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Sample records for enhances viral protein

  1. VPDB: Viral Protein Structural Database

    PubMed Central

    Sharma, Om Prakash; Jadhav, Ankush; Hussain, Afzal; Kumar, Muthuvel Suresh

    2011-01-01

    Viral Protein Database is an interactive database for three dimensional viral proteins. Our aim is to provide a comprehensive resource to the community of structural virology, with an emphasis on the description of derived data from structural biology. Currently, VPDB includes ˜1,670 viral protein structures from >277 viruses with more than 465 virus strains. The whole database can be easily accessed through the user convenience text search. Interactivity has been enhanced by using Jmol, WebMol and Strap to visualize the viral protein molecular structure. Availability The database is available for free at http://www.vpdb.bicpu.edu.in PMID:21769196

  2. Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity.

    PubMed

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

    2011-11-01

    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.

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

    PubMed Central

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

    2016-01-01

    Summary 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 (JEV) NS1s in the blood of infected interferon alpha and gamma 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

  4. Viruses and viral proteins.

    PubMed

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

    2014-11-01

    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.

  5. 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

  6. Improved silencing suppression and enhanced heterologous protein expression are achieved using an engineered viral helper component proteinase.

    PubMed

    Haikonen, T; Rajamäki, M-L; Valkonen, J P T

    2013-11-01

    RNA silencing limits transient expression of heterologous proteins in plants. Co-expression of viral silencing suppressor proteins can increase and prolong protein expression, but highly efficient silencing suppressors may stress plant tissue and be detrimental to protein yields. Little is known whether silencing suppression could be improved without harm to plant tissues. This study reports development of enhanced silencing suppressors by engineering the helper component proteinase (HCpro) of Potato virus A (PVA). Mutations were introduced to a short region of HCpro (positions 330-335 in PVA HCpro), which is hypervariable among potyviruses. Three out of the four HCpro mutants suppressed RNA silencing more efficiently and sustained expression of co-expressed jellyfish green fluorescent protein for a longer time than wild-type HCpro in agroinfiltrated leaves of Nicotiana benthamiana. Leaf tissues remained healthy-looking without any visible signs of stress.

  7. Intein-mediated backbone cyclization of VP1 protein enhanced protection of CVB3-induced viral myocarditis

    PubMed Central

    Qi, Xingmei; Xiong, Sidong

    2017-01-01

    CVB3 is a common human pathogen to be highly lethal to newborns and causes viral myocarditis and pancreatitis in adults. However, there is no vaccine available for clinical use. CVB3 capsid protein VP1 is an immunodominant structural protein, containing several B- and T-cell epitopes. However, immunization of mice with VP1 protein is ineffective. Cyclization of peptide is commonly used to improve their in vivo stability and biological activity. Here, we designed and synthesizd cyclic VP1 protein by using engineered split Rma DnaB intein and the cyclization efficiency was 100% in E. coli. As a result, the cyclic VP1 was significantly more stable against irreversible aggregation upon heating and against carboxypeptidase in vitro and the degradation rate was more slowly in vivo. Compared with linear VP1, immunization mice with circular VP1 significantly increased CVB3-specific serum IgG level and augmented CVB3-specific cellular immune responses, consequently afforded better protection against CVB3-induced viral myocarditis. The cyclic VP1 may be a novel candidate protein vaccine for preventing CVB3 infection and similar approaches could be employed to a variety of protein vaccines to enhance their protection effect. PMID:28148910

  8. Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses.

    PubMed

    Patel, Jaina M; Kim, Min-Chul; Vartabedian, Vincent F; Lee, Yu-Na; He, Sara; Song, Jae-Min; Choi, Hyo-Jick; Yamanaka, Satoshi; Amaram, Nikhil; Lukacher, Anna; Montemagno, Carlo D; Compans, Richard W; Kang, Sang-Moo; Selvaraj, Periasamy

    2015-07-01

    Recombinant virus-like nanoparticles (VLPs) are a promising nanoparticle platform to develop safe vaccines for many viruses. Herein, we describe a novel and rapid protein transfer process to enhance the potency of enveloped VLPs by decorating influenza VLPs with exogenously added glycosylphosphatidylinositol-anchored immunostimulatory molecules (GPI-ISMs). With protein transfer, the level of GPI-ISM incorporation onto VLPs is controllable by varying incubation time and concentration of GPI-ISMs added. ISM incorporation was dependent upon the presence of a GPI-anchor and incorporated proteins were stable and functional for at least 4weeks when stored at 4°C. Vaccinating mice with GPI-granulocyte macrophage colony-stimulating factor (GM-CSF)-incorporated-VLPs induced stronger antibody responses and better protection against a heterologous influenza virus challenge than unmodified VLPs. Thus, VLPs can be enriched with ISMs by protein transfer to increase the potency and breadth of the immune response, which has implications in developing effective nanoparticle-based vaccines against a broad spectrum of enveloped viruses. The inherent problem with current influenza vaccines is that they do not generate effective cross-protection against heterologous viral strains. In this article, the authors described the development of virus-like nanoparticles (VLPs) as influenza vaccines with enhanced efficacy for cross-protection, due to an easy protein transfer modification process. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2016-01-01

    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

  10. Association of Human Papillomavirus 16 E2 with Rad50-Interacting Protein 1 Enhances Viral DNA Replication

    PubMed Central

    Campos-León, Karen; Wijendra, Kalpanee; Siddiqa, Abida; Pentland, Ieisha; Feeney, Katherine M.; Knapman, Alison; Davies, Rachel

    2016-01-01

    ABSTRACT Rad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication. IMPORTANCE HPV infections are an important driver of many epithelial cancers, including those within the anogenital and oropharyngeal tracts. The HPV life cycle is tightly regulated and intimately linked to the differentiation of the epithelial cells that it infects. HPV replication factories formed in the nucleus are locations where viral DNA is copied to support virus persistence and amplification

  11. Association of Human Papillomavirus 16 E2 with Rad50-Interacting Protein 1 Enhances Viral DNA Replication.

    PubMed

    Campos-León, Karen; Wijendra, Kalpanee; Siddiqa, Abida; Pentland, Ieisha; Feeney, Katherine M; Knapman, Alison; Davies, Rachel; Androphy, Elliot J; Parish, Joanna L

    2017-03-01

    Rad50-interacting protein 1 (Rint1) associates with the DNA damage response protein Rad50 during the transition from the S phase to the G2/M phase and functions in radiation-induced G2 checkpoint control. It has also been demonstrated that Rint1 is essential in vesicle trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) through an interaction with Zeste-White 10 (ZW10). We have isolated a novel interaction between Rint1 and the human papillomavirus 16 (HPV16) transcription and replication factor E2. E2 binds to Rint1 within its ZW10 interaction domain, and we show that in the absence of E2, Rint1 is localized to the ER and associates with ZW10. E2 expression results in a disruption of the Rint1-ZW10 interaction and an accumulation of nuclear Rint1, coincident with a significant reduction in vesicle movement from the ER to the Golgi apparatus. Interestingly, nuclear Rint1 and members of the Mre11/Rad50/Nbs1 (MRN) complex were found in distinct E2 nuclear foci, which peaked during mid-S phase, indicating that the recruitment of Rint1 to E2 foci within the nucleus may also result in the recruitment of this DNA damage-sensing protein complex. We show that exogenous Rint1 expression enhances E2-dependent virus replication. Conversely, the overexpression of a truncated Rint1 protein that retains the E2 binding domain but not the Rad50 binding domain acts as a dominant negative inhibitor of E2-dependent HPV replication. Put together, these experiments demonstrate that the interaction between Rint1 and E2 has an important function in HPV replication.IMPORTANCE HPV infections are an important driver of many epithelial cancers, including those within the anogenital and oropharyngeal tracts. The HPV life cycle is tightly regulated and intimately linked to the differentiation of the epithelial cells that it infects. HPV replication factories formed in the nucleus are locations where viral DNA is copied to support virus persistence and amplification of

  12. 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.

