CAB) indicated that polyethylene glycol (PEG) significantly enhanced the recovery of active protein by reducing aggregation. To further test the ability of PEG to enhance refolding, three recombinant human proteins, deoxyribonuclease (rhDNAse), tissue plasminogen activator (rhtPA), and interferon-gamma (rhIFN-gamma) were refolded in the presence of PEG (3350 MW). rhDNAse produced from CHO cells was denatured in 7.2 M urea and refolded by rapid dilution to 4.0 M urea and 0.20 mg/ml protein. When a final PEG to rhDNAse molar ratio of 5 to 1 (0.1 milligram PEG, 3350 MW) was used in the dilution buffer, refolding was improved by 30% to yield complete recovery of active protein. Impure E. coli derived inclusion body preparations of rhDNAse were solubilized in 8 M urea and refolded by dilution to 4 M urea and 0.10 mg/ml protein. Refolding with a dilution buffer which yielded a final PEG to rhDNAse molar ratio of 10 to 1 (0.1 milligram PEG, 3350 MW) resulted in a three-fold increase in the recovery of active protein. When PEG was used in the dilution buffer, aggregation of rhDNAse did not occur during refolding in either case. rhtPA produced from CHO cells was denatured in 5 M guanidine hydrochloride (GuHCl) and refolded by rapid dilution to 0.10 M GuHCl and 0.20 mg/ml protein.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1368998

  13. SDS-PAGE and IR spectroscopy to evaluate modifications in the viral protein profile induced by a cationic porphyrinic photosensitizer.

    PubMed

    Costa, Liliana; Esteves, Ana Cristina; Correia, António; Moreirinha, Catarina; Delgadillo, Ivonne; Cunha, Ângela; Neves, Maria G P S; Faustino, Maria A F; Almeida, Adelaide

    2014-12-01

    Reactive oxygen species can be responsible for microbial photodynamic inactivation due to its toxic effects, which include severe damage to proteins, lipids and nucleic acids. In this study, the photo-oxidative modifications of the proteins of a non-enveloped T4-like bacteriophage, induced by the cationic porphyrin 5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin tri-iodide were evaluated. Two methods were used: sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and infrared spectroscopy. SDS-PAGE analysis showed that the phage protein profile was considerably altered after photodynamic treatment. Seven protein bands putatively corresponding to capsid and tail tube proteins were attenuated and two other were enhanced. Infrared spectroscopy confirmed the time-dependent alteration on the phage protein profile detected by SDS-PAGE, indicative of a response to oxidative damage. Infrared analysis showed to be a promising and rapid screening approach for the analysis of the modifications induced on viral proteins by photosensitization. In fact, one single infrared spectrum can highlight the changes induced to all viral molecular structures, overcoming the delays and complex protocols of the conventional methods, in a much simple and cost effective way. PMID:25241141

  14. The Unfolded Protein Response Is Triggered by a Plant Viral Movement Protein1[W][OA

    PubMed Central

    Ye, Changming; Dickman, Martin B.; Whitham, Steven A.; Payton, Mark; Verchot, Jeanmarie

    2011-01-01

    Infection with Potato virus X (PVX) in Nicotiana benthamiana plants leads to increased transcript levels of several stress-related host genes, including basic-region leucine zipper 60 (bZIP60), SKP1, ER luminal binding protein (BiP), protein disulfide isomerase (PDI), calreticulin (CRT), and calmodulin (CAM). bZIP60 is a key transcription factor that responds to endoplasmic reticulum (ER) stress and induces the expression of ER-resident chaperones (BiP, PDI, CRT, and CAM). SKP1 is a component of SCF (for SKP1-Cullin-F box protein) ubiquitin ligase complexes that target proteins for proteasomal degradation. Expression of PVX TGBp3 from a heterologous vector induces the same set of genes in N. benthamiana and Arabidopsis (Arabidopsis thaliana) leaves. Virus-induced gene silencing was employed to knock down the expression of bZIP60 and SKP1, and the number of infection foci on inoculated leaves was reduced and systemic PVX accumulation was altered. Silencing bZIP60 led to the suppression of BiP and SKP1 transcript levels, suggesting that bZIP60 might be an upstream signal transducer. Overexpression of TGBp3 led to localized necrosis, but coexpression of TGBp3 with BiP abrogated necrosis, demonstrating that the unfolded protein response alleviates ER stress-related cell death. Steady-state levels of PVX replicase and TGBp2 (which reside in the ER) proteins were unaltered by the presence of TGBp3, suggesting that TGBp3 does not contribute to their turnover. Taken together, PVX TGBp3-induced ER stress leads to up-regulation of bZIP60 and unfolded protein response-related gene expression, which may be important to regulate cellular cytotoxicity that could otherwise lead to cell death if viral proteins reach high levels in the ER. PMID:21474436

  15. 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

  16. Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

    SciTech Connect

    Asenjo, Ana; Gonzalez-Armas, Juan C.; Villanueva, Nieves

    2008-10-10

    The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) {beta} and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects.

  17. 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. PMID:25575816

  18. Vaccinia virus telomeres: interaction with the viral I1, I6, and K4 proteins.

    PubMed

    DeMasi, J; Du, S; Lennon, D; Traktman, P

    2001-11-01

    The 192-kb linear DNA genome of vaccinia virus has covalently closed hairpin termini that are extremely AT rich and contain 12 extrahelical bases. Vaccinia virus telomeres have previously been implicated in the initiation of viral genome replication; therefore, we sought to determine whether the telomeres form specific protein-DNA complexes. Using an electrophoretic mobility shift assay, we found that extracts prepared from virions and from the cytoplasm of infected cells contain telomere binding activity. Four shifted complexes were detected using hairpin probes representing the viral termini, two of which represent an interaction with the "flip" isoform and two with the "flop" isoform. All of the specificity for protein binding lies within the terminal 65-bp hairpin sequence. Viral hairpins lacking extrahelical bases cannot form the shifted complexes, suggesting that DNA structure is crucial for complex formation. Using an affinity purification protocol, we purified the proteins responsible for hairpin-protein complex formation. The vaccinia virus I1 protein was identified as being necessary and sufficient for the formation of the upper doublet of shifted complexes, and the vaccinia virus I6 protein was shown to form the lower doublet of shifted complexes. Competition and challenge experiments confirmed that the previously uncharacterized I6 protein binds tightly and with great specificity to the hairpin form of the viral telomeric sequence. Incubation of viral hairpins with extracts from infected cells also generates a smaller DNA fragment that is likely to reflect specific nicking at the apex of the hairpin; we show that the vaccinia virus K4 protein is necessary and sufficient for this reaction. We hypothesize that these telomere binding proteins may play a role in the initiation of vaccinia virus genome replication and/or genome encapsidation. PMID:11581377

  19. The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state.

    PubMed

    Alarcon, Valentina; Hernández, Sergio; Rubio, Lorena; Alvarez, Francisca; Flores, Yvo; Varas-Godoy, Manuel; De Ferrari, Giancarlo V; Kann, Michael; Villanueva, Rodrigo A; Loyola, Alejandra

    2016-01-01

    With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients. PMID:27174370

  20. The enzymes LSD1 and Set1A cooperate with the viral protein HBx to establish an active hepatitis B viral chromatin state

    PubMed Central

    Alarcon, Valentina; Hernández, Sergio; Rubio, Lorena; Alvarez, Francisca; Flores, Yvo; Varas-Godoy, Manuel; De Ferrari, Giancarlo V.; Kann, Michael; Villanueva, Rodrigo A.; Loyola, Alejandra

    2016-01-01

    With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients. PMID:27174370

  1. Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization

    PubMed Central

    Chiu, Elaine; Hijnen, Marcel; Bunker, Richard D.; Boudes, Marion; Rajendran, Chitra; Aizel, Kaheina; Oliéric, Vincent; Schulze-Briese, Clemens; Mitsuhashi, Wataru; Young, Vivienne; Ward, Vernon K.; Bergoin, Max; Metcalf, Peter; Coulibaly, Fasséli

    2015-01-01

    The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides. PMID:25787255

  2. Enhancement of the efficiency of non-viral gene delivery by application of pulsed magnetic field

    PubMed Central

    Kamau, Sarah W.; Hassa, Paul O.; Steitz, Benedikt; Petri-Fink, Alke; Hofmann, Heinrich; Hofmann-Amtenbrink, Margarethe; von Rechenberg, Brigitte; Hottiger, Michael O.

    2006-01-01

    New approaches to increase the efficiency of non-viral gene delivery are still required. Here we report a simple approach that enhances gene delivery using permanent and pulsating magnetic fields. DNA plasmids and novel DNA fragments (PCR products) containing sequence encoding for green fluorescent protein were coupled to polyethylenimine coated superparamagnetic nanoparticles (SPIONs). The complexes were added to cells that were subsequently exposed to permanent and pulsating magnetic fields. Presence of these magnetic fields significantly increased the transfection efficiency 40 times more than in cells not exposed to the magnetic field. The transfection efficiency was highest when the nanoparticles were sedimented on the permanent magnet before the application of the pulsating field, both for small (50 nm) and large (200–250 nm) nanoparticles. The highly efficient gene transfer already within 5 min shows that this technique is a powerful tool for future in vivo studies, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs. PMID:16540591

  3. Robust RNAi enhancement via human Argonaute-2 overexpression from plasmids, viral vectors and cell lines

    PubMed Central

    Börner, Kathleen; Niopek, Dominik; Cotugno, Gabriella; Kaldenbach, Michaela; Pankert, Teresa; Willemsen, Joschka; Zhang, Xian; Schürmann, Nina; Mockenhaupt, Stefan; Serva, Andrius; Hiet, Marie-Sophie; Wiedtke, Ellen; Castoldi, Mirco; Starkuviene, Vytaute; Erfle, Holger; Gilbert, Daniel F.; Bartenschlager, Ralf; Boutros, Michael; Binder, Marco; Streetz, Konrad; Kräusslich, Hans-Georg; Grimm, Dirk

    2013-01-01

    As the only mammalian Argonaute protein capable of directly cleaving mRNAs in a small RNA-guided manner, Argonaute-2 (Ago2) is a keyplayer in RNA interference (RNAi) silencing via small interfering (si) or short hairpin (sh) RNAs. It is also a rate-limiting factor whose saturation by si/shRNAs limits RNAi efficiency and causes numerous adverse side effects. Here, we report a set of versatile tools and widely applicable strategies for transient or stable Ago2 co-expression, which overcome these concerns. Specifically, we engineered plasmids and viral vectors to co-encode a codon-optimized human Ago2 cDNA along with custom shRNAs. Furthermore, we stably integrated this Ago2 cDNA into a panel of standard human cell lines via plasmid transfection or lentiviral transduction. Using various endo- or exogenous targets, we demonstrate the potential of all three strategies to boost mRNA silencing efficiencies in cell culture by up to 10-fold, and to facilitate combinatorial knockdowns. Importantly, these robust improvements were reflected by augmented RNAi phenotypes and accompanied by reduced off-targeting effects. We moreover show that Ago2/shRNA-co-encoding vectors can enhance and prolong transgene silencing in livers of adult mice, while concurrently alleviating hepatotoxicity. Our customizable reagents and avenues should broadly improve future in vitro and in vivo RNAi experiments in mammalian systems. PMID:24049077

  4. Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization.

    PubMed

    Chiu, Elaine; Hijnen, Marcel; Bunker, Richard D; Boudes, Marion; Rajendran, Chitra; Aizel, Kaheina; Oliéric, Vincent; Schulze-Briese, Clemens; Mitsuhashi, Wataru; Young, Vivienne; Ward, Vernon K; Bergoin, Max; Metcalf, Peter; Coulibaly, Fasséli

    2015-03-31

    The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides. PMID:25787255

  5. Vaccination with a Fusion Protein That Introduces HIV-1 Gag Antigen into a Multitrimer CD40L Construct Results in Enhanced CD8+ T Cell Responses and Protection from Viral Challenge by Vaccinia-Gag

    PubMed Central

    Gupta, Sachin; Termini, James M.; Raffa, Francesca N.; Williams, Cindi-Ann; Kornbluth, Richard S.

    2014-01-01

    CD40 ligand (CD40L, CD154) is a membrane protein that is important for the activation of dendritic cells (DCs) and DC-induced CD8+ T cell responses. To be active, CD40L must cluster CD40 receptors on responding cells. To produce a soluble form of CD40L that clusters CD40 receptors necessitates the use of a multitrimer construct. With this in mind, a tripartite fusion protein was made from surfactant protein D (SPD), HIV-1 Gag as a test antigen, and CD40L, where SPD serves as a scaffold for the multitrimer protein complex. This SPD-Gag-CD40L protein activated CD40-bearing cells and bone marrow-derived DCs in vitro. Compared to a plasmid for Gag antigen alone (pGag), DNA vaccination of mice with pSPD-Gag-CD40L induced an increased number of Gag-specific CD8+ T cells with increased avidity for major histocompatibility complex class I-restricted Gag peptide and improved vaccine-induced protection from challenge by vaccinia-Gag virus. The importance of the multitrimeric nature of the complex was shown using a plasmid lacking the N terminus of SPD that produced a single trimer fusion protein. This plasmid, pTrimer-Gag-CD40L, was only weakly active on CD40-bearing cells and did not elicit strong CD8+ T cell responses or improve protection from vaccinia-Gag challenge. An adenovirus 5 (Ad5) vaccine incorporating SPD-Gag-CD40L was much stronger than Ad5 expressing Gag alone (Ad5-Gag) and induced complete protection (i.e., sterilizing immunity) from vaccinia-Gag challenge. Overall, these results show the potential of a new vaccine design in which antigen is introduced into a construct that expresses a multitrimer soluble form of CD40L, leading to strongly protective CD8+ T cell responses. PMID:24227853

  6. 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

  7. Circovirus Transport Proceeds via Direct Interaction of the Cytoplasmic Dynein IC1 Subunit with the Viral Capsid Protein

    PubMed Central

    Cao, Jingjing; Lin, Cui; Wang, Huijuan; Wang, Lun; Zhou, Niu; Jin, Yulan; Liao, Min

    2014-01-01

    ABSTRACT Microtubule transport of circovirus from the periphery of the cell to the nucleus is essential for viral replication in early infection. How the microtubule is recruited to the viral cargo remains unclear. In this study, we observed that circovirus trafficking is dependent on microtubule polymerization and that incoming circovirus particles colocalize with cytoplasmic dynein and endosomes. However, circovirus binding to dynein was independent of the presence of microtubular α-tubulin and translocation of cytoplasmic dynein into the nucleus. The circovirus capsid (Cap) subunit enhanced microtubular acetylation and directly interacted with intermediate chain 1 (IC1) of dynein. N-terminal residues 42 to 100 of the Cap viral protein were required for efficient binding to the dynein IC1 subunit and for retrograde transport. Knockdown of IC1 decreased virus transport and replication. These results demonstrate that Cap is a direct ligand of the cytoplasmic dynein IC1 subunit and an inducer of microtubule α-tubulin acetylation. Furthermore, Cap recruits the host dynein/microtubule machinery to facilitate transport toward the nucleus by an endosomal mechanism distinct from that used for physiological dynein cargo. IMPORTANCE Incoming viral particles hijack the intracellular trafficking machinery of the host in order to migrate from the cell surface to the replication sites. Better knowledge of the interaction between viruses and virus proteins and the intracellular trafficking machinery may provide new targets for antiviral therapies. Currently, little is known about the molecular mechanisms of circovirus transport. Here, we report that circovirus particles enter early endosomes and utilize the microtubule-associated molecular motor dynein to travel along microtubules. The circovirus capsid subunit enhances microtubular acetylation, and N-terminal residues 42 to 100 directly interact with the dynein IC1 subunit during retrograde transport. These findings

  8. Viral protein R of human immunodeficiency virus type-1 induces retrotransposition of long interspersed element-1

    PubMed Central

    2013-01-01

    Background Viral protein R (Vpr), a protein of human immunodeficiency virus type-1 (HIV-1) with various biological functions, was shown to be present in the blood of HIV-1-positive patients. However, it remained unclear whether circulating Vpr in patients’ blood is biologically active. Here, we examined the activity of blood Vpr using an assay system by which retrotransposition of long interspersed element-1 (L1-RTP) was detected. We also investigated the in vivo effects of recombinant Vpr (rVpr) by administrating it to transgenic mice harboring human L1 as a transgene (hL1-Tg mice). Based on our data, we discuss the involvement of blood Vpr in the clinical symptoms of acquired immunodeficiency syndrome (AIDS). Results We first discovered that rVpr was active in induction of L1-RTP. Biochemical analyses revealed that rVpr-induced L1-RTP depended on the aryl hydrocarbon receptor, mitogen-activated protein kinases, and CCAAT/enhancer-binding protein β. By using a sensitive L1-RTP assay system, we showed that 6 of the 15 blood samples from HIV-1 patients examined were positive for induction of L1-RTP. Of note, the L1-RTP-inducing activity was blocked by a monoclonal antibody specific for Vpr. Moreover, L1-RTP was reproducibly induced in various organs, including the kidney, when rVpr was administered to hL1-Tg mice. Conclusions Blood Vpr is biologically active, suggesting that its monitoring is worthwhile for clarification of the roles of Vpr in the pathogenesis of AIDS. This is the first report to demonstrate a soluble factor in patients’ blood active for L1-RTP activity, and implies the involvement of L1-RTP in the development of human diseases. PMID:23915234

  9. A Herpesviral Lytic Protein Regulates the Structure of Latent Viral Chromatin

    PubMed Central

    Raja, Priya; Lee, Jennifer S.; Pan, Dongli; Pesola, Jean M.; Coen, Donald M.