  13. High-level expression of alternative oxidase protein sequences enhances the spread of viral vectors in resistant and susceptible plants.

    PubMed

    Murphy, Alex M; Gilliland, Androulla; York, Caroline J; Hyman, Belinda; Carr, John P

    2004-12-01

    The alternative oxidase (AOX) is the terminal oxidase of the cyanide-resistant alternative respiratory pathway in plants and has been implicated in resistance to viruses. When tobacco mosaic virus (TMV) vectors were used to drive very high levels of expression of either AOX or AOX mutated in its active site (AOX-E), virus spread was enhanced. This was visualized as the induction of larger hypersensitive-response lesions after inoculation onto NN-genotype tobacco than those produced by vectors bearing sequences of comparable length [the green fluorescent protein (gfp) gene sequence or antisense aox] or the 'empty' viral vector. Also, in the highly susceptible host Nicotiana benthamiana, systemic movement of TMV vectors expressing AOX or AOX-E was faster than that of TMV constructs bearing gfp or antisense aox sequences. Notably, in N. benthamiana, TMV.AOX and TMV.AOX-E induced symptoms that were severe and ultimately included cell death, whereas the empty vector, TMV.GFP and the TMV vector expressing antisense aox sequences never induced necrosis. The results show that, if expressed at sufficiently high levels, active and inactive AOX proteins can affect virus spread and symptomology in plants.

  14. Going Viral with Fluorescent Proteins.

    PubMed

    Costantini, Lindsey M; Snapp, Erik L

    2015-10-01

    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.

  15. 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

  16. Viral Organization of Human Proteins

    PubMed Central

    Wuchty, Stefan; Siwo, Geoffrey; Ferdig, Michael T.

    2010-01-01

    Although maps of intracellular interactions are increasingly well characterized, little is known about large-scale maps of host-pathogen protein interactions. The investigation of host-pathogen interactions can reveal features of pathogenesis and provide a foundation for the development of drugs and disease prevention strategies. A compilation of experimentally verified interactions between HIV-1 and human proteins and a set of HIV-dependency factors (HDF) allowed insights into the topology and intricate interplay between viral and host proteins on a large scale. We found that targeted and HDF proteins appear predominantly in rich-clubs, groups of human proteins that are strongly intertwined among each other. These assemblies of proteins may serve as an infection gateway, allowing the virus to take control of the human host by reaching protein pathways and diversified cellular functions in a pronounced and focused way. Particular transcription factors and protein kinases facilitate indirect interactions between HDFs and viral proteins. Discerning the entanglement of directly targeted and indirectly interacting proteins may uncover molecular and functional sites that can provide novel perspectives on the progression of HIV infection and highlight new avenues to fight this virus. PMID:20827298

  17. Disruption of Src homology 3-binding motif within non-structural protein 1 of influenza B virus unexpectedly enhances viral replication in human cells.

    PubMed

    Sadewasser, Anne; Saenger, Sandra; Paki, Katharina; Schwecke, Torsten; Wolff, Thorsten

    2016-11-01

    The influenza virus non-structural protein 1 (NS1) is a multifunctional virulence factor that plays a crucial role during infection by blocking the innate antiviral immune response of infected cells. In contrast to the well-studied NS1 protein of influenza A virus, knowledge about structure and functions of the influenza B virus homologue B/NS1, which shares less than 25 % sequence identity, is still limited. Here, we report on a reverse genetic analysis to study the role of a highly conserved class II Src homology 3 domain-binding motif matching the consensus PxxPx(K/R) that we identified at positions 122-127 of the B/NS1 protein. Surprisingly, glycine substitutions in the Src homology 3 domain-binding motif increased virus replication up to three orders of magnitude in human lung cells. Enhanced mutant virus propagation was accompanied by increased gene expression and apoptosis induction linking this motif to the control of programmed cell death. A MS-based interactome study revealed that the glycine substitutions facilitate binding of B/NS1 to heat shock protein 90-beta (HSP90β). Moreover, recruitment of the viral polymerase basic protein 2 to the B/NS1-HSP90β complex was observed. Pharmacological inhibition of HSP90 reduced mutant virus propagation suggesting that the mutation-induced involvement of HSP90β enhanced viral replication. This study not only functionally characterizes a conserved motif within the B/NS1 protein, but also illustrates a rare example in which mutation of a highly conserved sequence within a viral protein does not result in high fitness costs, but rather increases viral replication via recruitment of a host factor.

  18. A complex comprising phosphatidylinositol 4-kinase IIIβ, ACBD3, and Aichi virus proteins enhances phosphatidylinositol 4-phosphate synthesis and is critical for formation of the viral replication complex.

    PubMed

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

    2014-06-01

    Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for the replication of certain picornavirus genomes. We previously showed that nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB of Aichi virus (AiV), a picornavirus, interact with the Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3), which interacts with PI4KB. These five viral proteins, ACBD3, PI4KB, and the PI4KB product phosphatidylinositol 4-phosphate (PI4P) colocalize to the AiV RNA replication sites (J. Sasaki et al., EMBO J. 31:754-766, 2012). We here examined the roles of these viral and cellular molecules in the formation of AiV replication complexes. Immunofluorescence microscopy revealed that treatment of AiV polyprotein-expressing cells with a small interfering RNA targeting ACBD3 abolished colocalization of the viral 2B, 2C, and 3A proteins with PI4KB. A PI4KB-specific inhibitor also prevented their colocalization. Virus RNA replication increased the level of cellular PI4P without affecting that of PI4KB, and individual expression of 2B, 2BC, 2C, 3A, or 3AB stimulated PI4P generation. These results suggest that the viral protein/ACBD3/PI4KB complex plays an important role in forming the functional replication complex by enhancing PI4P synthesis. Of the viral proteins, 3A and 3AB were shown to stimulate the in vitro kinase activity of PI4KB through forming a 3A or 3AB/ACBD3/PI4KB complex, whereas the ACBD3-mediated PI4KB activation by 2B and 2C remains to be demonstrated. The phosphatidylinositol 4-kinase PI4KB is a host factor required for the replication of certain picornavirus genomes. Aichi virus, a picornavirus belonging to the genus Kobuvirus, forms a complex comprising one of the viral nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB, the Golgi protein ACBD3, and PI4KB to synthesize PI4P at the sites for viral RNA replication. However, the roles of this protein complex in forming the replication complex are unknown. This study showed that virus RNA replication

  19. The Herpes Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs Independently of Suppressing Protein Kinase R and Stress Granule Formation.

    PubMed

    Dauber, Bianca; Poon, David; Dos Santos, Theodore; Duguay, Brett A; Mehta, Ninad; Saffran, Holly A; Smiley, James R

    2016-07-01

    The herpes simplex virus (HSV) virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double-stranded RNA-activated protein kinase R (PKR) much more strongly than the wild-type virus does in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2α phosphorylation. The herpes simplex virus vhs RNase plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but at best, it plays a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least two distinct aspects

  20. The Herpes Simplex Virus Virion Host Shutoff Protein Enhances Translation of Viral True Late mRNAs Independently of Suppressing Protein Kinase R and Stress Granule Formation

    PubMed Central

    Dauber, Bianca; Poon, David; dos Santos, Theodore; Duguay, Brett A.; Mehta, Ninad; Saffran, Holly A.

    2016-01-01

    ABSTRACT The herpes simplex virus (HSV) virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double-stranded RNA-activated protein kinase R (PKR) much more strongly than the wild-type virus does in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2α phosphorylation. IMPORTANCE The herpes simplex virus vhs RNase plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but at best, it plays a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least

  1. UGGT1 enhances enterovirus 71 pathogenicity by promoting viral RNA synthesis and viral replication

    PubMed Central

    Huang, Peng-Nien; Cameron, Craig E.

    2017-01-01

    Positive-strand RNA virus infections can induce the stress-related unfolded protein response (UPR) in host cells. This study found that enterovirus A71 (EVA71) utilizes host UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1), a key endoplasmic reticulum protein (ER) involved in UPR, to enhance viral replication and virulence. EVA71 forms replication complexes (RCs) on cellular membranes that contain a mix of host and viral proteins to facilitate viral replication, but the components and processes involved in the assembly and function of RCs are not fully understood. Using EVA71 as a model, this study found that host UGGT1 and viral 3D polymerase co-precipitate along with other factors on membranous replication complexes to enhance viral replication. Increased UGGT1 levels elevated viral growth rates, while viral pathogenicity was observed to be lower in heterozygous knockout mice (Uggt1 +/- mice). These findings provide important insight on the role of UPR and host UGGT1 in regulating RNA virus replication and pathogenicity. PMID:28545059

  2. Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs.

    PubMed

    Grubor, Branka; Gallup, Jack M; Meyerholz, David K; Crouch, Erika C; Evans, Richard B; Brogden, Kim A; Lehmkuhl, Howard D; Ackermann, Mark R

    2004-05-01

    Defensins and surfactant protein A (SP-A) and SP-D are antimicrobial components of the pulmonary innate immune system. The purpose of this study was to determine the extent to which parainfluenza type 3 virus infection in neonatal lambs alters expression of sheep beta-defensin 1 (SBD-1), SP-A, and SP-D, all of which are constitutively transcribed by respiratory epithelia. Parainfluenza type 3 viral antigen was detected by immunohistochemistry (IHC) in the bronchioles of all infected lambs 3 days postinoculation and at diminished levels 6 days postinoculation, but it was absent 17 days postinoculation. At all times postinoculation, lung homogenates from parainfluenza type 3 virus-inoculated animals had increased SBD-1, SP-A, and SP-D mRNA levels as detected by fluorogenic real-time reverse transcriptase PCR. Protein levels of SP-A in lung homogenates detected by quantitative-competitive enzyme-linked immunosorbent assay and protein antigen of SP-A detected by IHC were not altered. These studies demonstrate that parainfluenza type 3 virus infection results in enhanced expression of constitutively transcribed innate immune factors expressed by respiratory epithelia and that this increased expression occurs concurrently with decreased viral replication.

  3. Inducible heat shock protein 70 enhances HPV31 viral genome replication and virion production during the differentiation-dependent life cycle in human keratinocytes.

    PubMed

    Song, Hebin; Moseley, Pope L; Lowe, Stephanie L; Ozbun, Michelle A

    2010-01-01

    Increasing data indicate heat shock proteins (HSPs) including inducible HSP70 (HSP70i) are involved in the replicative cycles of various viruses including adenoviruses (Ads), polyomaviruses (PyVs), and some RNA viruses. Cell-free system studies implicate HSP70i in human papillomavirus type 11 (HPV11) genome replication with E1 and E2 proteins, and there is evidence that HSP70 is involved in capsid assembly and disassembly for PyVs and HPVs. HSP70 expression is increased in HPV16 E6/E7 gene transduced human primary keratinocytes, and frequently detected in early stage uterine cervical cancer at levels in conjunction with lesion severity. In this study we carry out analyses in the natural host epithelial tissues to assess the role of inducible HSP70 (HSP70i) in the HPV infectious life cycle. For these studies we used the organotypic (raft) culture system to recapitulate the full viral life cycle of the high-risk HPV31. Upon heat shock of HPV31-infected organotypic tissues, we find high and sustained expression of HSP70i coincident with enhanced HPV genome replication and virion production. Whereas there is no clear effect on L1 expression levels, we find HSP70i and L1 interact and HSP70i colocalizes with and enhances the nuclear localization of L1 in differentiated cells. Ad-mediated gene transfer was used to study the effects of HSP70i in naturally HPV-infected differentiating tissues and showed results similar to those in heat shocked rafts. These results indicate that increased HSP70i augments late activities in the viral life cycle. We conclude that HSP70i contributes directly to HPV replicative viral activities and the production of infectious virions.

  4. Kaposi's sarcoma-associated herpesvirus ORF57 protein interacts with PYM to enhance translation of viral intronless mRNAs

    PubMed Central

    Boyne, James R; Jackson, Brian R; Taylor, Adam; Macnab, Stuart A; Whitehouse, Adrian

    2010-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) expresses numerous intronless mRNAs that are unable to access splicing-dependent cellular mRNA nuclear export pathways. To circumvent this problem, KSHV encodes the open reading frame 57 (ORF57) protein, which orchestrates the formation of an export-competent virus ribonucleoprotein particle comprising the nuclear export complex hTREX, but not the exon-junction complex (EJC). Interestingly, EJCs stimulate mRNA translation, which raises the intriguing question of how intronless KSHV transcripts are efficiently translated. Herein, we show that ORF57 associates with components of the 48S pre-initiation complex and co-sediments with the 40S ribosomal subunits. Strikingly, we observed a direct interaction between ORF57 and PYM, a cellular protein that enhances translation by recruiting the 48S pre-initiation complex to newly exported mRNAs, through an interaction with the EJC. Moreover, detailed biochemical analysis suggests that ORF57 recruits PYM to intronless KSHV mRNA and PYM then facilitates the association of ORF57 and the cellular translation machinery. We, therefore, propose a model whereby ORF57 interacts directly with PYM to enhance translation of intronless KSHV transcripts. PMID:20436455

  5. Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex.