    2016-01-01

    ABSTRACT Latent infections by viruses usually involve minimizing viral protein expression so that the host immune system cannot recognize the infected cell through the viral peptides presented on its cell surface. Herpes simplex virus (HSV), for example, is thought to express noncoding RNAs such as latency-associated transcripts (LATs) and microRNAs (miRNAs) as the only abundant viral gene products during latent infection. Here we describe analysis of HSV-1 mutant viruses, providing strong genetic evidence that HSV-infected cell protein 0 (ICP0) is expressed during establishment and/or maintenance of latent infection in murine sensory neurons in vivo. Studies of an ICP0 nonsense mutant virus showed that ICP0 promotes heterochromatin and latent and lytic transcription, arguing that ICP0 is expressed and functional. We propose that ICP0 promotes transcription of LATs during establishment or maintenance of HSV latent infection, much as it promotes lytic gene transcription. This report introduces the new concept that a lytic viral protein can be expressed during latent infection and can serve dual roles to regulate viral chromatin to optimize latent infection in addition to its role in epigenetic regulation during lytic infection. An additional implication of the results is that ICP0 might serve as a target for an antiviral therapeutic acting on lytic and latent infections. PMID:27190217

  10. Viral Evolved Inhibition Mechanism of the RNA Dependent Protein Kinase PKR's Kinase Domain, a Structural Perspective

    PubMed Central

    Krishna, K. Hari; Vadlamudi, Yallamandayya; Kumar, Muthuvel Suresh

    2016-01-01

    The protein kinase PKR activated by viral dsRNA, phosphorylates the eIF2α, which inhibit the mechanism of translation initiation. Viral evolved proteins mimicking the eIF2α block its phosphorylation and help in the viral replication. To decipher the molecular basis for the PKR’s substrate and inhibitor interaction mechanisms, we carried the molecular dynamics studies on the catalytic domain of PKR in complex with substrate eIF2α, and inhibitors TAT and K3L. The studies conducted show the altered domain movements of N lobe, which confers open and close state to the substrate-binding cavity. In addition, PKR exhibits variations in the secondary structural transition of the activation loop residues, and inter molecular contacts with the substrate and the inhibitors. Phosphorylation of the P+1 loop at the Thr-451 increases the affinity of the binding proteins exhibiting its role in the phosphorylation events. The implications of structural mechanisms uncovered will help to understand the basis of the evolution of the host-viral and the viral replication mechanisms. PMID:27088597

  11. Heterogeneous nuclear ribonuclear protein K interacts with Sindbis virus nonstructural proteins and viral subgenomic mRNA

    SciTech Connect

    Burnham, Andrew J.; Gong, Lei; Hardy, Richard W.

    2007-10-10

    Alphaviruses are a group of arthropod-borne human and animal pathogens that can cause epidemics of significant public health and economic consequence. Alphavirus RNA synthesis requires four virally encoded nonstructural proteins and probably a number of cellular proteins. Using comparative two-dimensional electrophoresis we were able to identify proteins enriched in cytoplasmic membrane fractions containing viral RNA synthetic complexes following infection with Sindbis virus. Our studies demonstrated the following: (i) the host protein hnRNP K is enriched in cytoplasmic membrane fractions following Sindbis virus infection, (ii) viral nonstructural proteins co-immunoprecipitate with hnRNP K, (iii) nsP2 and hnRNP K co-localize in the cytoplasm of Sindbis virus infected cells, (iv) Sindbis virus subgenomic mRNA, but not genomic RNA co-immunoprecipitates with hnRNP K, (v) viral RNA does not appear to be required for the interaction of hnRNP K with the nonstructural proteins. Potential functions of hnRNP K during virus replication are discussed.

  12. Sigma 1 protein of mammalian reoviruses extends from the surfaces of viral particles

    SciTech Connect

    Furlong, D.B.; Nibert, M.L.; Fields, B.N.

    1988-01-01

    Electron microscopy revealed structures consisting of long fibers topped with knobs extending from the surfaces of virions of mammalian reoviruses. The morphology of these structures was reminiscent of the fiber protein of adenovirus. Fibers were also seen extending from the reovirus top component and intermediate subviral particles but not from cores, suggesting that the fibers consist of either the ..mu..1C or sigma1 outer capsid protein. Amino acid sequence analysis predicts that the reovirus cell attachment protein sigma1 contains an extended fiber domain. When sigma1 protein was released from viral particles with mild heat and subsequently obtained in isolation, it was found to have a morphology identical to that of the fiber structures seen extending from the viral particles. The identification of an extended form of sigma1 has important implications for its function in cell attachment. Other evidence suggest that sigma1 protein may occur in virions in both an extended and an unextended state.

  13. Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

    SciTech Connect

    Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian . E-mail: bzheng@hkucc.hku.hk

    2007-07-20

    The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection.

  14. LL37 and Cationic Peptides Enhance TLR3 Signaling by Viral Double-stranded RNAs

    PubMed Central

    Lai, Yvonne; Adhikarakunnathu, Sreedevi; Bhardwaj, Kanchan; Ranjith-Kumar, C. T.; Wen, Yahong; Jordan, Jarrat L.; Wu, Linda H.; Dragnea, Bogdan; Mateo, Lani San; Kao, C. Cheng

    2011-01-01

    Background Toll-like Receptor 3 (TLR3) detects viral dsRNA during viral infection. However, most natural viral dsRNAs are poor activators of TLR3 in cell-based systems, leading us to hypothesize that TLR3 needs additional factors to be activated by viral dsRNAs. The anti-microbial peptide LL37 is the only known human member of the cathelicidin family of anti-microbial peptides. LL37 complexes with bacterial lipopolysaccharide (LPS) to prevent activation of TLR4, binds to ssDNA to modulate TLR9 and ssRNA to modulate TLR7 and 8. It synergizes with TLR2/1, TLR3 and TLR5 agonists to increase IL8 and IL6 production. This work seeks to determine whether LL37 enhances viral dsRNA recognition by TLR3. Methodology/Principal Findings Using a human bronchial epithelial cell line (BEAS2B) and human embryonic kidney cells (HEK 293T) transiently transfected with TLR3, we found that LL37 enhanced poly(I:C)-induced TLR3 signaling and enabled the recognition of viral dsRNAs by TLR3. The presence of LL37 also increased the cytokine response to rhinovirus infection in BEAS2B cells and in activated human peripheral blood mononuclear cells. Confocal microscopy determined that LL37 could co-localize with TLR3. Electron microscopy showed that LL37 and poly(I:C) individually formed globular structures, but a complex of the two formed filamentous structures. To separate the effects of LL37 on TLR3 and TLR4, other peptides that bind RNA and transport the complex into cells were tested and found to activate TLR3 signaling in response to dsRNAs, but had no effect on TLR4 signaling. This is the first demonstration that LL37 and other RNA-binding peptides with cell penetrating motifs can activate TLR3 signaling and facilitate the recognition of viral ligands. Conclusions/Significance LL37 and several cell-penetrating peptides can enhance signaling by TLR3 and enable TLR3 to respond to viral dsRNA. PMID:22039520

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

    PubMed Central

    Ou, Horng D.; May, Andrew P.

    2010-01-01

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

  16. 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

  17. Effect of metal catalyzed oxidation in recombinant viral protein assemblies

    PubMed Central

    2014-01-01

    Background Protein assemblies, such as virus-like particles, have increasing importance as vaccines, delivery vehicles and nanomaterials. However, their use requires stable assemblies. An important cause of loss of stability in proteins is oxidation, which can occur during their production, purification and storage. Despite its importance, very few studies have investigated the effect of oxidation in protein assemblies and their structural units. In this work, we investigated the role of in vitro oxidation in the assembly and stability of rotavirus VP6, a polymorphic protein. Results The susceptibility to oxidation of VP6 assembled into nanotubes (VP6NT) and unassembled VP6 (VP6U) was determined and compared to bovine serum albumin (BSA) as control. VP6 was more resistant to oxidation than BSA, as determined by measuring protein degradation and carbonyl content. It was found that assembly protected VP6 from in vitro metal-catalyzed oxidation. Oxidation provoked protein aggregation and VP6NT fragmentation, as evidenced by dynamic light scattering and transmission electron microscopy. Oxidative damage of VP6 correlated with a decrease of its center of fluorescence spectral mass. The in vitro assembly efficiency of VP6U into VP6NT decreased as the oxidant concentration increased. Conclusions Oxidation caused carbonylation, quenching, and destruction of aromatic amino acids and aggregation of VP6 in its assembled and unassembled forms. Such modifications affected protein functionality, including its ability to assemble. That assembly protected VP6 from oxidation shows that exposure of susceptible amino acids to the solvent increases their damage, and therefore the protein surface area that is exposed to the solvent is determinant of its susceptibility to oxidation. The inability of oxidized VP6 to assemble into nanotubes highlights the importance of avoiding this modification during the production of proteins that self-assemble. This is the first time that the role of

  18. Bovine viral diarrhea virus structural protein E2 as a complement regulatory protein.

    PubMed

    Ostachuk, Agustín

    2016-07-01

    Bovine viral diarrhea virus (BVDV) is a member of the genus Pestivirus, family Flaviviridae, and is one of the most widely distributed viruses in cattle worldwide. Approximately 60 % of cattle in endemic areas without control measures are infected with BVDV during their lifetime. This wide prevalence of BVDV in cattle populations results in significant economic losses. BVDV is capable of establishing persistent infections in its host due to its ability to infect fetuses, causing immune tolerance. However, this cannot explain how the virus evades the innate immune system. The objective of the present work was to test the potential activity of E2 as a complement regulatory protein. E2 glycoprotein, produced both in soluble and transmembrane forms in stable CHO-K1 cell lines, was able to reduce complement-mediated cell lysis up to 40 % and complement-mediated DNA fragmentation by 50 %, in comparison with cell lines not expressing the glycoprotein. This work provides the first evidence of E2 as a complement regulatory protein and, thus, the finding of a mechanism of immune evasion by BVDV. Furthermore, it is postulated that E2 acts as a self-associated molecular pattern (SAMP), enabling the virus to avoid being targeted by the immune system and to be recognized as self. PMID:27038454

  19. Towards protein-based viral mimetics for cancer therapies.

    PubMed

    Unzueta, Ugutz; Céspedes, María Virtudes; Vázquez, Esther; Ferrer-Miralles, Neus; Mangues, Ramón; Villaverde, Antonio

    2015-05-01

    High resistance and recurrence rates, together with elevated drug clearance, compel the use of maximum-tolerated drug doses in cancer therapy, resulting in high-grade toxicities and limited clinical applicability. Promoting active drug accumulation in tumor tissues would minimize such issues and improve therapeutic outcomes. A new class of therapeutic drugs suitable for the task has emerged based on the concept of virus-mimetic nanocarriers, or 'artificial viruses'. Among the spectrum of materials under exploration in nanocarrier research, proteins offer unparalleled structural and functional versatility for designing virus-like molecular vehicles. By exhibiting 'smart' functions and biomimetic traits, protein-based nanocarriers will be a step ahead of the conventional drug-protein conjugates already in the clinic in ensuring efficient delivery of passenger antitumor drugs. PMID:25805413

  20. Singapore Grouper Iridovirus ORF75R is a Scaffold Protein Essential for Viral Assembly

    PubMed Central

    Wang, Fan; Liu, Yang; Zhu, Yi; Ngoc Tran, Bich; Wu, Jinlu; Leong Hew, Choy

    2015-01-01

    Singapore Grouper Iridovirus (SGIV) is a member of nucleo cytoplasmic large DNA viruses (NCLDV). This paper reports the functional analysis of ORF75R, a major structural protein of SGIV. Immuno fluorescence studies showed that the protein was accumulated in the viral assembly site. Immunogold-labelling indicated that it was localized between the viral capsid shell and DNA core. Knockdown of ORF75R by morpholinos resulted in the reduction of coreshell thickness, the failure of DNA encapsidation, and the low yield of infectious particles. Comparative proteomics further identified the structural proteins affected by ORF75R knockdown. Two-dimensional gel electrophoresis combined with proteomics demonstrated that ORF75R was phosphorylated at multiple sites in SGIV-infected cell lysate and virions, but the vast majority of ORF75R in virions was the dephosphorylated isoform. A kinase assay showed that ORF75R could be phosphorylated in vitro by the SGIV structural protein ORF39L. Addition of ATP and Mg2+ into purified virions prompted extensive phosphorylation of structural proteins and release of ORF75R from virions. These data suggest that ORF75R is a novel scaffold protein important for viral assembly and DNA encapsidation, but its phosphorylation facilitates virion disassembly. Compared to proteins from other viruses, we found that ORF75R shares common features with herpes simplex virus VP22. PMID:26286371

  1. Structural basis for viral 5′-PPP-RNA recognition by human IFIT proteins

    PubMed Central

    Abbas, Yazan M.; Pichlmair, Andreas; Górna, Maria W.; Superti-Furga, Giulio; Nagar, Bhushan

    2016-01-01

    IFIT proteins are interferon-inducible, innate immune effector molecules that are thought to confer antiviral defence through disruption of protein-protein interactions in the host translation initiation machinery. However, recently it was discovered that IFITs could directly recognize viral RNA bearing a 5′-triphosphate group (PPP-RNA), which is a molecular signature that distinguishes it from host RNA. Here, we report crystal structures of human IFIT5, its complex with PPP-RNAs, and an N-terminal fragment of IFIT1. The structures reveal a new helical domain that houses a positively charged cavity designed to specifically engage only single stranded PPP-RNA, thus distinguishing it from the canonical cytosolic sensor of double stranded viral PPP-RNA, RIG-I. Mutational analysis, proteolysis and gel-shift assays reveal that PPP-RNA is bound in a non-sequence specific manner and requires approximately a 3-nucleotide 5′-overhang. Abrogation of PPP-RNA binding in IFIT1 and IFIT5 were found to cause a defect in the anti-viral response by HEK cells. These results demonstrate the mechanism by which IFIT proteins selectively recognize viral RNA and lend insight into their downstream effector function. PMID:23334420

  2. Viral AlkB proteins repair RNA damage by oxidative demethylation

    PubMed Central

    van den Born, Erwin; Omelchenko, Marina V.; Bekkelund, Anders; Leihne, Vibeke; Koonin, Eugene V.; Dolja, Valerian V.; Falnes, Pål Ø.

    2008-01-01

    Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant. PMID:18718927

  3. Viral protein requirements for assembly and release of human parainfluenza virus type 3 virus-like particles.

    PubMed

    Bracken, Megan K; Hayes, Brandon C; Kandel, Suresh R; Scott-Shemon, Deja; Ackerson, Larissa; Hoffman, Michael A

    2016-06-01

    To understand the roles of human parainfluenza virus 3 (HPIV3) proteins in assembly and release, viral proteins were expressed individually and in combination in 293T cells. Expression of the matrix (M) protein triggered release of enveloped, matrix-containing virus-like particles (VLPs) from cells. When M was co-expressed with the nucleocapsid (N), fusion (F) or haemagglutinin-neuraminidase (HN) proteins, VLPs that contained M+N, M+F and M+HN, respectively, were generated, suggesting that M can independently interact with each protein to facilitate assembly and release. Additionally, expression of N protein enabled incorporation of the phosphoprotein (P) into VLPs, likely due to known N-P interactions. Finally, the HPIV3 C protein did not enhance VLP release, in contrast to observations with the related Sendai virus. These findings reinforce the central importance of the M protein in virus assembly and release, but also illustrate the variable roles of other paramyxovirus proteins during these processes. PMID:26960133

  4. 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

  5. Viral-Host Protein Interaction Studies Using Yeast Two-Hybrid Screening Method.

    PubMed

    Dudha, Namrata; Gupta, Sanjay

    2016-01-01

    Yeast two-hybrid (Y2H) assay is one of the earliest methods developed to study protein-protein interactions. In the proteomics era, Y2H has created a niche of its own by providing protein interaction maps for various organisms. Owing to limited coding capacities of their genomes, viruses are dependent on their host cellular machinery for successful infection. Identification of the key players orchestrating the survival of virus in their host is essential for understanding viral life cycle and devising strategies to prevent interactions resulting in pathogenesis. In this chapter, Y2H assay will be explained in detail for studying viral-host protein interactions of Chikungunya virus (CHIKV). PMID:27233270

  6. The virally encoded killer proteins from Ustilago maydis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several strains of Ustilago maydis, a causal agent of corn smut disease, exhibit a 'killer' phenotype that is due to persistent infection by double-stranded RNA Totiviruses. These viruses produce potent killer proteins that are secreted by the host. This is a rare example of virus/host symbiosis in ...

  7. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins

    PubMed Central

    Hedil, Marcio; Kormelink, Richard

    2016-01-01

    The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far. PMID:27455310

  8. Viral RNA Silencing Suppression: The Enigma of Bunyavirus NSs Proteins.

    PubMed

    Hedil, Marcio; Kormelink, Richard

    2016-01-01

    The Bunyaviridae is a family of arboviruses including both plant- and vertebrate-infecting representatives. The Tospovirus genus accommodates plant-infecting bunyaviruses, which not only replicate in their plant host, but also in their insect thrips vector during persistent propagative transmission. For this reason, they are generally assumed to encounter antiviral RNA silencing in plants and insects. Here we present an overview on how tospovirus nonstructural NSs protein counteracts antiviral RNA silencing in plants and what is known so far in insects. Like tospoviruses, members of the related vertebrate-infecting bunyaviruses classified in the genera Orthobunyavirus, Hantavirus and Phlebovirus also code for a NSs protein. However, for none of them RNA silencing suppressor activity has been unambiguously demonstrated in neither vertebrate host nor arthropod vector. The second part of this review will briefly describe the role of these NSs proteins in modulation of innate immune responses in mammals and elaborate on a hypothetical scenario to explain if and how NSs proteins from vertebrate-infecting bunyaviruses affect RNA silencing. If so, why this discovery has been hampered so far. PMID:27455310

  9. Enhanced Antiretroviral Therapy in Rhesus Macaques Improves RT-SHIV Viral Decay Kinetics

    PubMed Central

    North, Thomas W.; Villalobos, Andradi; Hurwitz, Selwyn J.; Deere, Jesse D.; Higgins, Joanne; Chatterjee, Payel; Tao, Sijia; Kauffman, Robert C.; Luciw, Paul A.; Kohler, James J.

    2014-01-01

    Using an established nonhuman primate model, rhesus macaques were infected intravenously with a chimeric simian immunodeficiency virus (SIV) consisting of SIVmac239 with the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase from clone HXBc2 (RT-SHIV). The impacts of two enhanced (four- and five-drug) highly active antiretroviral therapies (HAART) on early viral decay and rebound were determined. The four-drug combination consisted of an integrase inhibitor, L-870-812 (L-812), together with a three-drug regimen comprising emtricitabine [(−)-FTC], tenofovir (TFV), and efavirenz (EFV). The five-drug combination consisted of one analog for each of the four DNA precursors {using TFV, (−)-FTC, (−)-β-d-(2R,4R)-1,3-dioxolane-2,6-diaminopurine (amdoxovir [DAPD]), and zidovudine (AZT)}, together with EFV. A cohort treated with a three-drug combination of (−)-FTC, TFV, and EFV served as treated controls. Daily administration of a three-, four-, or five-drug combination of antiretroviral agents was initiated at week 6 or 8 after inoculation and continued up to week 50, followed by a rebound period. Plasma samples were collected routinely, and drug levels were monitored using liquid chromatography-tandem mass spectrometry (LC–MS-MS). Viral loads were monitored with a standard TaqMan quantitative reverse transcriptase PCR (qRT-PCR) assay. Comprehensive analyses of replication dynamics were performed. RT-SHIV infection in rhesus macaques produced typical viral infection kinetics, with untreated controls establishing persistent viral loads of >104 copies of RNA/ml. RT-SHIV loads at the start of treatment (V0) were similar in all treated cohorts (P > 0.5). All antiretroviral drug levels were measureable in plasma. The four-drug and five-drug combination regimens (enhanced HAART) improved suppression of the viral load (within 1 week; P < 0.01) and had overall greater potency (P < 0.02) than the three-drug regimen (HAART). Moreover, rebound viremia occurred

  10. Genetic disruption of KSHV major latent nuclear antigen LANA enhances viral lytic transcriptional program

    SciTech Connect

    Li Qiuhua; Zhou Fuchun; Ye Fengchun; Gao Shoujiang

    2008-09-30

    Following primary infection, KSHV establishes a lifelong persistent latent infection in the host. The mechanism of KSHV latency is not fully understood. The latent nuclear antigen (LANA or LNA) encoded by ORF73 is one of a few viral genes expressed during KSHV latency, and is consistently detected in all KSHV-related malignancies. LANA is essential for KSHV episome persistence, and regulates the expression of viral lytic genes through epigenetic silencing, and inhibition of the expression and transactivation function of the key KSHV lytic replication initiator RTA (ORF50). In this study, we used a genetic approach to examine the role of LANA in regulating KSHV lytic replication program. Deletion of LANA did not affect the expression of its adjacent genes vCyclin (ORF72) and vFLIP (ORF71). In contrast, the expression levels of viral lytic genes including immediate-early gene RTA, early genes MTA (ORF57), vIL-6 (ORF-K2) and ORF59, and late gene ORF-K8.1 were increased before and after viral lytic induction with 12-O-tetradecanoyl-phorbol-13-acetate and sodium butyrate. This enhanced expression of viral lytic genes was also observed following overexpression of RTA with or without simultaneous chemical induction. Consistent with these results, the LANA mutant cells produced more infectious virions than the wild-type virus cells did. Furthermore, genetic repair of the mutant virus reverted the phenotypes to those of wild-type virus. Together, these results have demonstrated that, in the context of viral genome, LANA contributes to KSHV latency by regulating the expression of RTA and its downstream genes.

  11. Cloning of a cDNA encoding a plasma membrane-associated, uronide binding phosphoprotein with physical properties similar to viral movement proteins.

    PubMed Central

    Reymond, P; Kunz, B; Paul-Pletzer, K; Grimm, R; Eckerskorn, C; Farmer, E E

    1996-01-01

    Oligogalacturonides are structural and regulatory homopolymers from the extracellular pectic matrix of plants. In vitro micromolar concentrations of oligogalacturonates and polygalacturonates were shown previously to stimulate the phosphorylation of a small plasma membrane-associated protein in potato. Immunologically cross-reactive proteins were detected in plasma membrane-enriched fractions from all angiosperm subclasses in the Cronquist system. Polygalacturonate-enhanced phosphorylation of the protein was observed in four of the six dicotyledon subclasses but not in any of the five monocotyledon subclasses. A cDNA for the protein was cloned from potato. The deduced protein is extremely hydrophilic and has a proline-rich N terminus. The C-terminal half of the protein was predicted to be a coiled coil, suggesting that the protein interacts with other macromolecules. The recombinant protein was found to bind both simple and complex galacturonides. The behavior of the protein suggests several parallels with viral proteins involved in intercellular communication. PMID:8989883

  12. DNA-templated assembly of viral protein hydrogel

    NASA Astrophysics Data System (ADS)

    Xu, Xin; Tao, Ailin; Xu, Yun

    2014-11-01

    Hydrogels are a promising class of biomaterials that can be easily tailored to produce a native extracellular matrix that exhibits desirable mechanical and chemical properties. Here we report the construction of a hydrogel via the assembly of cucumber mosaic virus (CMV) capsid protein and Y-shaped and cross-shaped DNAs.Hydrogels are a promising class of biomaterials that can be easily tailored to produce a native extracellular matrix that exhibits desirable mechanical and chemical properties. Here we report the construction of a hydrogel via the assembly of cucumber mosaic virus (CMV) capsid protein and Y-shaped and cross-shaped DNAs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr02414a

  13. MicroRNA-binding viral protein interferes with Arabidopsis development.

    PubMed

    Chellappan, Padmanabhan; Vanitharani, Ramachandran; Fauquet, Claude M

    2005-07-19

    MicroRNAs (miRNAs) are small (approximately 21 nt), noncoding RNAs that negatively regulate target mRNAs at the posttranscriptional level that are involved in development. In plants, virus-induced disease symptoms often result in developmental abnormalities resembling perturbation of miRNA-mediated function. Here, we report that expression in transgenic plants of a geminivirus-encoded AC4 protein from African cassava mosaic virus Cameroon Strain (ACMV), a suppressor of posttranscriptional gene silencing, was correlated with decreased accumulation of host miRNAs and increased development abnormalities in Arabidopsis. Down-regulation of miRNA correlated with an up-regulation of target mRNA level. In vitro binding assays revealed the ability of AC4 of ACMV (A-AC4) but not East African cassava mosaic Cameroon virus AC2 to bind single-stranded forms of miRNAs and short interfering RNAs but not double-stranded RNA forms. Normally, a labile intermediate during the miRNA biogenesis/RNA-induced silencing complex assembly, miRNA*, was below the level of detection, indicating that AC4 might interfere at a point downstream of the miRNA duplex unwinding process. The association of AC4 with miRNA was demonstrated by the association of A-AC4-GFP fusion protein, extracted from Arabidopsis protoplasts, with 2'-O-methyloligonucleotide complementary to miR159 (miR159*) and by the presence of miRNA with the A-AC4-GFP fusion protein after immunoprecipitation with antibody against GFP. In both assays, A-AC4 protein and miRNA complexes were copurified. These results provide direct evidence that AC4 is a unique virus-encoded posttranscriptional gene-silencing suppressor protein that binds to and presumably inactivates mature miRNAs and thus blocks the normal miRNA-mediated regulation of target mRNAs, resulting in developmental defects in Arabidopsis. PMID:16006510

  14. Assembly of vaccinia virus: effects of rifampin on the intracellular distribution of viral protein p65.

    PubMed Central

    Sodeik, B; Griffiths, G; Ericsson, M; Moss, B; Doms, R W

    1994-01-01

    The cytoplasmic assembly of vaccinia virus is reversibly blocked by the antibiotic rifampin, leading to the accumulation of partially membrane-delineated rifampin bodies in infected cells. Rifampin-resistant vaccinia virus mutants have point mutations in the D13L gene, which is controlled by a late promoter and expresses a 65-kDa protein, designated p65. To further characterize the mechanism of rifampin inhibition and the function of p65 in virus assembly, we raised antibodies to this protein. Immunoreactive p65 was expressed at late times of infection, and neither its expression nor its turnover was affected by rifampin. Virus-associated p65 could be extracted only with denaturing detergents from purified virions, suggesting that it is an integral viral component. Immunofluorescence studies showed that p65 is localized to the sites of virus assembly. Also, immunoelectron microscopy showed p65 to be associated with viral crescents as well as spherical, immature virions, in both cases predominantly on the inner or concave surface. In the presence of rifampin, p65 was found in large, cytoplasmic inclusion bodies that were distinct from rifampin bodies. The rifampin bodies themselves were labeled with p65 antibodies only after reversal of the rifampin block, predominantly on the viral crescents which rapidly formed following removal of the drug. We propose that p65 functions as an internal scaffold in the formation of viral crescents and immature virions, analogously to the matrix proteins of other viruses. Images PMID:8289340

  15. Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

    PubMed Central

    Wu, Nicholas C.; Olson, C. Anders; Du, Yushen; Le, Shuai; Tran, Kevin; Remenyi, Roland; Gong, Danyang; Al-Mawsawi, Laith Q.; Qi, Hangfei; Wu, Ting-Ting; Sun, Ren

    2015-01-01

    Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available. PMID:26132554

  16. Herpes simplex virus glycoprotein C: molecular mimicry of complement regulatory proteins by a viral protein.

    PubMed

    Huemer, H P; Wang, Y; Garred, P; Koistinen, V; Oppermann, S

    1993-08-01

    Herpes simplex virus (HSV) encodes a protein, glycoprotein C (gC), which binds to the third complement component, the central mediator of complement activation. In this study the structural and functional relationships of gC from HSV type 1 (HSV-1) and known human complement regulatory proteins factor H, properdin, factor B, complement receptor 1 (CR1) and 2 (CR2) were investigated. The interaction of gC with C3b was studied using purified complement components, synthetic peptides, antisera against different C3 fragments and anti-C3 monoclonal antibodies (mAb) with known inhibitory effects on C3-ligand interactions. All the mAb that inhibited gC/C3b interactions, in a differential manner, also prevented binding of C3 fragments to factors H, B, CR1 or CR2. No blocking was observed with synthetic peptides representing different C3 regions or with factor B and C3d, whereas C3b, C3c and factor H were inhibitory, as well as purified gC. There was no binding of gC to cobra venom factor (CVF), a C3c-like fragment derived from cobra gland. Purified gC bound to iC3, iC3b and C3c, but failed to bind to C3d. Glycoprotein C bound only weakly to iC3 derived from bovine and porcine plasma, thus indicating a preference of the viral protein for the appropriate host. Binding of gC was also observed to proteolytic C3 fragments, especially to the beta-chain, thus suggesting the importance of the C3 region as a binding site. Purified gC from HSV-1, but not HSV-2, inhibited the binding of factor H and properdin but not of CR1 to C3b. The binding of iC3b to CR2, a molecule involved in B-cell activation and binding of the Epstein-Barr virus, was also inhibited by the HSV-1 protein. As factor H and properdin, the binding of which was inhibited by gC, are important regulators of the alternative complement pathway, these data further support a role of gC in the evasion of HSV from a major first-line host defence mechanism, i.e. the complement system. In addition, the inhibition of the C3/CR

  17. Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits

    PubMed Central

    Shi, Jiong; Friedman, David B.

    2013-01-01

    The host restriction factors TRIM5α and TRIMCyp potently inhibit retrovirus infection by binding to the incoming retrovirus capsid. TRIM5 proteins are dimeric, and their association with the viral capsid appears to be enhanced by avidity effects owing to formation of higher-order oligomeric complexes. We examined the stoichiometric requirement for TRIM5 functional recognition by quantifying the efficiencies of restriction of HIV-1 and murine leukemia virus (MLV) particles containing various proportions of restriction-sensitive and -insensitive CA subunits. Both TRIMCyp and TRIM5α inhibited infection of retrovirus particles containing as little as 25% of the restriction-sensitive CA protein. Accordingly, we also observed efficient binding of TRIMCyp in vitro to capsid assemblies containing as little as one-fourth wild-type CA protein. Paradoxically, the ability of HIV-1 particles to abrogate TRIMCyp restriction in trans was more strongly dependent on the fraction of wild-type CA than was restriction of infection. Collectively, our results indicate that TRIM5 restriction factors bind to retroviral capsids in a highly cooperative manner and suggest that TRIM5 can engage a capsid lattice containing a minimum of three or fewer recognizable subunits per hexamer. Our study supports a model in which localized binding of TRIM5 to the viral capsid nucleates rapid polymerization of a TRIM5 lattice on the capsid surface. PMID:23785198

  18. Graph theoretic network analysis reveals protein pathways underlying cell death following neurotropic viral infection.

    PubMed

    Ghosh, Sourish; Kumar, G Vinodh; Basu, Anirban; Banerjee, Arpan

    2015-01-01

    Complex protein networks underlie any cellular function. Certain proteins play a pivotal role in many network configurations, disruption of whose expression proves fatal to the cell. An efficient method to tease out such key proteins in a network is still unavailable. Here, we used graph-theoretic measures on protein-protein interaction data (interactome) to extract biophysically relevant information about individual protein regulation and network properties such as formation of function specific modules (sub-networks) of proteins. We took 5 major proteins that are involved in neuronal apoptosis post Chandipura Virus (CHPV) infection as seed proteins in a database to create a meta-network of immediately interacting proteins (1(st) order network). Graph theoretic measures were employed to rank the proteins in terms of their connectivity and the degree upto which they can be organized into smaller modules (hubs). We repeated the analysis on 2(nd) order interactome that includes proteins connected directly with proteins of 1(st) order. FADD and Casp-3 were connected maximally to other proteins in both analyses, thus indicating their importance in neuronal apoptosis. Thus, our analysis provides a blueprint for the detection and validation of protein networks disrupted by viral infections. PMID:26404759

  19. Graph theoretic network analysis reveals protein pathways underlying cell death following neurotropic viral infection

    PubMed Central

    Ghosh, Sourish; Kumar, G. Vinodh; Basu, Anirban; Banerjee, Arpan

    2015-01-01

    Complex protein networks underlie any cellular function. Certain proteins play a pivotal role in many network configurations, disruption of whose expression proves fatal to the cell. An efficient method to tease out such key proteins in a network is still unavailable. Here, we used graph-theoretic measures on protein-protein interaction data (interactome) to extract biophysically relevant information about individual protein regulation and network properties such as formation of function specific modules (sub-networks) of proteins. We took 5 major proteins that are involved in neuronal apoptosis post Chandipura Virus (CHPV) infection as seed proteins in a database to create a meta-network of immediately interacting proteins (1st order network). Graph theoretic measures were employed to rank the proteins in terms of their connectivity and the degree upto which they can be organized into smaller modules (hubs). We repeated the analysis on 2nd order interactome that includes proteins connected directly with proteins of 1st order. FADD and Casp-3 were connected maximally to other proteins in both analyses, thus indicating their importance in neuronal apoptosis. Thus, our analysis provides a blueprint for the detection and validation of protein networks disrupted by viral infections. PMID:26404759

  20. Host protein Snapin interacts with human cytomegalovirus pUL130 and affects viral DNA replication.

    PubMed

    Wang, Guili; Ren, Gaowei; Cui, Xin; Lu, Zhitao; Ma, Yanpin; Qi, Ying; Huang, Yujing; Liu, Zhongyang; Sun, Zhengrong; Ruan, Qiang

    2016-06-01

    The interplay between the host and Human cytomegalovirus (HCMV) plays a pivotal role in the outcome of an infection. HCMV growth in endothelial and epithelial cells requires expression of viral proteins UL128, UL130, and UL131 proteins (UL128-131), of which UL130 is the largest gene and the only one that is not interrupted by introns.Mutation of the C terminus of the UL130 protein causes reduced tropism of endothelial cells (EC). However, very few host factors have been identified that interact with the UL130 protein. In this study, HCMV UL130 protein was shown to directly interact with the human protein Snapin in human embryonic kidney HEK293 cells by Yeast two-hybrid screening, in vitro glutathione S-transferase (GST) pull-down, and co-immunoprecipitation. Additionally, heterologous expression of protein UL130 revealed co-localization with Snapin in the cell membrane and cytoplasm of HEK293 cells using fluorescence confocal microscopy. Furthermore, decreasing the level of Snapin via specific small interfering RNAs decreased the number of viral DNA copies and titer inHCMV-infected U373-S cells. Taken together, these results suggest that Snapin, the pUL130 interacting protein, has a role in modulating HCMV DNA synthesis. PMID:27240978

  1. Flavivirus NS1: a multifaceted enigmatic viral protein.

    PubMed

    Rastogi, Meghana; Sharma, Nikhil; Singh, Sunit Kumar

    2016-01-01

    Flaviviruses are emerging arthropod-borne viruses representing an immense global health problem. The prominent viruses of this group include dengue virus, yellow fever virus, Japanese encephalitis virus, West Nile virus tick borne encephalitis virus and Zika Virus. These are endemic in many parts of the world. They are responsible for the illness ranging from mild flu like symptoms to severe hemorrhagic, neurologic and cognitive manifestations leading to death. NS1 is a highly conserved non-structural protein among flaviviruses, which exist in diverse forms. The intracellular dimer form of NS1 plays role in genome replication, whereas, the secreted hexamer plays role in immune evasion. The secreted NS1 has been identified as a potential diagnostic marker for early detection of the infections caused by flaviviruses. In addition to the diagnostic marker, the importance of NS1 has been reported in the development of therapeutics. NS1 based subunit vaccines are at various stages of development. The structural details and diverse functions of NS1 have been discussed in detail in this review. PMID:27473856

  2. Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

    PubMed Central

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; Edwards, Robert A.; Burgin, Alex B.; Segall, Anca M.; Rohwer, Forest

    2015-01-01

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented. PMID:26132888

  3. Phage phenomics: Physiological approaches to characterize novel viral proteins

    DOE PAGESBeta

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; et al

    2015-06-11

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

  4. Phage phenomics: Physiological approaches to characterize novel viral proteins

    SciTech Connect

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; Liang, Tiffany Y.; Pivaroff, Cullen G.; Haynes, Matthew R.; Nulton, Jim; Felts, Ben; Bailey, Barbara A.; Salamon, Peter; Edwards, Robert A.; Burgin, Alex B.; Segall, Anca M.; Rohwer, Forest

    2015-06-11

    Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

  5. Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins

    NASA Astrophysics Data System (ADS)

    Champeimont, Raphaël; Laine, Elodie; Hu, Shuang-Wei; Penin, Francois; Carbone, Alessandra

    2016-05-01

    A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks.

  6. Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins

    PubMed Central

    Champeimont, Raphaël; Laine, Elodie; Hu, Shuang-Wei; Penin, Francois; Carbone, Alessandra

    2016-01-01

    A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks. PMID:27198619

  7. Coevolution analysis of Hepatitis C virus genome to identify the structural and functional dependency network of viral proteins.