    PubMed

    Lin, Jihui; Wang, Chengbao; Zhang, Longxiang; Wang, Tao; Zhang, Jing; Liang, Wulong; Li, Cheng; Qian, Gui; Ouyang, Yueling; Guo, Kangkang; Zhang, Yanming

    2017-01-01

    Classical swine fever virus (CSFV) is a fatal pig pestivirus and causes serious financial losses to the pig industry. CSFV NS4B protein is one of the most important viral replicase proteins. Rab5, a member of the small Rab GTPase family, is involved in infection and replication of numerous viruses including hepatitis C virus and dengue virus. Until now, the effects of Rab5 on the proliferation of CSFV are poorly defined. In the present study, we showed that Rab5 could enhance CSFV proliferation by utilizing lentivirus-mediated constitutive overexpression and eukaryotic plasmid transient overexpression approaches. On the other hand, lentivirus-mediated short hairpin RNA knockdown of Rab5 dramatically inhibited virus production. Co-immunoprecipitation, glutathione S-transferase pulldown and laser confocal microscopy assays further confirmed the interaction between Rab5 and CSFV NS4B protein. In addition, intracellular distribution of NS4B-Red presented many granular fluorescent signals (GFS) in CSFV infected PK-15 cells. Inhibition of basal Rab5 function with Rab5 dominant negative mutant Rab5S34N resulted in disruption of the GFS. These results indicate that Rab5 plays a critical role in facilitating the formation of the NS4B related complexes. Furthermore, it was observed that NS4B co-localized with viral NS3 and NS5A proteins in the cytoplasm, suggesting that NS3 and NS5A might be components of the NS4B related complex. Taken together, these results demonstrate that Rab5 positively modulates CSFV propagation and interacts with NS4B protein to facilitate the NS4B related complexes formation.

  6. Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex

    PubMed Central

    Lin, Jihui; Wang, Chengbao; Zhang, Longxiang; Wang, Tao; Zhang, Jing; Liang, Wulong; Li, Cheng; Qian, Gui; Ouyang, Yueling; Guo, Kangkang; Zhang, Yanming

    2017-01-01

    Classical swine fever virus (CSFV) is a fatal pig pestivirus and causes serious financial losses to the pig industry. CSFV NS4B protein is one of the most important viral replicase proteins. Rab5, a member of the small Rab GTPase family, is involved in infection and replication of numerous viruses including hepatitis C virus and dengue virus. Until now, the effects of Rab5 on the proliferation of CSFV are poorly defined. In the present study, we showed that Rab5 could enhance CSFV proliferation by utilizing lentivirus-mediated constitutive overexpression and eukaryotic plasmid transient overexpression approaches. On the other hand, lentivirus-mediated short hairpin RNA knockdown of Rab5 dramatically inhibited virus production. Co-immunoprecipitation, glutathione S-transferase pulldown and laser confocal microscopy assays further confirmed the interaction between Rab5 and CSFV NS4B protein. In addition, intracellular distribution of NS4B-Red presented many granular fluorescent signals (GFS) in CSFV infected PK-15 cells. Inhibition of basal Rab5 function with Rab5 dominant negative mutant Rab5S34N resulted in disruption of the GFS. These results indicate that Rab5 plays a critical role in facilitating the formation of the NS4B related complexes. Furthermore, it was observed that NS4B co-localized with viral NS3 and NS5A proteins in the cytoplasm, suggesting that NS3 and NS5A might be components of the NS4B related complex. Taken together, these results demonstrate that Rab5 positively modulates CSFV propagation and interacts with NS4B protein to facilitate the NS4B related complexes formation. PMID:28848503

  7. 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.

  8. 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.

    PubMed

    Östberg, Sara; Törmänen Persson, Heidi; Akusjärvi, Göran

    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' 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.

  9. 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

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

    2014-01-20

    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.

  10. 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

  11. 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

  12. 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.

  13. Protein-Protein Interfaces in Viral Capsids Are Structurally Unique.

    PubMed

    Cheng, Shanshan; Brooks, Charles L

    2015-11-06

    Viral capsids exhibit elaborate and symmetrical architectures of defined sizes and remarkable mechanical properties not seen with cellular macromolecular complexes. Given the uniqueness of the higher-order organization of viral capsid proteins in the virosphere, we explored the question of whether the patterns of protein-protein interactions within viral capsids are distinct from those in generic protein complexes. Our comparative analysis involving a non-redundant set of 551 inter-subunit interfaces in viral capsids from VIPERdb and 20,014 protein-protein interfaces in non-capsid protein complexes from the Protein Data Bank found 418 generic protein-protein interfaces that share similar physicochemical patterns with some protein-protein interfaces in the capsid set, using the program PCalign we developed for comparing protein-protein interfaces. This overlap in the structural space of protein-protein interfaces is significantly small, with a p-value <0.0001, based on a permutation test on the total set of protein-protein interfaces. Furthermore, the generic protein-protein interfaces that bear similarity in their spatial and chemical arrangement with capsid ones are mostly small in size with fewer than 20 interfacial residues, which results from the relatively limited choices of natural design for small interfaces rather than having significant biological implications in terms of functional relationships. We conclude based on this study that protein-protein interfaces in viral capsids are non-representative of patterns in the smaller, more compact cellular protein complexes. Our finding highlights the design principle of building large biological containers from repeated, self-assembling units and provides insights into specific targets for antiviral drug design for improved efficacy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Viral Protein VP4 Is a Target of Human Antibodies Enhancing Coxsackievirus B4- and B3-Induced Synthesis of Alpha Interferon

    PubMed Central

    Chehadeh, Wassim; Lobert, Pierre-Emmanuel; Sauter, Pierre; Goffard, Anne; Lucas, Bernadette; Weill, Jacques; Vantyghem, Marie-Christine; Alm, Gunnar; Pigny, Pascal; Hober, Didier

    2005-01-01

    Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-α) by peripheral blood mononuclear cells (PBMC) is enhanced in vitro by nonneutralizing anti-CVB4 antibodies from healthy subjects and, to a higher extent, from patients with insulin-dependent diabetes mellitus. In this study, we focused on identification of the viral target of these antibodies in CVB systems. High levels of IFN-α were obtained in supernatants of PBMC incubated with CVB4E2 or CVB3 and plasma from healthy subjects and, to a higher extent, from patients. The VP4 capsid proteins dissociated by heating at 56°C from CVB4E2 (VP4CVB4) and CVB3 (VP4CVB3) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis. There was no cross-reaction between VP4CVB4 and VP4CVB3 in the inhibiting effect. IFN-α levels in culture supernatants showed dose-dependent correlation with anti-VP4 antibodies eluted from plasma specimens using VP4-coated plates. There were higher index values for anti-VP4 antibodies detected by enzyme-linked immunosorbent assay (ELISA) and higher proportions of positive detection in 40 patients than in 40 healthy subjects (80% versus 15% for anti-VP4CVB4). There was no relationship between the levels of anti-CVB neutralizing antibodies and the detection of anti-VP4 antibodies by ELISA. The CVB plasma-induced IFN-α levels obtained in PBMC cultures in the anti-VP4 antibody-positive groups were significantly higher than those obtained in the anti-VP4 antibody-negative groups regardless of the titers of anti-CVB neutralizing antibodies. These results show that VP4 is the target of antibodies involved in the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis by PBMC. PMID:16254324

  15. Enhanced Viral Replication by Cellular Replicative Senescence

    PubMed Central

    Kim, Ji-Ae; Seong, Rak-Kyun

    2016-01-01

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

  16. 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

  17. Enhancement of viral fusion by nonadsorbing polymers.

    PubMed Central

    Herrmann, A; Clague, M J; Blumenthal, R

    1993-01-01

    Nonadsorbing polymers such as dextran and poly(ethylene glycol) enhance binding as well as extents of fusion of influenza virus with erythrocytes. Kinetics and extent of viral membrane fusion were measured using an assay based on lipid mixing of a fluorescent dye. The effects of nonadsorbing polymers were in the concentration range from 0 to 10 wt%, far below the concentration required to overcome hydration repulsion forces. The enhancing effects were dependent on the molecular weight of nonadsorbing polymer, and only occurred at molecular weight > 1500; this links the phenomena we observe to the so-called "excluded volume effect" of nonadsorbing polymers. The time delay between triggering and the onset of influenza virus fusion was significantly reduced in the presence of nonadsorbing polymers. High molecular weight poly(ethylene glycol) also induced fusion of vesicular stomatitis virus with intact erythrocytes, which do not serve as target of vesicular stomatitis virus fusion in the absence of the polymer. The forces between membranes which determine rate-limiting processes in viral fusion and how they are affected by nonadsorbing polymers are discussed. PMID:7690263