    PubMed

    Champeimont, Raphaël; Laine, Elodie; Hu, Shuang-Wei; Penin, Francois; Carbone, Alessandra

    2016-01-01

    A novel computational approach of coevolution analysis allowed us to reconstruct the protein-protein interaction network of the Hepatitis C Virus (HCV) at the residue resolution. For the first time, coevolution analysis of an entire viral genome was realized, based on a limited set of protein sequences with high sequence identity within genotypes. The identified coevolving residues constitute highly relevant predictions of protein-protein interactions for further experimental identification of HCV protein complexes. The method can be used to analyse other viral genomes and to predict the associated protein interaction networks. PMID:27198619

  8. Interferon-Gamma Enhances TLR3 Expression and Anti-Viral Activity in Keratinocytes.

    PubMed

    Kajita, Ai; Morizane, Shin; Takiguchi, Tetsuya; Yamamoto, Takenobu; Yamada, Masao; Iwatsuki, Keiji

    2015-08-01

    Toll-like receptors (TLRs) recognize specific microbial products in the innate immune response. TLR3, a double-stranded RNA sensor, is thought to have an important role in viral infections, but the regulation of TLR3 expression and its function in keratinocytes are not fully understood. Here we show the Th1 cytokine IFN-γ increased the TLR3 expression via STAT1 in cultured normal human epidermal keratinocytes (NHEKs). Co-stimulation with IFN-γ and the TLR3 ligand poly (I:C) synergistically increased the expression of IFN-β, IL-6, IL-8, and human β-defensin-2 in NHEKs compared with poly (I:C) or IFN-γ alone. These synergistic inductions were significantly inhibited by an endosomal acidification inhibitor, chloroquine, and by TLR3 siRNA. Co-stimulation with IFN-γ and poly (I:C) also significantly enhanced the anti-viral activity against herpes simplex virus type-1 in NHEKs compared with poly (I:C) or IFN-γ alone. In addition to the in vitro findings, an immunohistochemical analysis revealed IFN-γ-positive cells surrounding herpetic vesicles. These findings indicate that IFN-γ might contribute to the innate immune response to cutaneous viral infections by enhancing TLR3 expression and function in keratinocytes. PMID:25822580

  9. In vivo imaging of alphaherpesvirus infection reveals synchronized activity dependent on axonal sorting of viral proteins

    PubMed Central

    Granstedt, Andrea E.; Bosse, Jens B.; Thiberge, Stephan Y.; Enquist, Lynn W.

    2013-01-01

    A clinical hallmark of human alphaherpesvirus infections is peripheral pain or itching. Pseudorabies virus (PRV), a broad host range alphaherpesvirus, causes violent pruritus in many different animals, but the mechanism is unknown. Previous in vitro studies have shown that infected, cultured peripheral nervous system (PNS) neurons exhibited aberrant electrical activity after PRV infection due to the action of viral membrane fusion proteins, yet it is unclear if such activity occurs in infected PNS ganglia in living animals and if it correlates with disease symptoms. Using two-photon microscopy, we imaged autonomic ganglia in living mice infected with PRV strains expressing GCaMP3, a genetically encoded calcium indicator, and used the changes in calcium flux to monitor the activity of many neurons simultaneously with single-cell resolution. Infection with virulent PRV caused these PNS neurons to fire synchronously and cyclically in highly correlated patterns among infected neurons. This activity persisted even when we severed the presynaptic axons, showing that infection-induced firing is independent of input from presynaptic brainstem neurons. This activity was not observed after infections with an attenuated PRV recombinant used for circuit tracing or with PRV mutants lacking either viral glycoprotein B, required for membrane fusion, or viral membrane protein Us9, required for sorting virions and viral glycoproteins into axons. We propose that the viral fusion proteins produced by virulent PRV infection induce electrical coupling in unmyelinated axons in vivo. This action would then give rise to the synchronous and cyclical activity in the ganglia and contribute to the characteristic peripheral neuropathy. PMID:23980169

  10. New capillary gel electrophoresis method for fast and accurate identification and quantification of multiple viral proteins in influenza vaccines.

    PubMed

    van Tricht, Ewoud; Geurink, Lars; Pajic, Bojana; Nijenhuis, Johan; Backus, Harold; Germano, Marta; Somsen, Govert W; Sänger-van de Griend, Cari E

    2015-11-01

    Current methods for the identification and/or quantification of viral proteins in influenza virus and virosome samples suffer from long analysis times, limited protein coverage and/or low accuracy and precision. We studied and optimized capillary gel electrophoresis (CGE) in order to achieve faster and enhanced characterization and quantification of viral proteins. Sample preparation as well the composition of the gel buffer was investigated in order to achieve adequate protein separation in relatively short times. The total sample preparation (reduction and deglycosylation) could be carried out efficiently within two hours. Hydrodynamic injection, separation voltage, and capillary temperature were optimized in full factorial design. The final method was validated and showed good performance for hemagglutinin fragment 1 (HA1), hemagglutinin fragment 2 (HA2), matrix protein (M) and nucleoprotein (NP). The CGE method allowed identification of different virus strains based on their specific protein profile. B/Brisbane inactivated virus and virosome samples could be analyzed within one day. The CGE results (titers) were comparable to single radial immune-diffusion (SRID), but the method has the advantage of a much faster time to results. CGE analysis of A/Christchurch from upstream process demonstrated the applicability of the method to samples of high complexity. The CGE method could be used in the same analyte concentration range as the RP-HPLC method, but showed better precision and accuracy. Overall, the total analysis time for the CGE method was much shorter, allowing analysis of 100 samples in 4 days instead of 10 days for SRID. PMID:26452923

  11. Specific interaction between hnRNP H and HPV16 L1 proteins: Implications for late gene auto-regulation enabling rapid viral capsid protein production

    SciTech Connect

    Zheng, Zi-Zheng; Sun, Yuan-Yuan; Zhao, Min; Huang, Hui; Zhang, Jun; Xia, Ning-Shao; Miao, Ji; Zhao, Qinjian

    2013-01-18

    Highlights: ► The RNA-binding hnRNP H regulates late viral gene expression. ► hnRNP H activity was inhibited by a late viral protein. ► Specific interaction between HPV L1 and hnRNP H was demonstrated. ► Co-localization of HPV L1 and hnRNP H inside cells was observed. ► Viral capsid protein production, enabling rapid capsid assembly, was implicated. -- Abstract: Heterogeneous nuclear ribonucleoproteins (hnRNPs), including hnRNP H, are RNA-binding proteins that function as splicing factors and are involved in downstream gene regulation. hnRNP H, which binds to G triplet regions in RNA, has been shown to play an important role in regulating the staged expression of late proteins in viral systems. Here, we report that the specific association between hnRNP H and a late viral capsid protein, human papillomavirus (HPV) L1 protein, leads to the suppressed function of hnRNP H in the presence of the L1 protein. The direct interaction between the L1 protein and hnRNP H was demonstrated by complex formation in solution and intracellularly using a variety of biochemical and immunochemical methods, including peptide mapping, specific co-immunoprecipitation and confocal fluorescence microscopy. These results support a working hypothesis that a late viral protein HPV16 L1, which is down regulated by hnRNP H early in the viral life cycle may provide an auto-regulatory positive feedback loop that allows the rapid production of HPV capsid proteins through suppression of the function of hnRNP H at the late stage of the viral life cycle. In this positive feedback loop, the late viral gene products that were down regulated earlier themselves disable their suppressors, and this feedback mechanism could facilitate the rapid production of capsid proteins, allowing staged and efficient viral capsid assembly.

  12. Artificial Neural Networks Trained to Detect Viral and Phage Structural Proteins

    PubMed Central

    Seguritan, Victor; Raymond, Amy; Lorimer, Don; Burgin, Alex B.; Salamon, Peter; Segall, Anca M.

    2012-01-01

    Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conversion factors, and in nutrient redistribution, via cell lysis. Analyses of phage- and viral-encoded genes in environmental samples provide insights into the physiological impact of viruses on microbial communities and human health. However, phage ORFs are extremely diverse of which over 70% of them are dissimilar to any genes with annotated functions in GenBank. Better identification of viruses would also aid in better detection and diagnosis of disease, in vaccine development, and generally in better understanding the physiological potential of any environment. In contrast to enzymes, viral structural protein function can be much more challenging to detect from sequence data because of low sequence conservation, few known conserved catalytic sites or sequence domains, and relatively limited experimental data. We have designed a method of predicting phage structural protein sequences that uses Artificial Neural Networks (ANNs). First, we trained ANNs to classify viral structural proteins using amino acid frequency; these correctly classify a large fraction of test cases with a high degree of specificity and sensitivity. Subsequently, we added estimates of protein isoelectric points as a feature to ANNs that classify specialized families of proteins, namely major capsid and tail proteins. As expected, these more specialized ANNs are more accurate than the structural ANNs. To experimentally validate the ANN predictions, several ORFs with no significant similarities to known sequences that are ANN-predicted structural proteins were examined by transmission electron microscopy. Some of these self-assembled into structures strongly resembling virion structures. Thus, our ANNs are new tools for identifying phage and potential prophage structural proteins that are difficult or impossible to detect by other bioinformatic analysis. The networks will be valuable when sequence is

  13. Artificial neural networks trained to detect viral and phage structural proteins.

    PubMed

    Seguritan, Victor; Alves, Nelson; Arnoult, Michael; Raymond, Amy; Lorimer, Don; Burgin, Alex B; Salamon, Peter; Segall, Anca M

    2012-01-01

    Phages play critical roles in the survival and pathogenicity of their hosts, via lysogenic conversion factors, and in nutrient redistribution, via cell lysis. Analyses of phage- and viral-encoded genes in environmental samples provide insights into the physiological impact of viruses on microbial communities and human health. However, phage ORFs are extremely diverse of which over 70% of them are dissimilar to any genes with annotated functions in GenBank. Better identification of viruses would also aid in better detection and diagnosis of disease, in vaccine development, and generally in better understanding the physiological potential of any environment. In contrast to enzymes, viral structural protein function can be much more challenging to detect from sequence data because of low sequence conservation, few known conserved catalytic sites or sequence domains, and relatively limited experimental data. We have designed a method of predicting phage structural protein sequences that uses Artificial Neural Networks (ANNs). First, we trained ANNs to classify viral structural proteins using amino acid frequency; these correctly classify a large fraction of test cases with a high degree of specificity and sensitivity. Subsequently, we added estimates of protein isoelectric points as a feature to ANNs that classify specialized families of proteins, namely major capsid and tail proteins. As expected, these more specialized ANNs are more accurate than the structural ANNs. To experimentally validate the ANN predictions, several ORFs with no significant similarities to known sequences that are ANN-predicted structural proteins were examined by transmission electron microscopy. Some of these self-assembled into structures strongly resembling virion structures. Thus, our ANNs are new tools for identifying phage and potential prophage structural proteins that are difficult or impossible to detect by other bioinformatic analysis. The networks will be valuable when sequence is

  14. T cell inactivation by poxviral B22 family proteins increases viral virulence.

    PubMed

    Alzhanova, Dina; Hammarlund, Erika; Reed, Jason; Meermeier, Erin; Rawlings, Stephanie; Ray, Caroline A; Edwards, David M; Bimber, Ben; Legasse, Alfred; Planer, Shannon; Sprague, Jerald; Axthelm, Michael K; Pickup, David J; Lewinsohn, David M; Gold, Marielle C; Wong, Scott W; Sacha, Jonah B; Slifka, Mark K; Früh, Klaus

    2014-05-01

    Infections with monkeypox, cowpox and weaponized variola virus remain a threat to the increasingly unvaccinated human population, but little is known about their mechanisms of virulence and immune evasion. We now demonstrate that B22 proteins, encoded by the largest genes of these viruses, render human T cells unresponsive to stimulation of the T cell receptor by MHC-dependent antigen presentation or by MHC-independent stimulation. In contrast, stimuli that bypass TCR-signaling are not inhibited. In a non-human primate model of monkeypox, virus lacking the B22R homologue (MPXVΔ197) caused only mild disease with lower viremia and cutaneous pox lesions compared to wild type MPXV which caused high viremia, morbidity and mortality. Since MPXVΔ197-infected animals displayed accelerated T cell responses and less T cell dysregulation than MPXV US2003, we conclude that B22 family proteins cause viral virulence by suppressing T cell control of viral dissemination. PMID:24832205

  15. Targeting of Pseudorabies Virus Structural Proteins to Axons Requires Association of the Viral Us9 Protein with Lipid Rafts

    PubMed Central

    Lyman, Mathew G.; Curanovic, Dusica; Enquist, Lynn W.

    2008-01-01

    The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 null mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system. PMID:18483549

  16. The full-length E1-circumflexE4 protein of human papillomavirus type 18 modulates differentiation-dependent viral DNA amplification and late gene expression

    SciTech Connect

    Wilson, Regina; Ryan, Gordon B.; Knight, Gillian L.; Laimins, Laimonis A.; Roberts, Sally . E-mail: s.roberts@bham.ac.uk

    2007-06-05

    Activation of the productive phase of the human papillomavirus (HPV) life cycle in differentiated keratinocytes is coincident with high-level expression of E1-circumflexE4 protein. To determine the role of E1-circumflexE4 in the HPV replication cycle, we constructed HPV18 mutant genomes in which expression of the full-length E1-circumflexE4 protein was abrogated. Undifferentiated keratinocytes containing mutant genomes showed enhanced proliferation when compared to cells containing wildtype genomes, but there were no differences in maintenance of viral episomes. Following differentiation, cells with mutant genomes exhibited reduced levels of viral DNA amplification and late gene expression, compared to wildtype genome-containing cells. This indicates that HPV18 E1-circumflexE4 plays an important role in regulating HPV late functions, and it may also function in the early phase of the replication cycle. Our finding that full-length HPV18 E1-circumflexE4 protein plays a significant role in promoting viral genome amplification concurs with a similar report with HPV31, but is in contrast to an HPV11 study where viral DNA amplification was not dependent on full-length E1-circumflexE4 expression, and to HPV16 where only C-terminal truncations in E1-circumflexE4 abrogated vegetative genome replication. This suggests that type-specific differences exist between various E1-circumflexE4 proteins.

  17. Host Tissue and Glycan Binding Specificities of Avian Viral Attachment Proteins Using Novel Avian Tissue Microarrays

    PubMed Central

    Ambepitiya Wickramasinghe, Iresha N.; de Vries, Robert P.; Eggert, Amber M.; Wandee, Nantaporn; de Haan, Cornelis A. M.; Gröne, Andrea; Verheije, Monique H.

    2015-01-01

    The initial interaction between viral attachment proteins and the host cell is a critical determinant for the susceptibility of a host for a particular virus. To increase our understanding of avian pathogens and the susceptibility of poultry species, we developed novel avian tissue microarrays (TMAs). Tissue binding profiles of avian viral attachment proteins were studied by performing histochemistry on multi-species TMA, comprising of selected tissues from ten avian species, and single-species TMAs, grouping organ systems of each species together. The attachment pattern of the hemagglutinin protein was in line with the reported tropism of influenza virus H5N1, confirming the validity of TMAs in profiling the initial virus-host interaction. The previously believed chicken-specific coronavirus (CoV) M41 spike (S1) protein displayed a broad attachment pattern to respiratory tissues of various avian species, albeit with lower affinity than hemagglutinin, suggesting that other avian species might be susceptible for chicken CoV. When comparing tissue-specific binding patterns of various avian coronaviral S1 proteins on the single-species TMAs, chicken and partridge CoV S1 had predominant affinity for the trachea, while pigeon CoV S1 showed marked preference for lung of their respective hosts. Binding of all coronaviral S1 proteins was dependent on sialic acids; however, while chicken CoV S1 preferred sialic acids type I lactosamine (Gal(1-3)GlcNAc) over type II (Gal(1-4)GlcNAc), the fine glycan specificities of pigeon and partridge CoVs were different, as chicken CoV S1-specific sialylglycopolymers could not block their binding to tissues. Taken together, TMAs provide a novel platform in the field of infectious diseases to allow identification of binding specificities of viral attachment proteins and are helpful to gain insight into the susceptibility of host and organ for avian pathogens. PMID:26035584

  18. Rubella virus capsid protein modulation of viral genomic and subgenomic RNA synthesis

    SciTech Connect

    Tzeng, W.-P.; Frey, Teryl K. . E-mail: tfrey@gsu.edu

    2005-07-05

    The ratio of the subgenomic (SG) to genome RNA synthesized by rubella virus (RUB) replicons expressing the green fluorescent protein reporter gene (RUBrep/GFP) is substantially higher than the ratio of these species synthesized by RUB (4.3 for RUBrep/GFP vs. 1.3-1.4 for RUB). It was hypothesized that this modulation of the viral RNA synthesis was by one of the virus structural protein genes and it was found that introduction of the capsid (C) protein gene into the replicons as an in-frame fusion with GFP resulted in an increase of genomic RNA production (reducing the SG/genome RNA ratio), confirming the hypothesis and showing that the C gene was the moiety responsible for the modulation effect. The N-terminal one-third of the C gene was required for the effect of be exhibited. A similar phenomenon was not observed with the replicons of Sindbis virus, a related Alphavirus. Interestingly, modulation was not observed when RUBrep/GFP was co-transfected with either other RUBrep or plasmid constructs expressing the C gene, demonstrating that modulation could occur only when the C gene was provided in cis. Mutations that prevented translation of the C protein failed to modulate RNA synthesis, indicating that the C protein was the moiety responsible for modulation; consistent with this conclusion, modulation of RNA synthesis was maintained when synonymous codon mutations were introduced at the 5' end of the C gene that changed the C gene sequence without altering the amino acid sequence of the C protein. These results indicate that C protein translated in proximity of viral replication complexes, possibly from newly synthesized SG RNA, participate in regulating the replication of viral RNA.

  19. Transcriptional enhancer from milk protein genes

    SciTech Connect

    Casperson, G.F.; Schmidhauser, C.T.; Bissell, M.J.

    1999-12-21

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  20. Transcriptional enhancer from milk protein genes

    SciTech Connect

    Casperson, Gerald F.; Schmidhauser, Christian T.; Bissell, Mina J.

    1999-01-01

    The invention relates to novel enhancer nucleotide sequences which stimulate transcription of heterologous DNA in cells in culture. The enhancers are derived from major milk protein genes by the process of deletion mapping and functional analysis. The invention also relates to expression vectors containing the novel enhancers.

  1. Transient viral DNA replication and repression of viral transcription are supported by the C-terminal domain of the bovine papillomavirus type 1 E1 protein.

    PubMed

    Ferran, M C; McBride, A A

    1998-01-01

    The bovine papillomavirus type 1 E1 protein is important for viral DNA replication and transcriptional repression. It has been proposed that the full-length E1 protein consists of a small N-terminal and a larger C-terminal domain. In this study, it is shown that an E1 polypeptide containing residues 132 to 605 (which represents the C-terminal domain) is able to support transient viral DNA replication, although at a level lower than that supported by the wild-type protein. This domain can also repress E2-mediated transactivation from the P89 promoter as well as the wild-type E1 protein can. PMID:9420289

  2. Applying horizontal gene transfer phenomena to enhance non-viral gene therapy

    PubMed Central

    Elmer, Jacob J.; Christensen, Matthew D.; Rege, Kaushal

    2014-01-01

    Horizontal gene transfer (HGT) is widespread amongst prokaryotes, but eukaryotes tend to be far less promiscuous with their genetic information. However, several examples of HGT from pathogens into eukaryotic cells have been discovered and mimicked to improve non-viral gene delivery techniques. For example, several viral proteins and DNA sequences have been used to significantly increase cytoplasmic and nuclear gene delivery. Plant genetic engineering is routinely performed with the pathogenic bacterium Agrobacterium tumefaciens and similar pathogens (e.g. Bartonella henselae) may also be able to transform human cells. Intracellular parasites like Trypanosoma cruzi may also provide new insights into overcoming cellular barriers to gene delivery. Finally, intercellular nucleic acid transfer between host cells will also be briefly discussed. This article will review the unique characteristics of several different viruses and microbes and discuss how their traits have been successfully applied to improve non-viral gene delivery techniques. Consequently, pathogenic traits that originally caused diseases may eventually be used to treat many genetic diseases. PMID:23994344

  3. Viral Expression Cassette Elements to Enhance Transgene Target Specificity and Expression in Gene Therapy

    PubMed Central

    Powell, Sara Kathleen; Rivera-Soto, Ricardo; Gray, Steven James

    2015-01-01

    Over the last five years, the number of clinical trials involving AAV (adeno-associated virus) and lentiviral vectors continue to increase by about 150 trials each year. For continued success, AAV and lentiviral expression cassettes need to be designed to meet each disease's specific needs. This review discusses how viral vector expression cassettes can be engineered with elements to enhance target specificity and increase transgene expression. The key differences relating to target specificity between ubiquitous and tissue-specific promoters are discussed, as well as how endogenous miRNAs and their target sequences have been used to restrict transgene expression. Specifically, relevant studies indicating how cis-acting elements such as introns, WPRE, polyadenylation signals, and the CMV enhancer are highlighted to show their utility for enhancing transgene expression in gene therapy applications. All discussion bears in mind that expression cassettes have space constraints. In conclusion, this review can serve as a menu of vector genome design elements and their cost in terms of space to thoughtfully engineer viral vectors for gene therapy. PMID:25636961

  4. Evaluation of the immune response elicited by vaccination with viral vectors encoding FMDV capsid proteins and boosted with inactivated virus.

    PubMed

    Romanutti, Carina; D'Antuono, Alejandra; Palacios, Carlos; Quattrocchi, Valeria; Zamorano, Patricia; La Torre, Jose; Mattion, Nora

    2013-08-30

    The aim of the present study was to assess the effect of introducing a priming step with replication-defective viral vectors encoding the capsid proteins of FMDV, followed by a boost with killed virus vaccines, using a suitable BALB/c mice model. Additionally, the immune response to other combined vector immunization regimens was studied. For this purpose, we analyzed different prime-boost immunizations with recombinant adenovirus (Ad), herpesvirus amplicons (Hs) and/or killed virus (KV) vaccines. The highest antibody titers were found in the group that received two doses of adjuvanted KV (P<0.002). Antibody titers were higher in those groups receiving a mixed regimen of vectors, compared to immunization with either vector alone (P<0.0001). Priming with any of the viral vectors induced a shift of the cytokine balance toward a Th1 type immune response regardless of the delivery system used for boosting. The highest IgG1 titer was induced by two doses of adjuvanted KV (P=0.0002) and the highest IgG2a titer corresponded to the group primed with Ad and boosted with KV (P=0.01). Re-stimulation of all groups of mice with 0.5 μg of inactivated virus five months later resulted in a fast increase of antibody titers in all the groups tested. After virus stimulation, antibody titers in the groups that received KV alone or Ad prime-KV boost, were indistinguishable (P=0.800). Protection from challenge was similar (75%) in the groups of animals that received Ad prime-Hs boost or Ad prime-KV boost, or two doses of oil-adjuvanted KV. The data presented in this study suggest that sequential immunization with viral vectors-based vaccines combined with protein-based vaccines have the potential to enhance the quality of the immune response against FMDV. PMID:23683999

  5. Cytoskeletal proteins participate in conserved viral strategies across kingdoms of life.

    PubMed

    Erb, Marcella L; Pogliano, Joe

    2013-12-01

    The discovery of tubulin-like cytoskeletal proteins carried on the genomes of bacteriophages that are actively used for phage propagation during both the lytic and lysogenic cycle have revealed that there at least two ways that viruses can utilize a cytoskeleton; co-opt the host cytoskeleton or bring their own homologues. Either strategy underscores the deep evolutionary relationship between viruses and cytoskeletal proteins and points to a conservation of viral strategies that crosses the kingdoms of life. Here we review some of the most recent discoveries about tubulin cytoskeletal elements encoded by phages and compare them to some of the strategies utilized by the gammaherpesvirues of mammalian cells. PMID:24055040

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

    PubMed Central

    Bienz, K; Egger, D; Troxler, M; Pasamontes, L

    1990-01-01

    Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed. Images PMID:2154600

  7. The non-structural protein μNS of piscine orthoreovirus (PRV) forms viral factory-like structures.

    PubMed

    Haatveit, Hanne Merethe; Nyman, Ingvild B; Markussen, Turhan; Wessel, Øystein; Dahle, Maria Krudtaa; Rimstad, Espen

    2016-01-01

    Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation in farmed Atlantic salmon. The virus is ubiquitous and found in both farmed and wild salmonid fish. It belongs to the family Reoviridae, closely related to the genus Orthoreovirus. The PRV genome comprises ten double-stranded RNA segments encoding at least eight structural and two non-structural proteins. Erythrocytes are the major target cells for PRV. Infected erythrocytes contain globular inclusions resembling viral factories; the putative site of viral replication. For the mammalian reovirus (MRV), the non-structural protein μNS is the primary organizer in factory formation. The analogous PRV protein was the focus of the present study. The subcellular location of PRV μNS and its co-localization with the PRV σNS, µ2 and λ1 proteins was investigated. We demonstrated that PRV μNS forms dense globular cytoplasmic inclusions in transfected fish cells, resembling the viral factories of MRV. In co-transfection experiments with μNS, the σNS, μ2 and λ1 proteins were recruited to the globular structures. The ability of μNS to recruit other PRV proteins into globular inclusions indicates that it is the main viral protein involved in viral factory formation and pivotal in early steps of viral assembly. PMID:26743679

  8. 3C protein of feline coronavirus inhibits viral replication independently of the autophagy pathway.

    PubMed

    Hsieh, Li-En; Huang, Wei-Pang; Tang, Da-Jay; Wang, Ying-Ting; Chen, Ching-Tang; Chueh, Ling-Ling

    2013-12-01

    Feline coronavirus (FCoV) can cause either asymptomatic enteric infection or fatal peritonitis in cats. Although the mutation of FCoV accessory gene 3c has been suggested to be related to the occurrence of feline infectious peritonitis (FIP), how the 3C protein is involved in this phenomenon remains unknown. To investigate the role of the 3C protein, a full-length 3c gene was transiently expressed and the cytoplasmic distribution of the protein was found to be primarily in the perinuclear region. Using 3c-stable expression cells, the replication of a 3c-defective FCoV strain was titrated and a significant decrease in replication (p<0.05) was observed. The mechanism underlying the decreased FIPV replication caused by the 3C protein was further investigated; neither the induction nor inhibition of autophagy rescued the viral replication. Taken together, our data suggest that the 3C protein might have a virulence-suppressing effect in FCoV-infected cats. Deletion of the 3c gene could therefore cause more efficient viral replication, which leads to a fatal infection. PMID:24050534

  9. Viral terminal protein directs early organization of phage DNA replication at the bacterial nucleoid

    PubMed Central

    Muñoz-Espín, Daniel; Holguera, Isabel; Ballesteros-Plaza, David; Carballido-López, Rut; Salas, Margarita

    2010-01-01

    The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages ϕ29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage ϕ29 revealed that the TP N-terminal domain (residues 1–73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of ϕ29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid. PMID:20823229

  10. Viral potassium channels as a robust model system for studies of membrane-protein interaction.

    PubMed

    Braun, Christian J; Lachnit, Christine; Becker, Patrick; Henkes, Leonhard M; Arrigoni, Cristina; Kast, Stefan M; Moroni, Anna; Thiel, Gerhard; Schroeder, Indra

    2014-04-01

    The viral channel KcvNTS belongs to the smallest K(+) channels known so far. A monomer of a functional homotetramer contains only 82 amino acids. As a consequence of the small size the protein is almost fully submerged into the membrane. This suggests that the channel is presumably sensitive to its lipid environment. Here we perform a comparative analysis for the function of the channel protein embedded in three different membrane environments. 1. Single-channel currents of KcvNTS were recorded with the patch clamp method on the plasma membrane of HEK293 cells. 2. They were also measured after reconstitution of recombinant channel protein into classical planar lipid bilayers and 3. into horizontal bilayers derived from giant unilamellar vesicles (GUVs). The recombinant channel protein was either expressed and purified from Pichia pastoris or from a cell-free expression system; for the latter a new approach with nanolipoprotein particles was used. The data show that single-channel activity can be recorded under all experimental conditions. The main functional features of the channel like a large single-channel conductance (80pS), high open-probability (>50%) and the approximate duration of open and closed dwell times are maintained in all experimental systems. An apparent difference between the approaches was only observed with respect to the unitary conductance, which was ca. 35% lower in HEK293 cells than in the other systems. The reason for this might be explained by the fact that the channel is tagged by GFP when expressed in HEK293 cells. Collectively the data demonstrate that the small viral channel exhibits a robust function in different experimental systems. This justifies an extrapolation of functional data from these systems to the potential performance of the channel in the virus/host interaction. This article is part of a Special Issue entitled: Viral Membrane Proteins-Channels for Cellular Networking. PMID:23791706

  11. Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466.

    PubMed

    Huang, Canhua; Zhang, Xiaobo; Lin, Qingsong; Xu, Xun; Hu, Zhihong; Hew, Choy-L

    2002-03-01

    White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV. PMID:12096122

  12. VIRUS INFECTION RAPIDLY ACTIVATES THE P58IPK PATHWAY, DELAYING PEAK KINASE ACTIVATION TO ENHANCE VIRAL REPLICATION

    PubMed Central

    GOODMAN, ALAN G.; TANNER, BERTRAND C. W.; CHANG, STEWART T.; ESTEBAN, MARIANO; KATZE, MICHAEL G.

    2011-01-01

    Previously we showed that the cellular protein P58IPK contributes to viral protein synthesis by decreasing the activity of the anti-viral protein, PKR. Here, we constructed a mathematical model to examine the P58IPK pathway and investigated temporal behavior of this biological system. We find that influenza virus infection results in the rapid activation of P58IPK which delays and reduces maximal PKR and eIF2α phosphorylation, leading to increased viral protein levels. We confirmed that the model could accurately predict viral and host protein levels at extended time points by testing it against experimental data. Sensitivity analysis of relative reaction rates describing P58IPK activity and the downstream proteins through which it functions helped identify processes that may be the most beneficial targets to thwart virus replication. Together, our study demonstrates how computational modeling can guide experimental design to further understand a specific metabolic signaling pathway during viral infection in a mammalian system. PMID:21612809

  13. Proteomic approaches to uncovering virus–host protein interactions during the progression of viral infection

    PubMed Central

    Lum, Krystal K; Cristea, Ileana M

    2016-01-01

    The integration of proteomic methods to virology has facilitated a significant breadth of biological insight into mechanisms of virus replication, antiviral host responses and viral subversion of host defenses. Throughout the course of infection, these cellular mechanisms rely heavily on the formation of temporally and spatially regulated virus–host protein–protein interactions. Reviewed here are proteomic-based approaches that have been used to characterize this dynamic virus–host interplay. Specifically discussed are the contribution of integrative mass spectrometry, antibody-based affinity purification of protein complexes, cross-linking and protein array techniques for elucidating complex networks of virus–host protein associations during infection with a diverse range of RNA and DNA viruses. The benefits and limitations of applying proteomic methods to virology are explored, and the contribution of these approaches to important biological discoveries and to inspiring new tractable avenues for the design of antiviral therapeutics is highlighted. PMID:26817613

  14. Viral channel forming proteins - How to assemble and depolarize lipid membranes in silico.

    PubMed

    Fischer, Wolfgang B; Kalita, Monoj Mon; Heermann, Dieter

    2016-07-01

    Viral channel forming proteins (VCPs) have been discovered in the late 70s and are found in many viruses to date. Usually they are small and have to assemble to form channels which depolarize the lipid membrane of the host cells. Structural information is just about to emerge for just some of them. Thus, computational methods play a pivotal role in generating plausible structures which can be used in the drug development process. In this review the accumulation of structural data is introduced from a historical perspective. Computational performances and their predictive power are reported guided by biological questions such as the assembly, mechanism of function and drug-protein interaction of VCPs. An outlook of how coarse grained simulations can contribute to yet unexplored issues of these proteins is given. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov. PMID:26806161

  15. Regulation of viral gene expression by the herpes simplex virus 1UL24 protein (HSV-1UL24 inhibits accumulation of viral transcripts).

    PubMed

    Sanabria-Solano, Carolina; Gonzalez, Carmen Elena; Richerioux, Nicolas; Bertrand, Luc; Dridi, Slimane; Griffiths, Anthony; Langelier, Yves; Pearson, Angela

    2016-08-01

    UL24 is conserved among all Herpesviridae. In herpes simplex virus 1 (HSV-1), UL24 mutations lead to reduced viral titers both in cell culture and in vivo, and reduced pathogenicity. The human cytomegalovirus ortholog of UL24 has a gene regulatory function; however, it is not known whether other UL24 orthologs also affect gene expression. We discovered that in co-transfection experiments, expression of UL24 correlated with a reduction in the expression of several viral proteins and transcripts. Substitution mutations targeting conserved residues in UL24 impaired this function. Reduced transcript levels did not appear attributable to changes in mRNA stability. The UL24 ortholog of Herpes B virus exhibited a similar activity. An HSV-1 mutant that does not express UL24 produced more viral R1 and R2 transcripts than the wild type or rescue virus relative to the amount of viral DNA. These results reveal a new role for HSV-1UL24 in regulating viral mRNA accumulation. PMID:27214229

  16. Peripherally restricted viral challenge elevates extracellular glutamate and enhances synaptic transmission in the hippocampus.

    PubMed

    Hunsberger, Holly C; Wang, Desheng; Petrisko, Tiffany J; Alhowail, Ahmad; Setti, Sharay E; Suppiramaniam, Vishnu; Konat, Gregory W; Reed, Miranda N

    2016-07-01

    Peripheral infections increase the propensity and severity of seizures in susceptible populations. We have previously shown that intraperitoneal injection of a viral mimic, polyinosinic-polycytidylic acid (PIC), elicits hypersusceptibility of mice to kainic acid (KA)-induced seizures. This study was undertaken to determine whether this seizure hypersusceptibility entails alterations in glutamate signaling. Female C57BL/6 mice were intraperitoneally injected with PIC, and after 24 h, glutamate homeostasis in the hippocampus was monitored using the enzyme-based microelectrode arrays. PIC challenge robustly increased the level of resting extracellular glutamate. While pre-synaptic potassium-evoked glutamate release was not affected, glutamate uptake was profoundly impaired and non-vesicular glutamate release was augmented, indicating functional alterations of astrocytes. Electrophysiological examination of hippocampal slices from PIC-challenged mice revealed a several fold increase in the basal synaptic transmission as compared to control slices. PIC challenge also increased the probability of pre-synaptic glutamate release as seen from a reduction of paired-pulse facilitation and synaptic plasticity as seen from an enhancement of long-term potentiation. Altogether, our results implicate a dysregulation of astrocytic glutamate metabolism and an alteration of excitatory synaptic transmission as the underlying mechanism for the development of hippocampal hyperexcitability, and consequently seizure hypersusceptibility following peripheral PIC challenge. Peripheral infections/inflammations enhance seizure susceptibility. Here, we explored the effect of peritoneal inflammation induced by a viral mimic on glutamate homeostasis and glutamatergic neurotransmission in the mouse hippocampus. We found that peritoneal inflammation elevated extracellular glutamate concentration and enhanced the probability of pre-synaptic glutamate release resulting in hyperexcitability of

  17. Use of host-like peptide motifs in viral proteins is a prevalent strategy in host-virus interactions

    PubMed Central

    Hagai, Tzachi; Azia, Ariel; Babu, M. Madan; Andino, Raul

    2014-01-01

    Virus interact extensively with host proteins, but the mechanisms controlling these interactions are not well understood. We present a comprehensive analysis of eukaryotic linear-peptide motifs (ELMs) in 2,208 viral genomes and reveal that viruses exploit molecular mimicry of host-like ELMs to possibly assist in host-virus interactions. Using a statistical genomics approach, we identify a large number of potentially functional ELMs and observe that the occurrence of ELMs is often evolutionarily conserved but not uniform across virus families. Some viral proteins contain multiple types of ELMs, in striking similarity to complex regulatory modules in host proteins, suggesting that ELMs may act combinatorially to assist viral replication. Furthermore, a simple evolutionary model suggests that the inherent structural simplicity of ELMs often enables them to tolerate mutations and evolve quickly. Our findings suggest that ELMs may allow fast rewiring of host-virus interactions, which likely assists rapid viral evolution and adaptation to diverse environments. PMID:24882001

  18. Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions.