  18. 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

  19. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Protein intrinsic disorder toolbox for comparative analysis of viral proteins

    PubMed Central

    Goh, Gerard Kian-Meng; Dunker, A Keith; Uversky, Vladimir N

    2008-01-01

    To examine the usefulness of protein disorder predictions as a tool for the comparative analysis of viral proteins, a relational database has been constructed. The database includes proteins from influenza A and HIV-related viruses. Annotations include viral protein sequence, disorder prediction, structure, and function. Location of each protein within a virion, if known, is also denoted. Our analysis reveals a clear relationship between proximity to the RNA core and the percentage of predicted disordered residues for a set of influenza A virus proteins. Neuraminidases (NA) and hemagglutinin (HA) of major influenza A pandemics tend to pair in such a way that both proteins tend to be either ordered-ordered or disordered-disordered by prediction. This may be the result of these proteins evolving from being lipid-associated. High abundance of intrinsic disorder in envelope and matrix proteins from HIV-related viruses likely represents a mechanism where HIV virions can escape immune response despite the availability of antibodies for the HIV-related proteins. This exercise provides an example showing how the combined use of intrinsic disorder predictions and relational databases provides an improved understanding of the functional and structural behaviour of viral proteins. PMID:18831795

  1. 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

    2009-11-20

    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.

  2. APOBEC3 Proteins in Viral Immunity

    PubMed Central

    Stavrou, Spyridon; Ross, Susan R.

    2015-01-01

    Apolipoprotein B Editing Complex (APOBEC3) family members are cytidine deaminases that play important roles in intrinsic responses to infection by retroviruses and have also been implicated in the control of other viruses such as parvoviruses, herpesviruses, papillomaviruses, hepatitis B virus and retrotransposons. While their direct effect on modification of viral DNA has been clearly demonstrated, whether they play additional roles in innate and adaptive immunity to viruses is less clear. Here we review the data regarding the various steps in the innate and adaptive immune response to virus infection in which APOBEC3 proteins have been implicated. PMID:26546688

  3. 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

  4. The herpes simplex virus 1 virion host shutoff protein enhances translation of viral late mRNAs by preventing mRNA overload.

    PubMed

    Dauber, Bianca; Saffran, Holly A; Smiley, James R

    2014-09-01

    We recently demonstrated that the virion host shutoff (vhs) protein, an mRNA-specific endonuclease, is required for efficient herpes simplex virus 1 (HSV-1) replication and translation of viral true-late mRNAs, but not other viral and cellular mRNAs, in many cell types (B. Dauber, J. Pelletier, and J. R. Smiley, J. Virol. 85:5363-5373, 2011, http://dx.doi.org/10.1128/JVI.00115-11). Here, we evaluated whether the structure of true-late mRNAs or the timing of their transcription is responsible for the poor translation efficiency in the absence of vhs. To test whether the highly structured 5' untranslated region (5'UTR) of the true-late gC mRNA is the primary obstacle for translation initiation, we replaced it with the less structured 5'UTR of the γ-actin mRNA. However, this mutation did not restore translation in the context of a vhs-deficient virus. We then examined whether the timing of transcription affects translation efficiency at late times. To this end, we engineered a vhs-deficient virus mutant that transcribes the true-late gene US11 with immediate-early kinetics (IEUS11-ΔSma). Interestingly, IEUS11-ΔSma showed increased translational activity on the US11 transcript at late times postinfection, and US11 protein levels were restored to wild-type levels. These results suggest that mRNAs can maintain translational activity throughout the late stage of infection if they are present before translation factors and/or ribosomes become limiting. Taken together, these results provide evidence that in the absence of the mRNA-destabilizing function of vhs, accumulation of viral mRNAs overwhelms the capacity of the host translational machinery, leading to functional exclusion of the last mRNAs that are made during infection. The process of mRNA translation accounts for a significant portion of a cell's energy consumption. To ensure efficient use of cellular resources, transcription, translation, and mRNA decay are tightly linked and highly regulated. However, during

  5. Mutation of neutralizing/antibody-dependent enhancing epitope on spike protein and 7b gene of feline infectious peritonitis virus: influences of viral replication in monocytes/macrophages and virulence in cats.

    PubMed

    Takano, Tomomi; Tomiyama, Yoshika; Katoh, Yasuichiroh; Nakamura, Michiyo; Satoh, Ryoichi; Hohdatsu, Tsutomu

    2011-03-01

    We previously prepared neutralizing monoclonal antibody (MAb)-resistant (mar) mutant viruses using a laboratory strain feline infectious peritonitis virus (FIPV) 79-1146 (Kida et al., 1999). Mar mutant viruses are mutated several amino acids of the neutralizing epitope of Spike protein, compared with the parent strain, FIPV 79-1146. We clarified that MAb used to prepare mar mutant viruses also lost its activity to enhance homologous mar mutant viruses, strongly suggesting that neutralizing and antibody-dependent enhancing epitopes are present in the same region in the strain FIPV 79-1146. We also discovered that amino acid mutation in the neutralizing epitope reduced viral replication in monocytes/macrophages. We also demonstrated that the mutation or deletion of two nucleotides in 7b gene abrogate the virulence of strain FIPV 79-1146.

  6. Potyviral VPg enhances viral RNA Translation and inhibits reporter mRNA translation in planta.

    PubMed

    Eskelin, Katri; Hafrén, Anders; Rantalainen, Kimmo I; Mäkinen, Kristiina

    2011-09-01

    Viral protein genome-linked (VPg) plays a central role in several stages of potyvirus infection. This study sought to answer questions about the role of Potato virus A (PVA; genus Potyvirus) VPg in viral and host RNA expression. When expressed in Nicotiana benthamiana leaves in trans, a dual role of VPg in translation is observed. It repressed the expression of monocistronic luciferase (luc) mRNA and simultaneously induced a significant upregulation in the expression of both replicating and nonreplicating PVA RNAs. This enhanced viral gene expression was due at least to the 5' untranslated region (UTR) of PVA RNA, eukaryotic initiation factors 4E and iso 4E [eIF4E/eIF(iso)4E], and the presence of a sufficient amount of VPg. Coexpression of VPg with viral RNA increased the viral RNA amount, which was not the case with the monocistronic mRNA. Both mutations at certain lysine residues in PVA VPg and eIF4E/eIF(iso)4E depletion reduced its ability to upregulate the viral RNA expression. These modifications were also involved in VPg-mediated downregulation of monocistronic luc expression. These results suggest that VPg can titrate eIF4Es from capped monocistronic RNAs. Because VPg-mediated enhancement of viral gene expression required eIF4Es, it is possible that VPg directs eIF4Es to promote viral RNA expression. From this study it is evident that VPg can serve as a specific regulator of PVA expression by boosting the viral RNA amounts as well as the accumulation of viral translation products. Such a mechanism could function to protect viral RNA from being degraded and to secure efficient production of coat protein (CP) for virion formation.

  7. 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

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

    PubMed Central

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

    2015-01-01

    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 2Apro, 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.—Deng, H., Fung, G., Shi, J., Xu, S., Wang, C., Yin, M., Hou, J., Zhang, J., Jin, Z.-G., Luo, H. Enhanced enteroviral infectivity via viral protease-mediated cleavage of Grb2-associated binder 1. PMID:26183772

  9. Large Ribosomal Protein 4 Increases Efficiency of Viral Recoding Sequences

    PubMed Central

    Green, Lisa; Houck-Loomis, Brian; Yueh, Andrew

    2012-01-01

    Expression of retroviral replication enzymes (Pol) requires a controlled translational recoding event to bypass the stop codon at the end of gag. This recoding event occurs either by direct suppression of termination via the insertion of an amino acid at the stop codon (readthrough) or by alteration of the mRNA reading frame (frameshift). Here we report the effects of a host protein, large ribosomal protein 4 (RPL4), on the efficiency of recoding. Using a dual luciferase reporter assay, we found that transfection of cells with a plasmid encoding RPL4 cDNA increases recoding efficiency in a dose-dependent manner, with a maximal enhancement of nearly twofold. Expression of RPL4 increases recoding of reporters containing retroviral readthrough and frameshift sequences, as well as the Sindbis virus leaky termination signal. RPL4-induced enhancement of recoding is cell line specific and appears to be specific to RPL4 among ribosomal proteins. Cotransfection of RPL4 cDNA with Moloney murine leukemia proviral DNA results in Gag processing defects and a reduction of viral particle formation, presumably caused by the RPL4-dependent alteration of the Gag-to-Gag-Pol ratio required for virion assembly and release. PMID:22718819

  10. 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

  11. Cleavage of Grb2-Associated Binding Protein 2 by Viral Proteinase 2A during Coxsackievirus Infection

    PubMed Central

    Deng, Haoyu; Fung, Gabriel; Qiu, Ye; Wang, Chen; Zhang, Jingchun; Jin, Zheng-Gen; Luo, Honglin

    2017-01-01

    Coxsackievirus type B3 (CV-B3), an enterovirus associated with the pathogenesis of several human diseases, subverts, or employs the host intracellular signaling pathways to support effective viral infection. We have previously demonstrated that Grb2-associated binding protein 1 (GAB1), a signaling adaptor protein that serves as a platform for intracellular signaling assembly and transduction, is cleaved upon CV-B3 infection, resulting in a gain-of-pro-viral-function via the modification of GAB1-mediated ERK1/2 pathway. GAB2 is a mammalian homolog of GAB1. In this study, we aim to address whether GAB2 plays a synergistic role with GAB1 in the regulation of CV-B3 replication. Here, we reported that GAB2 is also a target of CV-B3-encoded viral proteinase. We showed that GAB2 is cleaved at G238 during CV-B3 infection by viral proteinase 2A, generating two cleaved fragments of GAB2-N1−237 and GAB2-C238−676. Moreover, knockdown of GAB2 significantly inhibits the synthesis of viral protein and subsequent viral progeny production, accompanied by reduced levels of phosphorylated p38, suggesting a pro-viral function for GAB2 linked to p38 activation. Finally, we examined whether the cleavage of GAB2 can promote viral replication as observed for GAB1 cleavage. We showed that expression of neither GAB2-N1−237 nor GAB2-C238−676 results in enhanced viral infectivity, indicating a loss-of-function, rather than a gain-of-function of GAB2 cleavage in mediating virus replication. Taken together, our findings in this study suggest a novel host defense machinery through which CV-B3 infection is limited by the cleavage of a pro-viral protein. PMID:28361043

  12. 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.

  13. VZV ORF47 serine protein kinase and its viral substrates.

    PubMed

    Kenyon, Teri K; Grose, Charles

    2010-01-01

    ORF47, a serine protein kinase of varicella-zoster virus (VZV) and homolog of herpes simplex virus UL13, is an interesting modulator of VZV pathogenesis. This chapter summarizes research showing that ORF47 protein kinase activity, by virtue of phosphorylation of or binding to various viral substrates, regulates VZV proteins during all phases of viral infection and has a pronounced effect on the trafficking of gE, the predominant VZV glycoprotein, which in turn is critical for cell-to-cell spread of the virus. Casein kinase II, an ubiquitous cellular protein kinase, recognizes a similar but less stringent phosphorylation consensus sequence and can partially compensate for lack of ORF47 activity in VZV-infected cells. Differences between the phosphorylation consensus sites of the viral and cellular kinases are outlined in detail.

  14. RNA-protein interaction methods to study viral IRES elements.

    PubMed

    Francisco-Velilla, Rosario; Fernandez-Chamorro, Javier; Lozano, Gloria; Diaz-Toledano, Rosa; Martínez-Salas, Encarnación

    2015-12-01

    Translation control often takes place through the mRNA untranslated regions, involving direct interactions with RNA-binding proteins (RBPs). Internal ribosome entry site elements (IRESs) are cis-acting RNA regions that promote translation initiation using a cap-independent mechanism. A subset of positive-strand RNA viruses harbor IRESs as a strategy to ensure efficient viral protein synthesis. IRESs are organized in modular structural domains with a division of functions. However, viral IRESs vary in nucleotide sequence, secondary RNA structure, and transacting factor requirements. Therefore, in-depth studies are needed to understand how distinct types of viral IRESs perform their function. In this review we describe methods to isolate and identify RNA-binding proteins important for IRES activity, and to study the impact of RNA structure and RNA-protein interactions on IRES activity.

  15. Viral capsid proteins are segregated in structural fold space.

    PubMed

    Cheng, Shanshan; Brooks, Charles L

    2013-01-01

    Viral capsid proteins assemble into large, symmetrical architectures that are not found in complexes formed by their cellular counterparts. Given the prevalence of the signature jelly-roll topology in viral capsid proteins, we are interested in whether these functionally unique capsid proteins are also structurally unique in terms of folds. To explore this question, we applied a structure-alignment based clustering of all protein chains in VIPERdb filtered at 40% sequence identity to identify distinct capsid folds, and compared the cluster medoids with a non-redundant subset of protein domains in the SCOP database, not including the viral capsid entries. This comparison, using Template Modeling (TM)-score, identified 2078 structural "relatives" of capsid proteins from the non-capsid set, covering altogether 210 folds following the definition in SCOP. The statistical significance of the 210 folds shared by two sets of the same sizes, estimated from 10,000 permutation tests, is less than 0.0001, which is an upper bound on the p-value. We thus conclude that viral capsid proteins are segregated in structural fold space. Our result provides novel insight on how structural folds of capsid proteins, as opposed to their surface chemistry, might be constrained during evolution by requirement of the assembled cage-like architecture. Also importantly, our work highlights a guiding principle for virus-based nanoplatform design in a wide range of biomedical applications and materials science.

  16. Viral Capsid Proteins Are Segregated in Structural Fold Space

    PubMed Central

    Cheng, Shanshan; Brooks, Charles L.