    PubMed

    Antanasijevic, Aleksandar; Kingsley, Carolyn; Basu, Arnab; Bowlin, Terry L; Rong, Lijun; Caffrey, Michael

    2016-03-01

    The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins. PMID:26921030

  19. Application and correlation of nano resolution microscopy techniques to viral protein localization

    NASA Astrophysics Data System (ADS)

    Hodges, Jeffery Allen

    This dissertation is primarily focused on the application of super-resolution microscopy techniques to localization of viral proteins within envelope viruses. Advances in optical super-resolution microscopy techniques have enabled scientists to observe phenomena much smaller than the Abbe diffraction limit by stochastically limiting the number of molecules excited at a given instance and localizing their positions one at a time. Additionally, methods such as Atomic Force Microscopy (AFM) allow scientists to measure the topological features and material properties of samples through contact with a force probe. This dissertation describes the application of these two techniques to virology in order to localize internal viral proteins of enveloped virions, and measure their effect on the elastic properties of the virion. By utilizing super-resolution microscopy techniques such as Fluorescent Photo-Activated Localization Microscopy (fPALM) on virions, which have had their surface glycoproteins labeled with a photo-switchable label, the viral envelope may be accurately recovered. This dissertation describes the development and application of this technique as it applies to envelope recovery of Vesicular Stomatitis Virus (VSV) and Human Immunodeficiency Virus-1 (HIV-1). By fluorescently labeling proteins, which are internal to each of these viruses, I have been able to localize a variety of viral proteins within their recovered envelopes. This is done without significant damage to the virion, making this method a highly effective in vivo technique. In the case of VSV, an asymmetric localization along the central axis towards the blunt 5' end was found to exist for both the polymerase and phosphoproteins. These have been determined to occupy a region in the central cavity of ˜57 +/- 12 nm on the 5' end. This inhomogeneity of the underlying proteins such an asymmetry would predict that the Young's modulus would vary along the central axis of the virion. This dissertation

  20. Vaxfectin-formulated influenza DNA vaccines encoding NP and M2 viral proteins protect mice against lethal viral challenge.

    PubMed

    Jimenez, Gretchen S; Planchon, Rodrick; Wei, Qun; Rusalov, Denis; Geall, Andrew; Enas, Joel; Lalor, Peggy; Leamy, Vicky; Vahle, Ruth; Luke, Catherine J; Rolland, Alain; Kaslow, David C; Smith, Larry R

    2007-01-01

    Next generation influenza vaccines containing conserved antigens may enhance immunity against seasonal or pandemic influenza virus strains. Using a plasmid DNA (pDNA)-based vaccine approach, we systematically tested combinations of NP, M1, and M2 antigens derived from consensus sequences for protection against lethal influenza challenge and compared formulations for adjuvanting low pDNA vaccine doses. The highest level of protection at the lowest pDNA doses was provided by Vaxfectin-formulated NP + M2. Vaxfectin adjuvanticity was confirmed with a low dose of HA pDNA. These promising proof-of-concept data support the clinical development of Vaxfectin-formulated pDNA encoding NP + M2 consensus proteins. PMID:17637571

  1. Function of ubiquitin (Ub) specific protease 15 (USP15) in HIV-1 replication and viral protein degradation.

    PubMed

    Pyeon, Dohun; Timani, Khalid Amine; Gulraiz, Fahad; He, Johnny J; Park, In-Woo

    2016-09-01

    HIV-1 Nef is necessary and may be sufficient for HIV-1-associated AIDS pathogenicity, in that knockout of Nef alone can protect HIV-infected patients from AIDS. We therefore investigated the feasibility of physical knockout of Nef, using the host ubiquitin proteasome system in HIV-1-infected cells. Our co-immunoprecipitation analysis demonstrated that Nef interacted with ubiquitin specific protease 15 (USP15), and that USP15, which is known to stabilize cellular proteins, degraded Nef. Nef could also cause decay of USP15, although Nef-mediated degradation of USP15 was weaker than USP15-mediated Nef degradation. Direct interaction between Nef and USP15 was essential for the observed reciprocal decay of the proteins. Further, USP15 degraded not only Nef but also HIV-1 structural protein, Gag, thereby substantially inhibiting HIV-1 replication. However, Gag did not degrade USP15, indicating that the Nef and USP15 complex, in distinction to other viral proteins, play an integral role in coordinating viral protein degradation and hence HIV-1 replication. Moreover, Nef and USP15 globally suppressed ubiquitylation of cellular proteins, indicating that these proteins are major determinants for the stability of cellular as well as viral proteins. Taken together, these data indicate that Nef and USP15 are vital in regulating degradation of viral and cellular proteins and thus HIV-1 replication, and specific degradation of viral, not cellular proteins, by USP15 points to USP15 as a candidate therapeutic agent to combat AIDS by eliminating viral proteins from the infected cells via USP15-mediated proteosomal degradation. PMID:27460547

  2. Dengue E Protein Domain III-Based DNA Immunisation Induces Strong Antibody Responses to All Four Viral Serotypes

    PubMed Central

    Chan, Kuan Rong; Tan, Hwee Cheng; Bestagno, Marco; Ooi, Eng Eong; Burrone, Oscar R.

    2015-01-01

    Dengue virus (DENV) infection is a major emerging disease widely distributed throughout the tropical and subtropical regions of the world affecting several millions of people. Despite constants efforts, no specific treatment or effective vaccine is yet available. Here we show a novel design of a DNA immunisation strategy that resulted in the induction of strong antibody responses with high neutralisation titres in mice against all four viral serotypes. The immunogenic molecule is an engineered version of the domain III (DIII) of the virus E protein fused to the dimerising CH3 domain of the IgG immunoglobulin H chain. The DIII sequences were also codon-optimised for expression in mammalian cells. While DIII alone is very poorly secreted, the codon-optimised fusion protein is rightly expressed, folded and secreted at high levels, thus inducing strong antibody responses. Mice were immunised using gene-gun technology, an efficient way of intradermal delivery of the plasmid DNA, and the vaccine was able to induce neutralising titres against all serotypes. Additionally, all sera showed reactivity to a recombinant DIII version and the recombinant E protein produced and secreted from mammalian cells in a mono-biotinylated form when tested in a conformational ELISA. Sera were also highly reactive to infective viral particles in a virus-capture ELISA and specific for each serotype as revealed by the low cross-reactive and cross-neutralising activities. The serotype specific sera did not induce antibody dependent enhancement of infection (ADE) in non-homologous virus serotypes. A tetravalent immunisation protocol in mice showed induction of neutralising antibodies against all four dengue serotypes as well. PMID:26218926

  3. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    SciTech Connect

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  4. Translation Inhibition of Capped and Uncapped Viral RNAs Mediated by Ribosome-Inactivating Proteins.

    PubMed

    Vivanco, Jorge M; Tumer, Nilgun E

    2003-05-01

    ABSTRACT Ribosome-inactivating proteins (RIPs) are N-glycosidases that remove specific purine residues from the sarcin/ricin (S/R) loop of the large rRNA and arrest protein synthesis at the translocation step. In addition to their enzymatic activity, RIPs have been reputed to be potent antiviral agents against many plant, animal, and human viruses. We recently showed that pokeweed antiviral protein (PAP), an RIP from pokeweed, inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNAs and depurinating these RNAs at multiple sites downstream of the cap structure. In this study, we examined the activity of three different RIPs against capped and uncapped viral RNAs. PAP, Mirabilis expansa RIP (ME1), and the Saponaria officinalis RIP (saporin) depurinated the capped Tobacco mosaic virus and Brome mosaic virus RNAs, but did not depurinate the uncapped luciferase RNA, indicating that other type I RIPs besides PAP can distinguish between capped and uncapped RNAs. We did not detect depurination of Alfalfa mosaic virus (AMV) RNAs at multiple sites by PAP or ME1. Because AMV RNAs are capped, these results indicate that recognition of the cap structure alone is not sufficient for depurination of the RNA at multiple sites throughout its sequence. Furthermore, PAP did not cause detectable depurination of uncapped RNAs from Tomato bushy stunt virus (TBSV), Satellite panicum mosaic virus (SPMV), and uncapped RNA containing poliovirus internal ribosome entry site (IRES). However, in vitro translation experiments showed that PAP inhibited translation of AMV, TBSV, SPMV RNAs, and poliovirus IRES dependent translation. These results demonstrate that PAP does not depurinate every capped RNA and that PAP can inhibit translation of uncapped viral RNAs in vitro without causing detectable depurination at multiple sites. Thus, the cap structure is not the only determinant for inhibition of translation by PAP. PMID:18942981

  5. Singapore grouper iridovirus protein VP088 is essential for viral infectivity

    PubMed Central

    Yuan, Yongming; Wang, Yunzhi; Liu, Qizhi; Zhu, Feng; Hong, Yunhan

    2016-01-01

    Viral infection is a great challenge in healthcare and agriculture. The Singapore grouper iridovirus (SGIV) is highly infectious to numerous marine fishes and increasingly threatens mariculture and wildlife conservation. SGIV intervention is not available because little is known about key players and their precise roles in SGVI infection. Here we report the precise role of VP088 as a key player in SGIV infection. VP088 was verified as an envelope protein encoded by late gene orf088. We show that SGIV could be neutralized with an antibody against VP088. Depletion or deletion of VP088 significantly suppresses SGIV infection without altering viral gene expression and host responses. By precisely quantifying the genome copy numbers of host cells and virions, we reveal that VP088 deletion dramatically reduces SGIV infectivity through inhibiting virus entry without altering viral pathogenicity, genome stability and replication and progeny virus release. These results pinpoint that VP088 is a key player in SGIV entry and represents an ideal target for SGIV intervention. PMID:27498856

  6. Singapore grouper iridovirus protein VP088 is essential for viral infectivity.

    PubMed

    Yuan, Yongming; Wang, Yunzhi; Liu, Qizhi; Zhu, Feng; Hong, Yunhan

    2016-01-01

    Viral infection is a great challenge in healthcare and agriculture. The Singapore grouper iridovirus (SGIV) is highly infectious to numerous marine fishes and increasingly threatens mariculture and wildlife conservation. SGIV intervention is not available because little is known about key players and their precise roles in SGVI infection. Here we report the precise role of VP088 as a key player in SGIV infection. VP088 was verified as an envelope protein encoded by late gene orf088. We show that SGIV could be neutralized with an antibody against VP088. Depletion or deletion of VP088 significantly suppresses SGIV infection without altering viral gene expression and host responses. By precisely quantifying the genome copy numbers of host cells and virions, we reveal that VP088 deletion dramatically reduces SGIV infectivity through inhibiting virus entry without altering viral pathogenicity, genome stability and replication and progeny virus release. These results pinpoint that VP088 is a key player in SGIV entry and represents an ideal target for SGIV intervention. PMID:27498856

  7. [KIL-d] Protein Element Confers Antiviral Activity via Catastrophic Viral Mutagenesis.

    PubMed

    Suzuki, Genjiro; Weissman, Jonathan S; Tanaka, Motomasa

    2015-11-19

    Eukaryotic cells are targeted by pathogenic viruses and have developed cell defense mechanisms against viral infection. In yeast, the cellular extrachromosomal genetic element [KIL-d] alters killer activity of M double-stranded RNA killer virus and confers cell resistance against the killer virus. However, its underlying mechanism and the molecular nature of [KIL-d] are unknown. Here, we demonstrate that [KIL-d] is a proteinaceous prion-like aggregate with non-Mendelian cytoplasmic transmission. Deep sequencing analyses revealed that [KIL-d] selectively increases the rate of de novo mutation in the killer toxin gene of the viral genome, producing yeast harboring a defective mutant killer virus with a selective growth advantage over those with WT killer virus. These results suggest that a prion-like [KIL-d] element reprograms the viral replication machinery to induce mutagenesis and genomic inactivation via the long-hypothesized mechanism of "error catastrophe." The findings also support a role for prion-like protein aggregates in cellular defense and adaptation. PMID:26590718

  8. Flavivirus premembrane protein cleavage and spike heterodimer secretion require the function of the viral proteinase NS3.

    PubMed Central

    Lobigs, M

    1993-01-01

    Flavivirus protein biosynthesis involves the proteolytic processing of a single polyprotein precursor by host- and virus-encoded proteinases. In this study, the requirement for the proteolytic function of the viral proteinase NS3 for correct processing of a polyprotein segment encompassing the Murray Valley encephalitis virus structural proteins is shown. The NS3-mediated cleavage in the structural polyprotein region presumably releases the capsid protein from its membrane anchor and triggers the appearance of the premembrane (prM) protein. This suggests that cleavage of prM by signal peptidase in the lumen of the endoplasmic reticulum is under control of a cytoplasmic cleavage catalyzed by a viral proteinase. The function of the viral proteinase is also essential for secretion of flaviviral spike proteins when expressed from cDNA via vaccinia virus recombinants or in COS cell transfections. This has important implications for the design of flavivirus subunit vaccines. Images Fig. 1 Fig. 2 Fig. 3 PMID:8392191

  9. 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

  10. Protein kinase R reveals an evolutionary model for defeating viral mimicry

    PubMed Central

    Elde, Nels C.; Child, Stephanie J.; Geballe, Adam P.; Malik, Harmit S.

    2008-01-01

    Distinguishing self from non-self is a fundamental biological challenge. Many pathogens exploit the challenge of self discrimination by employing mimicry to subvert key cellular processes including the cell cycle, apoptosis, and cytoskeletal dynamics1-5. Other mimics interfere with immunity6, 7. Poxviruses encode K3L, a mimic of eIF2α, which is the substrate of Protein Kinase R (PKR), an important component of innate immunity in vertebrates8, 9. The PKR-K3L interaction exemplifies the conundrum imposed by viral mimicry. To be effective, PKR must recognize a conserved substrate (eIF2α) while avoiding rapidly evolving substrate mimics like K3L. Using the PKR-K3L system and a combination of phylogenetic and functional analyses, we uncover evolutionary strategies by which host proteins can overcome mimicry. We find that PKR has evolved under dramatic episodes of positive selection in primates. The ability of PKR to evade viral mimics is partly due to positive selection at sites most intimately involved in eIF2α recognition. We also find that adaptive changes on multiple surfaces of PKR produce combinations of substitutions that increase the odds of defeating mimicry. Thus, while it can appear that pathogens gain insurmountable advantages by mimicking cellular components, host factors like PKR can compete in molecular ‘arms races’ with mimics because of remarkable evolutionary flexibility at protein interaction interfaces challenged by mimicry. PMID:19043403

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

    PubMed Central

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

    2015-01-01

    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

  12. Importance of SARS-CoV spike protein Trp-rich region in viral infectivity

    SciTech Connect

    Lu Yanning; Neo, T.L.; Liu, D.Xi.; Tam, James P.

    2008-07-04

    SARS-CoV entry is mediated by spike glycoprotein. During the viral and host cellular membrane fusion, HR1 and HR2 form 6-helix bundle, positioning the fusion peptide closely to the C-terminal region of ectodomain to drive apposition and subsequent membrane fusion. Connecting to the HR2 region is a Trp-rich region which is absolutely conserved in members of coronaviruses. To investigate the importance of Trp-rich region in SARS-CoV entry, we produced different mutated S proteins using Alanine scan strategy. SARS-CoV pseudotyped with mutated S protein was used to measure viral infectivity. To restore the aromaticity of Ala-mutants, we performed rescue experiments using phenylalanine substitutions. Our results show that individually substituted Ala-mutants substantially decrease infectivity by >90%, global Ala-mutants totally abrogated infectivity. In contrast, Phe-substituted mutants are able to restore 10-25% infectivity comparing to the wild-type. The results suggest that the Trp-rich region of S protein is essential for SARS-CoV infectivity.

  13. Cellular transcription factor Oct-1 interacts with the Epstein-Barr virus BRLF1 protein to promote disruption of viral latency.

    PubMed

    Robinson, Amanda R; Kwek, Swee Sen; Hagemeier, Stacy R; Wille, Coral K; Kenney, Shannon C

    2011-09-01

    The Epstein-Barr virus (EBV) latent-to-lytic switch is an essential part of the viral life cycle, but the cellular factors that promote viral reactivation are not well defined. In this report, we demonstrate that the cellular transcription factor Oct-1 cooperates with the EBV immediate-early protein BRLF1 (R, Rta) to induce lytic viral reactivation. We show that cotransfected Oct-1 enhances the ability of BRLF1 to activate lytic gene expression in 293 cells stably infected with a BRLF1-defective EBV mutant (BRLF1-stop) and that Oct-1 increases BRLF1-mediated activation of lytic EBV promoters in reporter gene assays. We find that Oct-1 interacts directly with BRLF1 in vitro and that a mutant BRLF1 protein (the M140A mutant) attenuated for the ability to interact with Oct-1 in vitro is also resistant to Oct-1-mediated transcriptional enhancement in 293 BRLF1-stop cells. Furthermore, we show that cotransfected Oct-1 augments BRLF1 binding to a variety of lytic EBV promoters in chromatin immunoprecipitation (ChIP) assays (including the BZLF1, BMRF1, and SM promoters) and that BRLF1 tethers Oct-1 to lytic EBV promoters. In addition, we demonstrate that an Oct-1 mutant defective in DNA binding (the S335D mutant) still retains the ability to enhance BRLF1 transcriptional effects. Finally, we show that knockdown of endogenous Oct-1 expression reduces the level of constitutive lytic EBV gene expression in both EBV-positive B-cell and EBV-positive epithelial cell lines. These results suggest that Oct-1 acts as a positive regulator of EBV lytic gene expression and that this effect is at least partially mediated through its interaction with the viral protein BRLF1. PMID:21697476

  14. Expanding the proteome of an RNA virus by phosphorylation of an intrinsically disordered viral protein.

    PubMed

    Cordek, Daniel G; Croom-Perez, Tayler J; Hwang, Jungwook; Hargittai, Michele R S; Subba-Reddy, Chennareddy V; Han, Qingxia; Lodeiro, Maria Fernanda; Ning, Gang; McCrory, Thomas S; Arnold, Jamie J; Koc, Hasan; Lindenbach, Brett D; Showalter, Scott A; Cameron, Craig E

    2014-08-29

    The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using (15)N-, (13)C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome. PMID:25031324

  15. Expression of Multiple Functional RNAs or Proteins from One Viral Vector.

    PubMed

    Björklund, Tomas

    2016-01-01

    In this chapter, we will cover the available design choices for enabling expression of two functional protein or RNA sequences from a single viral vector. Such vectors are very useful in the neuroscience-related field of neuronal control and modulation, e.g., using optogenetics or DREADDs, but are also desirable in applications of CRISPR/Cas9 in situ genome editing and more refined therapeutic approaches. Each approach to achieving this combined expression has its own strengths and limitations, which makes them more or less suitable for different applications. In this chapter, we describe the available alternatives and provide tips on how they can be implemented. PMID:26611577

  16. 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

  17. p32 Is a Novel Target for Viral Protein ICP34.5 of Herpes Simplex Virus Type 1 and Facilitates Viral Nuclear Egress*

    PubMed Central

    Wang, Yu; Yang, Yin; Wu, Songfang; Pan, Shuang; Zhou, Chaodong; Ma, Yijie; Ru, Yongxin; Dong, Shuxu; He, Bin; Zhang, Cuizhu; Cao, Youjia

    2014-01-01

    As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles. PMID:25355318

  18. Gene Expression Driven by a Strong Viral Promoter in MVA Increases Vaccination Efficiency by Enhancing Antibody Responses and Unmasking CD8+ T Cell Epitope

    PubMed Central

    Becker, Pablo D.; Nörder, Miriam; Weissmann, Sebastian; Ljapoci, Ronny; Erfle, Volker; Drexler, Ingo; Guzmán, Carlos A.