    2013-01-01

    Viral capsid proteins assemble into large, symmetrical architectures that are not found in complexes formed by their cellular counterparts. Given the prevalence of the signature jelly-roll topology in viral capsid proteins, we are interested in whether these functionally unique capsid proteins are also structurally unique in terms of folds. To explore this question, we applied a structure-alignment based clustering of all protein chains in VIPERdb filtered at 40% sequence identity to identify distinct capsid folds, and compared the cluster medoids with a non-redundant subset of protein domains in the SCOP database, not including the viral capsid entries. This comparison, using Template Modeling (TM)-score, identified 2078 structural “relatives” of capsid proteins from the non-capsid set, covering altogether 210 folds following the definition in SCOP. The statistical significance of the 210 folds shared by two sets of the same sizes, estimated from 10,000 permutation tests, is less than 0.0001, which is an upper bound on the p-value. We thus conclude that viral capsid proteins are segregated in structural fold space. Our result provides novel insight on how structural folds of capsid proteins, as opposed to their surface chemistry, might be constrained during evolution by requirement of the assembled cage-like architecture. Also importantly, our work highlights a guiding principle for virus-based nanoplatform design in a wide range of biomedical applications and materials science. PMID:23408879

  17. The repressing and enhancing functions of the herpes simplex virus regulatory protein ICP27 map to C-terminal regions and are required to modulate viral gene expression very early in infection.

    PubMed Central

    McMahan, L; Schaffer, P A

    1990-01-01

    The phenotypic properties of ICP27 temperature-sensitive and deletion mutants and the results of transient expression assays have demonstrated that ICP27 has a modulatory effect on viral gene expression induced by ICPs 0 and 4. In order to identify the regions of the ICP27 molecule that are responsible for its enhancing and repressing activities, 10 nonsense and 3 in-frame deletion mutations were introduced into the coding sequence of the cloned ICP27 gene. These mutant genes were tested in transient expression assays for their ability to complement an ICP27 null mutant and to enhance and repress expression from a spectrum of herpes simplex virus type 1 promoters in reporter CAT genes when expression was induced by ICP0 or ICP4. The results of assays with cloned mutant genes demonstrate that the ICP27 polypeptide contains two regions, located between amino acid residues 327 and 407 and residues 465 and 511, that contribute to its repressing activity. The amino acid region located between the two repressing regions (residues 407 to 465) is able to interfere with ICP27 repressing activity. None of the mutant genes exhibited efficient enhancing activity for any of the herpes simplex type 1 promoters tested, demonstrating that amino acids comprising the carboxy-terminal half of the ICP27 molecule, including the terminal phenylalanine residue, are required for wild-type enhancement as well as for efficient complementation of an ICP27 null mutant. Phenotypic characterization of an in-frame deletion mutant, vd3, and a previously isolated null mutant, 5dl 1.2 (A. M. McCarthy, L. and P. A. Schaffer, J. Virol. 63:18-27, 1989), demonstrated that ICP27 is required to induce the expression of all classes of viral genes very early in infection and confirmed the requirement for ICP27 later in infection (i) to repress early gene expression, (ii) to induce wild-type levels of delayed-early or gamma 1 gene expression, and (iii) to induce true late or gamma 2 gene expression. The vd3

  18. Chimeric Flock House virus protein A with endoplasmic reticulum-targeting domain enhances viral replication and virus-like particle trans-encapsidation in plants.

    PubMed

    Zhou, Yiyang; Kearney, Christopher M

    2017-07-01

    Flock House virus (FHV) RNA can be trans-encapsidated, entirely in planta, by tobacco mosaic virus coat protein to form virus-like particles (VLPs). Vaccination with these VLPs leads to strong antigen expression in mice and immune-activation. We hypothesize that creating an additional cellular site for replication and/or trans-encapsidation might significantly improve the final output of trans-encapsidated product. FHV protein A was engineered to target the endoplasmic reticulum (ER) via a heterologous tobacco etch virus ER-targeting domain, and was expressed in cis or in trans relative to the replicating FHV RNA1. A strong increase in marker gene expression in plants was noted when ER-targeted protein A was supplied in trans. RNA fluorescence in situ hybridization revealed RNA1 replication in both the mitochondria and ER, and total RNA1 accumulation was increased. In support of our hypothesis, VLP yield was increased significantly by the addition of this single genetic component to the inoculum. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Fc receptors in antibody-dependent enhancement of viral infections.

    PubMed

    Taylor, Adam; Foo, Suan-Sin; Bruzzone, Roberto; Dinh, Luan Vu; King, Nicholas J C; Mahalingam, Suresh

    2015-11-01

    Sensitization of the humoral immune response to invading viruses and production of antiviral antibodies forms part of the host antiviral repertoire. Paradoxically, for a number of viral pathogens, under certain conditions, antibodies provide an attractive means of enhanced virus entry and replication in a number of cell types. Known as antibody-dependent enhancement (ADE) of infection, the phenomenon occurs when virus-antibody immunocomplexes interact with cells bearing complement or Fc receptors, promoting internalization of the virus and increasing infection. Frequently associated with exacerbation of viral disease, ADE of infection presents a major obstacle to the prevention of viral disease by vaccination and is thought to be partly responsible for the adverse effects of novel antiviral therapeutics such as intravenous immunoglobulins. There is a growing body of work examining the intracellular signaling pathways and epitopes responsible for mediating ADE, with a view to aiding rational design of antiviral strategies. With in vitro studies also confirming ADE as a feature of infection for a growing number of viruses, challenges remain in understanding the multilayered molecular mechanisms of ADE and its effect on viral pathogenesis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  20. Interactions between HIV-1 Gag and Viral RNA Genome Enhance Virion Assembly.

    PubMed

    Dilley, Kari A; Nikolaitchik, Olga A; Galli, Andrea; Burdick, Ryan C; Levine, Louis; Li, Kelvin; Rein, Alan; Pathak, Vinay K; Hu, Wei-Shau

    2017-08-15

    Most HIV-1 virions contain two copies of full-length viral RNA, indicating that genome packaging is efficient and tightly regulated. However, the structural protein Gag is the only component required for the assembly of noninfectious viruslike particles, and the viral RNA is dispensable in this process. The mechanism that allows HIV-1 to achieve such high efficiency of genome packaging when a packageable viral RNA is not required for virus assembly is currently unknown. In this report, we examined the role of HIV-1 RNA in virus assembly and found that packageable HIV-1 RNA enhances particle production when Gag is expressed at levels similar to those in cells containing one provirus. However, such enhancement is diminished when Gag is overexpressed, suggesting that the effects of viral RNA can be replaced by increased Gag concentration in cells. We also showed that the specific interactions between Gag and viral RNA are required for the enhancement of particle production. Taken together, these studies are consistent with our previous hypothesis that specific dimeric viral RNA-Gag interactions are the nucleation event of infectious virion assembly, ensuring that one RNA dimer is packaged into each nascent virion. These studies shed light on the mechanism by which HIV-1 achieves efficient genome packaging during virus assembly.IMPORTANCE Retrovirus assembly is a well-choreographed event, during which many viral and cellular components come together to generate infectious virions. The viral RNA genome carries the genetic information to new host cells, providing instructions to generate new virions, and therefore is essential for virion infectivity. In this report, we show that the specific interaction of the viral RNA genome with the structural protein Gag facilitates virion assembly and particle production. These findings resolve the conundrum that HIV-1 RNA is selectively packaged into virions with high efficiency despite being dispensable for virion assembly