    2014-01-01

    Viral vectors are promising tools for vaccination strategies and immunotherapies. However, CD8+ T cell responses against pathogen-derived epitopes are usually limited to dominant epitopes and antibody responses to recombinant encoded antigens (Ags) are mostly weak. We have previously demonstrated that the timing of viral Ag expression in infected professional Ag-presenting cells strongly shapes the epitope immunodominance hierarchy. T cells recognizing determinants derived from late viral proteins have a clear disadvantage to proliferate during secondary responses. In this work we evaluate the effect of overexpressing the recombinant Ag using the modified vaccinia virus early/late promoter H5 (mPH5). Although the Ag-expression from the natural promoter 7.5 (P7.5) and the mPH5 seemed similar, detailed analysis showed that mPH5 not only induces higher expression levels than P7.5 during early phase of infection, but also Ag turnover is enhanced. The strong overexpression during the early phase leads to broader CD8 T cell responses, while preserving the priming efficiency of stable Ags. Moreover, the increase in Ag-secretion favors the induction of strong antibody responses. Our findings provide the rationale to develop new strategies for fine-tuning the responses elicited by recombinant modified vaccinia virus Ankara by using selected promoters to improve the performance of this viral vector. PMID:26344747

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  20. Depressed chemiluminescence response by influenza virus is enhanced after conjugation of viral subunits to muramyl dipeptide.

    PubMed Central

    Masihi, K N; Lange, W; Rohde-Schulz, B; Chedid, L; Jolivet, M

    1985-01-01

    The effect on respiratory burst of murine spleen cells after in vitro exposure to influenza virus, subunits, or subunits conjugated to muramyl dipeptide (MDP) was studied by luminol-dependent chemiluminescence (CL) in response to stimulation by zymosan. CL induced by infectious influenza A virus was depressed but could be elevated to normal levels when MDP was added together with a low, but not with a high, dose of the virus. Profound depression of CL was induced by high doses of influenza A/Brazil, A/Bangkok, and B/Singapore subunits. The same amounts of viral subunits conjugated to MDP restored or even enhanced the CL responses of spleen cells from BALB/c and C57BL/6 mice. Splenic cells from BALB/c mice generated higher levels of CL than did cells from C57BL/6 mice. PMID:4044031

  1. Arterivirus Minor Envelope Proteins Are a Major Determinant of Viral Tropism in Cell Culture

    PubMed Central

    Tian, Debin; Wei, Zuzhang; Zevenhoven-Dobbe, Jessika C.; Liu, Runxia; Tong, Guangzhi

    2012-01-01

    Arteriviruses are enveloped positive-strand RNA viruses for which the attachment proteins and cellular receptors have remained largely controversial. Arterivirus particles contain at least eight envelope proteins, an unusually large number among RNA viruses. These appear to segregate into three groups: major structural components (major glycoprotein GP5 and membrane protein [M]), minor glycoproteins (GP2a, GP3, and GP4), and small hydrophobic proteins (E and the recently discovered ORF5a protein). Biochemical studies previously suggested that the GP5-M heterodimer of porcine reproductive and respiratory syndrome virus (PRRSV) interacts with porcine sialoadhesin (pSn) in porcine alveolar macrophages (PAM). However, another study proposed that minor protein GP4, along with GP2a, interacts with CD163, another reported cellular receptor for PRRSV. In this study, we provide genetic evidence that the minor envelope proteins are the major determinant of arterivirus entry into cultured cells. A PRRSV infectious cDNA clone was equipped with open reading frames (ORFs) encoding minor envelope and E proteins of equine arteritis virus (EAV), the only known arterivirus displaying a broad tropism in cultured cells. Although PRRSV and EAV are only distantly related and utilize diversified transcription-regulating sequences (TRSs), a viable chimeric progeny virus was rescued. Strikingly, this chimeric virus (vAPRRS-EAV2ab34) acquired the broad in vitro cell tropism of EAV, demonstrating that the minor envelope proteins play a critical role as viral attachment proteins. We believe that chimeric arteriviruses of this kind will be a powerful tool for further dissection of the arterivirus replicative cycle, including virus entry, subgenomic RNA synthesis, and virion assembly. PMID:22258262

  2. N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression.

    PubMed

    Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

    2016-01-01

    The internal N(6)-methyladenosine (m(6)A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m(6)A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1-3) bind to m(6)A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1-3 proteins recognize m(6)A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4(+) T-cells. We further mapped the YTHDF1-3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1-3 in cells had the opposite effects. Moreover, silencing the m(6)A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m(6)A erasers increased Gag expression. Our findings suggest an important role of m(6)A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. PMID:27371828

  3. N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    PubMed Central

    Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

    2016-01-01

    The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. DOI: http://dx.doi.org/10.7554/eLife.15528.001 PMID:27371828

  4. Viral Interactions with PDZ Domain-Containing Proteins-An Oncogenic Trait?

    PubMed

    James, Claire D; Roberts, Sally

    2016-01-01

    Many of the human viruses with oncogenic capabilities, either in their natural host or in experimental systems (hepatitis B and C, human T cell leukaemia virus type 1, Kaposi sarcoma herpesvirus, human immunodeficiency virus, high-risk human papillomaviruses and adenovirus type 9), encode in their limited genome the ability to target cellular proteins containing PSD95/ DLG/ZO-1 (PDZ) interaction modules. In many cases (but not always), the viruses have evolved to bind the PDZ domains using the same short linear peptide motifs found in host protein-PDZ interactions, and in some cases regulate the interactions in a similar fashion by phosphorylation. What is striking is that the diverse viruses target a common subset of PDZ proteins that are intimately involved in controlling cell polarity and the structure and function of intercellular junctions, including tight junctions. Cell polarity is fundamental to the control of cell proliferation and cell survival and disruption of polarity and the signal transduction pathways involved is a key event in tumourigenesis. This review focuses on the oncogenic viruses and the role of targeting PDZ proteins in the virus life cycle and the contribution of virus-PDZ protein interactions to virus-mediated oncogenesis. We highlight how many of the viral associations with PDZ proteins lead to deregulation of PI3K/AKT signalling, benefitting virus replication but as a consequence also contributing to oncogenesis. PMID:26797638

  5. Visualizing Viral Dissemination in the Mouse Nervous System, Using a Green Fluorescent Protein-Expressing Borna Disease Virus Vector▿

    PubMed Central

    Ackermann, Andreas; Guelzow, Timo; Staeheli, Peter; Schneider, Urs; Heimrich, Bernd

    2010-01-01

    Borna disease virus (BDV) frequently persists in the brain of infected animals. To analyze viral dissemination in the mouse nervous system, we generated a mouse-adapted virus that expresses green fluorescent protein (GFP). This viral vector supported GFP expression for up to 150 days and possessed an extraordinary staining capacity, visualizing complete dendritic arbors as well as individual axonal fibers of infected neurons. GFP-positive cells were first detected in cortical areas from where the virus disseminated through the entire central nervous system (CNS). Late in infection, GFP expression was found in the sciatic nerve, demonstrating viral spread from the central to the peripheral nervous system. PMID:20219925

  6. Quassinoids: Viral protein R inhibitors from Picrasma javanica bark collected in Myanmar for HIV infection.

    PubMed

    Win, Nwet Nwet; Ito, Takuya; Win, Yi Yi; Ngwe, Hla; Kodama, Takeshi; Abe, Ikuro; Morita, Hiroyuki

    2016-10-01

    Viral protein R (Vpr) is an accessory protein that plays important roles in the viral pathogenesis of Human Immunodeficiency Virus-1 (HIV-1). An assay for anti-Vpr activity, using TREx-HeLa-Vpr cells, is a promising strategy to discover Vpr inhibitors. The anti-Vpr assay revealed that the CHCl3-soluble extract of Picrasma javanica bark possesses potent anti-Vpr activity. Furthermore, studies of quassinoids (1-15) previously isolated from the extract demonstrated that all of the tested quassinoids exhibit anti-Vpr activity. Among the tested compounds, javanicin I (15) exhibited the most potent anti-Vpr activity ((***)p <0.001) in comparing with that of the positive control, damnacanthal. The structure-activity relationships of the active quassinoids suggested that the presence of a methyl group at C-13 in the 2,12,14-triene-1,11,16-trione-2,12-dimethoxy-18-norpicrasane quassinoids is the important factor for the potent inhibitory effect in TREx-HeLa-Vpr cells. PMID:27575477

  7. Evidence of apoptosis induced by viral protein 2 of chicken anaemia virus.

    PubMed

    Kaffashi, Amir; Pagel, Charles N; Noormohammadi, Amir H; Browning, Glenn F

    2015-10-01

    Although viral protein 3 (VP3) of chicken anaemia virus (CAV) has been well recognised as an inducer of apoptosis, viral protein 2 (VP2) of the virus has only been speculated to have apoptotic activity. This has not been verified because the open reading frame (ORF) encoding VP2 completely encompasses that encoding VP3, and thus the possibility of expression of VP3 cannot be excluded. The aim of this study was to elucidate the potential role of VP2 as an inducer of apoptosis. Site-directed mutagenesis was used to generate a point mutation that knocked out VP3 by early termination of its translation with a stop codon without imposing any change in the amino acid sequence of VP2. The mutated sequence was inserted into the pCAT plasmid preceded by a favorable Kozak's consensus sequence to create pCAT-VP2(+)VP3(-). The absence of VP3 expression in MSB1 cells transfected with this plasmid was confirmed using Western blotting, and DNA strand breaks and nuclear morphological changes were assessed to detect apoptosis. There was an increased level of apoptotic death in cells transfected with pCAT-VP2(+)VP3(-) compared to those transfected with the vector alone. This provides evidence that CAV VP2 can induce apoptosis. PMID:26233670

  8. The Varicella-Zoster Virus Portal Protein Is Essential for Cleavage and Packaging of Viral DNA

    PubMed Central

    Visalli, Melissa A.; House, Brittany L.; Selariu, Anca; Zhu, Hua

    2014-01-01

    ABSTRACT The varicella-zoster virus (VZV) open reading frame 54 (ORF54) gene encodes an 87-kDa monomer that oligomerizes to form the VZV portal protein, pORF54. pORF54 was hypothesized to perform a function similar to that of a previously described herpes simplex virus 1 (HSV-1) homolog, pUL6. pUL6 and the associated viral terminase are required for processing of concatemeric viral DNA and packaging of individual viral genomes into preformed capsids. In this report, we describe two VZV bacterial artificial chromosome (BAC) constructs with ORF54 gene deletions, Δ54L (full ORF deletion) and Δ54S (partial internal deletion). The full deletion of ORF54 likely disrupted essential adjacent genes (ORF53 and ORF55) and therefore could not be complemented on an ORF54-expressing cell line (ARPE54). In contrast, Δ54S was successfully propagated in ARPE54 cells but failed to replicate in parental, noncomplementing ARPE19 cells. Transmission electron microscopy confirmed the presence of only empty VZV capsids in Δ54S-infected ARPE19 cell nuclei. Similar to the HSV-1 genome, the VZV genome is composed of a unique long region (UL) and a unique short region (US) flanked by inverted repeats. DNA from cells infected with parental VZV (VZVLUC strain) contained the predicted UL and US termini, whereas cells infected with Δ54S contained neither. This result demonstrates that Δ54S is not able to process and package viral DNA, thus making pORF54 an excellent chemotherapeutic target. In addition, the utility of BAC constructs Δ54L and Δ54S as tools for the isolation of site-directed ORF54 mutants was demonstrated by recombineering single-nucleotide changes within ORF54 that conferred resistance to VZV-specific portal protein inhibitors. IMPORTANCE Antivirals with novel mechanisms of action would provide additional therapeutic options to treat human herpesvirus infections. Proteins involved in the herpesviral DNA encapsidation process have become promising antiviral targets

  9. Hepatitis C Virus NS2 Protein Triggers Endoplasmic Reticulum Stress and Suppresses its Own Viral Replication

    PubMed Central

    von dem Bussche, Annette; Machida, Raiki; Li, Ke; Loevinsohn, Gideon; Khander, Amrin; Wang, Jianguo; Wakita, Takaji; Wands, Jack R.; Li, Jisu

    2010-01-01

    Background & Aims We previously reported that the NS2 protein of hepatitis C virus (HCV) inhibits the expression of reporter genes driven by a variety of cellular and viral promoters. The aim of the study was to determine whether the broad transcriptional repression is caused by endoplasmic reticulum (ER) stress. Methods Phosphorylation of the translation initiation factor eIF2α and HCV replication were detected by Western and Northern blot, respectively. De novo protein synthesis was measured by metabolic labeling. Activation of ER stress responsive genes was determined by promoter reporter assay, as well as mRNA and protein measurement by real time PCR and Western blot. Results Transient or inducible NS2 protein expression increased eIF2α phosphorylation and reduced de novo protein synthesis. It up-regulated promoter activities and transcript levels of ER stress inducible genes including GRP78, ATF6, and GADD153, as well as GRP78 protein level. The same effect was observed when NS2 was synthesized as part of the core-E1-E2-p7-NS2 polypeptide. NS2 protein also inhibited reporter gene expression from the HCV internal ribosome entry site and consequently reduced HCV replication. The full-length HCV replicon activated GRP78, ATF6, and GADD153 promoters more efficiently than the subgenomic replicon lacking the coding sequence for both the structural proteins and NS2. Abrogation of HCV infection/replication, by an inhibitor of the NS3 protease, relieved ER stress. Conclusions HCV infection can induce ER stress, with NS2 protein being a major mediator. The stress can be relieved by a feedback mechanism. PMID:20801537

  10. Two Plant–Viral Movement Proteins Traffic in the Endocytic Recycling PathwayW⃞

    PubMed Central

    Haupt, Sophie; Cowan, Graham H.; Ziegler, Angelika; Roberts, Alison G.; Oparka, Karl J.; Torrance, Lesley

    2005-01-01

    Many plant viruses exploit a conserved group of proteins known as the triple gene block (TGB) for cell-to-cell movement. Here, we investigated the interaction of two TGB proteins (TGB2 and TGB3) of Potato mop-top virus (PMTV), with components of the secretory and endocytic pathways when expressed as N-terminal fusions to green fluorescent protein or monomeric red fluorescent protein (mRFP). Our studies revealed that fluorophore-labeled TGB2 and TGB3 showed an early association with the endoplasmic reticulum (ER) and colocalized in motile granules that used the ER-actin network for intracellular movement. Both proteins increased the size exclusion limit of plasmodesmata, and TGB3 accumulated at plasmodesmata in the absence of TGB2. TGB3 contains a putative Tyr-based sorting motif, mutations in which abolished ER localization and plasmodesmatal targeting. Later in the expression cycle, both fusion proteins were incorporated into vesicular structures. TGB2 associated with these structures on its own, but TGB3 could not be incorporated into the vesicles in the absence of TGB2. Moreover, in addition to localization to the ER and motile granules, mRFP-TGB3 was incorporated into vesicles when expressed in PMTV-infected epidermal cells, indicating recruitment by virus-expressed TGB2. The TGB fusion protein-containing vesicles were labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway. TGB2 also colocalized in vesicles with Ara7, a Rab5 ortholog that marks the early endosome. Protein interaction analysis revealed that recombinant TGB2 interacted with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, shown to be essential for endocytic trafficking in Caenorhabditis elegans and Drosophila melanogaster. Collectively, the data indicate the involvement of the endocytic pathway in viral intracellular movement, the implications of which are discussed. PMID:15608333

  11. The potato mop-top virus TGB2 protein and viral RNA associate with chloroplasts and viral infection induces inclusions in the plastids

    PubMed Central

    Cowan, Graham H.; Roberts, Alison G.; Chapman, Sean N.; Ziegler, Angelika; Savenkov, Eugene I.; Torrance, Lesley

    2012-01-01

    The potato mop-top virus (PMTV) triple gene block 2 (TGB2) movement proteins fused to monomeric red fluorescent protein (mRFP-TGB2) was expressed under the control of the PMTV subgenomic promoter from a PMTV vector. The subcellular localizations and interactions of mRFP-TGB2 were investigated using confocal imaging [confocal laser-scanning microscope, (CLSM)] and biochemical analysis. The results revealed associations with membranes of the endoplasmic reticulum (ER), mobile granules, small round structures (1–2 μm in diameter), and chloroplasts. Expression of mRFP-TGB2 in epidermal cells enabled cell-to-cell movement of a TGB2 defective PMTV reporter clone, indicating that the mRFP-TGB2 fusion protein was functional and required for cell-to-cell movement. Protein-lipid interaction assays revealed an association between TGB2 and lipids present in chloroplasts, consistent with microscopical observations where the plastid envelope was labeled later in infection. To further investigate the association of PMTV infection with chloroplasts, ultrastructural studies of thin sections of PMTV-infected potato and Nicotiana benthamiana leaves by electron microscopy revealed abnormal chloroplasts with cytoplasmic inclusions and terminal projections. Viral coat protein (CP), genomic RNA and fluorescently-labeled TGB2 were detected in plastid preparations isolated from the infected leaves, and viral RNA was localized to chloroplasts in infected tissues. The results reveal a novel association of TGB2 and vRNA with chloroplasts, and suggest viral replication is associated with chloroplast membranes, and that TGB2 plays a novel role in targeting the virus to chloroplasts. PMID:23269927

  12. Identification of the gC1qR sites for the HIV-1 viral envelope protein gp41 and the HCV core protein: Implications in viral-specific pathogenesis and therapy

    PubMed Central

    Pednekar, Lina; Valentino, Alisa; Ji, Yan; Tumma, Nithin; Valentino, Christopher; Kadoor, Adarsh; Hosszu, Kinga K.; Ramadass, Mahalakshmi; Kew, Richard R.; Kishore, Uday; Peerschke, Ellinor I.B.; Ghebrehiwet, Berhane

    2016-01-01

    A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogen-associated pattern recognition receptor (PRR). This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host’s immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4+ T cells, thereby rendering them susceptible to autologous destruction by NKp44 receptor expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 11 gC1qR deletion mutants. The results obtained from these studies have identified two major HCV core protein sites on a domain of gC1qR comprising of residues 144–148 and 196–202. Domain 196–202 in turn, is located in the last half of the larger gC1qR segment encoded by exons IV–VI (residues 159–282), which was proposed previously to contain the site for HCV core protein. The major gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174–180. Interestingly, gC1qR residues 174–180 also constitute the cell surface-binding site for soluble

  13. Identification of the gC1qR sites for the HIV-1 viral envelope protein gp41 and the HCV core protein: Implications in viral-specific pathogenesis and therapy.

    PubMed

    Pednekar, Lina; Valentino, Alisa; Ji, Yan; Tumma, Nithin; Valentino, Christopher; Kadoor, Adarsh; Hosszu, Kinga K; Ramadass, Mahalakshmi; Kew, Richard R; Kishore, Uday; Peerschke, Ellinor I B; Ghebrehiwet, Berhane

    2016-06-01

    A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogen-associated pattern recognition receptor (PRR). This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host's immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4(+) T cells, thereby rendering them susceptible to autologous destruction by NKp44 receptor expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 11 gC1qR deletion mutants. The results obtained from these studies have identified two major HCV core protein sites on a domain of gC1qR comprising of residues 144-148 and 196-202. Domain 196-202 in turn, is located in the last half of the larger gC1qR segment encoded by exons IV-VI (residues 159-282), which was proposed previously to contain the site for HCV core protein. The major gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174-180. Interestingly, gC1qR residues 174-180 also constitute the cell surface-binding site for soluble gC1qR (sgC1q

  14. Enhanced protein domain discovery using taxonomy

    PubMed Central

    Coin, Lachlan; Bateman, Alex; Durbin, Richard

    2004-01-01

    Background It is well known that different species have different protein domain repertoires, and indeed that some protein domains are kingdom specific. This information has not yet been incorporated into statistical methods for finding domains in sequences of amino acids. Results We show that by incorporating our understanding of the taxonomic distribution of specific protein domains, we can enhance domain recognition in protein sequences. We identify 4447 new instances of Pfam domains in the SP-TREMBL database using this technique, equivalent to the coverage increase given by the last 8.3% of Pfam families and to a 0.7% increase in the number of domain predictions. We use PSI-BLAST to cross-validate our new predictions. We also benchmark our approach using a SCOP test set of proteins of known structure, and demonstrate improvements relative to standard Hidden Markov model techniques. Conclusions Explicitly including knowledge about the taxonomic distribution of protein domains can enhance protein domain recognition. Our method can also incorporate other context-specific domain distributions – such as domain co-occurrence and protein localisation. PMID:15137915

  15. 1918 Influenza virus hemagglutinin (HA) and the viral RNA polymerase complex enhance viral pathogenicity, but only HA induces aberrant host responses in mice.

    PubMed

    Watanabe, Tokiko; Tisoncik-Go, Jennifer; Tchitchek, Nicolas; Watanabe, Shinji; Benecke, Arndt G; Katze, Michael G; Kawaoka, Yoshihiro

    2013-05-01

    The 1918 pandemic influenza virus was the most devastating infectious agent in human history, causing fatal pneumonia and an estimated 20 to 50 million deaths worldwide. Previous studies indicated a prominent role of the hemagglutinin (HA) gene in efficient replication and high virulence of the 1918 virus in mice. It is, however, still unclear whether the high replication ability or the 1918 influenza virus HA gene is required for 1918 virus to exhibit high virulence in mice. Here, we examined the biological properties of reassortant viruses between the 1918 virus and a contemporary human H1N1 virus (A/Kawasaki/173/2001 [K173]) in a mouse model. In addition to the 1918 influenza virus HA, we demonstrated the role of the viral RNA replication complex in efficient replication of viruses in mouse lungs, whereas only the HA gene is responsible for lethality in mice. Global gene expression profiling of infected mouse lungs revealed that the 1918 influenza virus HA was sufficient to induce transcriptional changes similar to those induced by the 1918 virus, despite difference in lymphocyte gene expression. Increased expression of genes associated with the acute-phase response and the protein ubiquitination pathway were enriched during infections with the 1918 and 1918HA/K173 viruses, whereas reassortant viruses bearing the 1918 viral RNA polymerase complex induced transcriptional changes similar to those seen with the K173 virus. Taken together, these data suggest that HA and the viral RNA polymerase complex are critical determinants of Spanish influenza pathogenesis, but only HA, and not the viral RNA polymerase complex and NP, is responsible for extreme host responses observed in mice infected with the 1918 influenza virus. PMID:23449804

  16. Mycobacterium avium infection in HIV-1-infected subjects increases monokine secretion and is associated with enhanced viral load and diminished immune response to viral antigens.

    PubMed Central

    Denis, M; Ghadirian, E

    1994-01-01

    The complex interaction between HIV-1 infection and Mycobacterium avium was studied. Viral burden was assessed, as well as immune response to HIV-1 in the context of Myco. avium infections. We also examined serum cytokine levels and cytokine release by blood mononuclear cells in HIV-1-infected subjects, infected or not with Myco. avium. Undetectable serum levels of IL-1, tumour necrosis factor-alpha (TNF-alpha) and IL-6 were found in normal controls and in groups I, II and III of HIV-1-infected subjects. Moderate levels of TNF-alpha, IL-1 and IL-6 were found in the sera of group IV patients. When group IV was subdivided into subjects with and without Myco. avium infections, subjects with Myco, avium infections were shown to have higher serum levels of TNF-alpha, IL-1 beta and IL-6 than those with other infections. Blood mononuclear cells from controls and HIV subjects were stimulated with bacterial lipopolysaccharide, and cytokine levels assessed. Cells from group II patients were shown to secrete normal levels of TNF-alpha and IL-6, and lower levels of IL-1 beta; group III subjects released higher levels of IL-6. Patients in group IV had blood cells that released elevated levels of IL-6 and TNF-alpha, and lower levels of IL-1 beta. Group IV subjects with Myco. avium infections had blood cells that released higher levels of TNF-alpha, IL-6 and IL-1 than group IV subjects with other infections. Assessment of viral burden in cells of HIV-1-infected subjects revealed that Myco. avium-infected subjects had a higher level of virus burden and a lower level of lymphoproliferative response to an inactivated gp120-depleted HIV-1 antigen than AIDS subjects with other infections. These data suggest that Myco. avium infections in HIV-1-infected subjects hasten the progression of viral disease, enhance cytokine release and contribute to the anergy to viral antigens. PMID:8033423

  17. Small glutamine-rich protein/viral protein U–binding protein is a novel cochaperone that affects heat shock protein 70 activity

    PubMed Central

    Angeletti, Peter C.; Walker, Doriann; Panganiban, Antonito T.

    2002-01-01

    Molecular chaperone complexes containing heat shock protein (Hsp) 70 and Hsp90 are regulated by cochaperones, including a subclass of regulators, such as Hsp70 interacting protein (Hip), C-terminus of Hsp70 interacting protein (CHIP), and Hsp70-Hsp90 organizing factor (Hop), that contain tetratricopeptide repeats (TPRs), where Hsp70 refers to Hsp70 and its nearly identical constitutive counterpart, Hsc70, together. These proteins interact with the Hsp70 to regulate adenosine triphosphatase (ATPase) and folding activities or to generate the chaperone complex. Here we provide evidence that small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) is a cochaperone that negatively regulates Hsp70. By “Far-Western” and pull-down assays, SGT/UBP was shown to interact directly with Hsp70 and weakly with Hsp90. The interaction of SGT/UBP with both these protein chaperones was mapped to 3 TPRs in SGT/UBP (amino acids 95–195) that are flanked by charged residues. Moreover, SGT/UBP caused an approximately 30% reduction in both the intrinsic ATPase activity of Hsc70 and the ability of Hsc70 to refold denatured luciferase in vitro. This negative effect of SGT/UBP on Hsc70 is similar in magnitude to that observed for the cochaperone CHIP. A role for SGT/UBP in protein folding is also supported by evidence that a yeast strain containing a deletion in the yeast homolog to SGT/UBP (ΔSGT/UBP) displays a 50-fold reduction in recovery from heat shock compared with the wild type parent. Together, these results are consistent with a regulatory role for SGT/UBP in the chaperone complex. PMID:12482202

  18. Bacterial expression of antigenic sites A and D in the spike protein of transmissible gastroenteritis virus and evaluation of their inhibitory effects on viral infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The spike (S) protein is a key structural protein of coronaviruses including, the porcine transmissible gastroenteritis virus (TGEV). The S protein is a type I membrane glycoprotein located in the viral envelope and is responsible for mediating the binding of viral particles to specific cell recepto...

  19. Residue 82 of the Chikungunya Virus E2 Attachment Protein Modulates Viral Dissemination and Arthritis in Mice

    PubMed Central

    Ashbrook, Alison W.; Burrack, Kristina S.; Silva, Laurie A.; Montgomery, Stephanie A.; Heise, Mark T.; Morrison, Thomas E.

    2014-01-01

    ABSTRACT Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that has reemerged to cause profound epidemics of fever, rash, and arthralgia throughout sub-Saharan Africa, Southeast Asia, and the Caribbean. Like other arthritogenic alphaviruses, mechanisms of CHIKV pathogenesis are not well defined. Using the attenuated CHIKV strain 181/25 and virulent strain AF15561, we identified a residue in the E2 viral attachment protein that is a critical determinant of viral replication in cultured cells and pathogenesis in vivo. Viruses containing an arginine at E2 residue 82 displayed enhanced infectivity in mammalian cells but reduced infectivity in mosquito cells and diminished virulence in a mouse model of CHIKV disease. Mice inoculated with virus containing an arginine at this position exhibited reduced swelling at the site of inoculation with a concomitant decrease in the severity of necrosis in joint-associated tissues. Viruses containing a glycine at E2 residue 82 produced higher titers in the spleen and serum at early times postinfection. Using wild-type and glycosaminoglycan (GAG)-deficient Chinese hamster ovary (CHO) cell lines and soluble GAGs, we found that an arginine at residue 82 conferred greater dependence on GAGs for infection of mammalian cells. These data suggest that CHIKV E2 interactions with GAGs diminish dissemination to lymphoid tissue, establishment of viremia, and activation of inflammatory responses early in infection. Collectively, these results suggest a function for GAG utilization in regulating CHIKV tropism and host responses that contribute to arthritis. IMPORTANCE CHIKV is a reemerging alphavirus of global significance with high potential to spread into new, immunologically naive populations. The severity of CHIKV disease, particularly its propensity for chronic musculoskeletal manifestations, emphasizes the need for identification of genetic determinants that dictate CHIKV virulence in the host. To better understand mechanisms of

  20. Far upstream element binding protein 2 interacts with enterovirus 71 internal ribosomal entry site and negatively regulates viral translation

    PubMed Central

    Lin, Jing-Yi; Li, Mei-Ling; Shih, Shin-Ru

    2009-01-01

    An internal ribosomal entry site (IRES) that directs the initiation of viral protein translation is a potential drug target for enterovirus 71 (EV71). Regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the internal ribosomal entry site. Biotinylated RNA-affinity chromatography and proteomic approaches were employed to identify far upstream element (FUSE) binding protein 2 (FBP2) as an ITAF for EV71. The interactions of FBP2 with EV71 IRES were confirmed by competition assay and by mapping the association sites in both viral IRES and FBP2 protein. During EV71 infection, FBP2 was enriched in cytoplasm where viral replication occurs, whereas FBP2 was localized in the nucleus in mock-infected cells. The synthesis of viral proteins increased in FBP2-knockdown cells that were infected by EV71. IRES activity in FBP2-knockdown cells exceeded that in the negative control (NC) siRNA-treated cells. On the other hand, IRES activity decreased when FBP2 was over-expressed in the cells. Results of this study suggest that FBP2 is a novel ITAF that interacts with EV71 IRES and negatively regulates viral translation. PMID:19010963

  1. 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

  2. Single-site cleavage in the 5'-untranslated region of Leishmaniavirus RNA is mediated by the viral capsid protein.

    PubMed Central

    MacBeth, K J; Patterson, J L

    1995-01-01

    Leishmaniavirus (LRV) is a double-stranded RNA virus that persistently infects the protozoan parasite Leishmania. LRV produces a short RNA transcript, corresponding to the 5' end of positive-sense viral RNA, both in vivo and in in vitro polymerase assays. The short transcript is generated by a single site-specific cleavage event in the 5' untranslated region of the 5.3-kb genome. This cleavage event can be reproduced in vitro with purified viral particles and a substrate RNA transcript possessing the viral cleavage site. A region of nucleotides required for cleavage was identified by analyzing the cleavage sites yielding the short transcripts of various LRV isolates. A 6-nt deletion at this cleavage site completely abolished RNA processing. In an in vitro cleavage assay, baculovirus-expressed capsid protein possessed an endonuclease activity identical to that of native virions, showing that the viral capsid protein is the RNA endonuclease. Identification of the LRV capsid protein as an RNA endonuclease is unprecedented among known viral capsid proteins. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:7568059

  3. 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

  4. Construction of a mutagenesis cartridge for poliovirus genome-linked viral protein: isolation and characterization of viable and nonviable mutants

    SciTech Connect

    Kuhn, R.J.; Tada, H.; Ypma-Wong, M.F.; Dunn, J.J.; Semler, B.L.; Wimmer, E.

    1988-01-01

    By following a strategy of genetic analysis of poliovirus, the authors have constructed a synthetic mutagenesis cartridge spanning the genome-linked viral protein coding region and flanking cleavage sites in an infectious cDNA clone of the type I (Mahoney) genome. The insertion of new restriction sites within the infectious clone has allowed them to replace the wild-type sequences with short complementary pairs of synthetic oligonucleotides containing various mutations. A set of mutations have been made that create methionine codons within the genome-linked viral protein region. The resulting viruses have growth characteristics similar to wild type. Experiments that led to an alteration of the tyrosine residue responsible for the linkage to RNA have resulted in nonviable virus. In one mutant, proteolytic processing assayed in vitro appeared unimpaired by the mutation. They suggest that the position of the tyrosine residue is important for genome-linked viral protein function(s).

  5. [Increased efficiency of recombinant proteins production in plants due to optimized translation of RNA of viral vector].

    PubMed

    Mardanova, E S; Kotliarov, R Iu; Ravin, N V

    2009-01-01

    One of the most efficient methods for fast and efficient production of the target proteins in plants is based on the use of self-replicating recombinant viral vectors. We constructed phytoviral vector based on the genome of potato X virus containing the sequence of 5'-untranslated region of RNA 4 of alfalfa mosaic virus immediately upstream of the target gene. We demonstrated that incorporation of this sequence into the viral vector results in 3-4 fold elevation of the level of production of the target protein in plant due to increased efficiency of translation of viral subgenomic RNA comprising the target gene. The new vector may be used for production of recombinant proteins in plants. PMID:19548543

  6. Surface-enhanced Raman scattering (SERS) detection of multiple viral antigens using magnetic capture of SERS-active nanoparticles

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A highly sensitive immunoassay based on surface-enhanced Raman scattering (SERS) spectroscopy has been developed for multiplex detection of surface envelope and capsid antigens of the viral zoonotic pathogens West Nile virus (WNV) and Rift Valley fever virus (RVFV). Detection was mediated by antibo...

  7. Subcellular localization of host and viral proteins associated with tobamovirus RNA replication.

    PubMed

    Hagiwara, Yuka; Komoda, Keisuke; Yamanaka, Takuya; Tamai, Atsushi; Meshi, Tetsuo; Funada, Ryo; Tsuchiya, Tomohiro; Naito, Satoshi; Ishikawa, Masayuki

    2003-01-15

    Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus-encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)-like structures in plant cells. In subcellular fractionation analyses, GFP-AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low-density tonoplast-rich fractions, whereas AtTOM1-GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast-rich fractions and partially into higher-buoyant-density fractions containing membranes from several other organelles. The tobamovirus-encoded replication proteins were co-fractionated with both NtTOM1 and viral RNA-dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non-membrane-bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1-containing membranes and is facilitated by TOM2A. PMID:12514140

  8. 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

  9. Conserved residues in Lassa fever virus Z protein modulate viral infectivity at the level of the ribonucleoprotein.

    PubMed

    Capul, Althea A; de la Torre, Juan Carlos; Buchmeier, Michael J

    2011-04-01

    Arenaviruses are negative-strand RNA viruses that cause human diseases such as lymphocytic choriomeningitis, Bolivian hemorrhagic fever, and Lassa hemorrhagic fever. No licensed vaccines exist, and current treatment is limited to ribavirin. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a model for dissecting virus-host interactions in persistent and acute disease. The RING finger protein Z has been identified as the driving force of arenaviral budding and acts as the viral matrix protein. While residues in Z required for viral budding have been described, residues that govern the Z matrix function(s) have yet to be fully elucidated. Because this matrix function is integral to viral assembly, we reasoned that this would be reflected in sequence conservation. Using sequence alignment, we identified several conserved residues in Z outside the RING and late domains. Nine residues were each mutated to alanine in Lassa fever virus Z. All of the mutations affected the expression of an LCMV minigenome and the infectivity of virus-like particles, but to greatly varying degrees. Interestingly, no mutations appeared to affect Z-mediated budding or association with viral GP. Our findings provide direct experimental evidence supporting a role for Z in the modulation of the activity of the viral ribonucleoprotein (RNP) complex and its packaging into mature infectious viral particles. PMID:21228230

  10. Conserved Residues in Lassa Fever Virus Z Protein Modulate Viral Infectivity at the Level of the Ribonucleoprotein▿

    PubMed Central

    Capul, Althea A.; de la Torre, Juan Carlos; Buchmeier, Michael J.

    2011-01-01

    Arenaviruses are negative-strand RNA viruses that cause human diseases such as lymphocytic choriomeningitis, Bolivian hemorrhagic fever, and Lassa hemorrhagic fever. No licensed vaccines exist, and current treatment is limited to ribavirin. The prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), is a model for dissecting virus-host interactions in persistent and acute disease. The RING finger protein Z has been identified as the driving force of arenaviral budding and acts as the viral matrix protein. While residues in Z required for viral budding have been described, residues that govern the Z matrix function(s) have yet to be fully elucidated. Because this matrix function is integral to viral assembly, we reasoned that this would be reflected in sequence conservation. Using sequence alignment, we identified several conserved residues in Z outside the RING and late domains. Nine residues were each mutated to alanine in Lassa fever virus Z. All of the mutations affected the expression of an LCMV minigenome and the infectivity of virus-like particles, but to greatly varying degrees. Interestingly, no mutations appeared to affect Z-mediated budding or association with viral GP. Our findings provide direct experimental evidence supporting a role for Z in the modulation of the activity of the viral ribonucleoprotein (RNP) complex and its packaging into mature infectious viral particles. PMID:21228230