  1. 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

  2. A predictor for toxin-like proteins exposes cell modulator candidates within viral genomes

    PubMed Central

    Naamati, Guy; Askenazi, Manor; Linial, Michal

    2010-01-01

    Motivation: Animal toxins operate by binding to receptors and ion channels. These proteins are short and vary in sequence, structure and function. Sporadic discoveries have also revealed endogenous toxin-like proteins in non-venomous organisms. Viral proteins are the largest group of quickly evolving proteomes. We tested the hypothesis that toxin-like proteins exist in viruses and that they act to modulate functions of their hosts. Results: We updated and improved a classifier for compact proteins resembling short animal toxins that is based on a machine-learning method. We applied it in a large-scale setting to identify toxin-like proteins among short viral proteins. Among the ∼26 000 representatives of such short proteins, 510 sequences were positively identified. We focused on the 19 highest scoring proteins. Among them, we identified conotoxin-like proteins, growth factors receptor-like proteins and anti-bacterial peptides. Our predictor was shown to enhance annotation inference for many ‘uncharacterized’ proteins. We conclude that our protocol can expose toxin-like proteins in unexplored niches including metagenomics data and enhance the systematic discovery of novel cell modulators for drug development. Availability: ClanTox is available at http://www.clantox.cs.huji.ac.il Contact: michall@cc.huji.ac.il PMID:20823311

  3. 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.

  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. Manipulation of plant host susceptibility: an emerging role for viral movement proteins?

    PubMed

    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.

  6. 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

  7. Assessing ubiquitination of viral proteins: Lessons from flavivirus NS5.

    PubMed

    Taylor, R Travis; Best, Sonja M

    2011-10-01

    Ubiquitin (Ub) conjugation to a substrate protein is a widely used cellular mechanism for control of protein stability and function, modulation of signal transduction pathways and antiviral responses. Identification and characterization of ubiquitinated viral proteins is an important step in understanding novel mechanisms of viral protein regulation as well as elucidating cellular antiviral strategies. Here we describe a protocol to easily detect and characterize the ubiquitination status of a viral substrate protein expressed either during infection or ectopically expressed as a fusion with a biotinylatable epitope tag. This tag provides advantages over current immunoprecipitation techniques by making use of the extremely tight biotin-streptavidin interaction. We provide an example of this protocol using the nonstructural protein 5 (NS5) from Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serocomplex within the Flavivirus genus. Using the protocols outlined here, we describe some of the pitfalls inherent in determination of Ub linkage and demonstrate that NS5 is modified by at least two distinct ubiquitination types, multiubiquitination and K48-linked polyubiquitin chains.

  8. Assessing ubiquitination of viral proteins: lessons from flavivirus NS5

    PubMed Central

    Taylor, R. Travis; Best, Sonja M.

    2011-01-01

    Ubiquitin (Ub) conjugation to a substrate protein is a widely used cellular mechanism for control of protein stability and function, modulation of signal transduction pathways and antiviral responses. Identification and characterization of ubiquitinated viral proteins is an important step in understanding novel mechanisms of viral protein regulation as well as elucidating cellular antiviral strategies. Here we describe a protocol to easily detect and characterize the ubiquitination status of a viral substrate protein expressed either during infection or ectopically expressed as a fusion with a biotinylatable epitope tag. This tag provides advantages over current immunoprecipitation techniques by making use of the extremely tight biotin-streptavidin interaction. We provide an example of this protocol using the nonstructural protein 5 (NS5) from Langat virus (LGTV), a member of the tick-borne encephalitis virus (TBEV) serocomplex within the Flavivirus genus. Using the protocols outlined here, we describe some of the pitfalls inherent in determination of Ub linkage and demonstrate that NS5 is modified by at least two distinct ubiquitination types, multiubiquitination and K48-linked polyubiquitin chains. PMID:21855635

  9. Effect of DNA Repair Protein Rad18 on Viral Infection

    PubMed Central

    Lloyd, Aliza G; Tateishi, Satoshi; Bieniasz, Paul D; Muesing, Mark A; Yamaizumi, Masaru; Mulder, Lubbertus C. F

    2006-01-01

    Host factors belonging to the DNA repair machineries are assumed to aid retroviruses in the obligatory step of integration. Here we describe the effect of DNA repair molecule Rad18, a component of the post-replication repair pathway, on viral infection. Contrary to our expectations, cells lacking Rad18 were consistently more permissive to viral transduction as compared to Rad18+/+ controls. Remarkably, such susceptibility was integration independent, since retroviruses devoid of integration activity also showed enhancement of the initial steps of infection. Moreover, the elevated sensitivity of the Rad18−/− cells was also observed with adenovirus. These data indicate that Rad18 suppresses viral infection in a non-specific fashion, probably by targeting incoming DNA. Furthermore, considering data published recently, it appears that the interactions between DNA repair components with incoming viruses, often result in inhibition of the infection rather than cooperation toward its establishment. PMID:16710452

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

    PubMed

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

    2015-12-01

    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. We identified two regions of the V protein that interact with NP and determined that one of these regions enhances viral RNA transcription via its interaction with NP. Our data suggest that a common host factor may be involved in the regulation of paramyxovirus replication and could be a target for broad antiviral drug development. Understanding the regulation of paramyxovirus replication will enable the rational design of

  11. 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

  12. Comprehensive Analysis of LANA Interacting Proteins Essential for Viral Genome Tethering and Persistence

    PubMed Central

    Verma, Subhash C.; Cai, Qiliang; Kreider, Edward; Lu, Jie; Robertson, Erle S.

    2013-01-01

    Kaposi’s sarcoma associated herpesvirus is tightly linked to multiple human malignancies including Kaposi’s sarcoma (KS), Primary Effusion Lymphoma (PEL) and Multicentric Castleman’s Disease (MCD). KSHV like other herpesviruses establishes life-long latency in the infected host by persisting as chromatin and tethering to host chromatin through the virally encoded protein Latency Associated Nuclear Antigen (LANA). LANA, a multifunctional protein, is capable of binding to a large number of cellular proteins responsible for transcriptional regulation of various cellular and viral pathways involved in blocking cell death and promoting cell proliferation. This leads to enhanced cell division and replication of the viral genome, which segregates faithfully in the dividing tumor cells. The mechanism of genome segregation is well known and the binding of LANA to nucleosomal proteins, throughout the cell cycle, suggests that these interactions play an important role in efficient segregation. Various biochemical methods have identified a large number of LANA binding proteins, including histone H2A/H2B, histone H1, MeCP2, DEK, CENP-F, NuMA, Bub1, HP-1, and Brd4. These nucleosomal proteins may have various functions in tethering of the viral genome during specific phases of the viral life cycle. Therefore, we performed a comprehensive analysis of their interaction with LANA using a number of different assays. We show that LANA binds to core nucleosomal histones and also associates with other host chromatin proteins including histone H1 and high mobility group proteins (HMGs). We used various biochemical assays including co-immunoprecipitation and in-vivo localization by split GFP and fluorescence resonance energy transfer (FRET) to demonstrate their association. PMID:24040311

  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.

  14. 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

  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. Transactivation of programmed ribosomal frameshifting by a viral protein

    PubMed Central

    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

    2014-01-01

    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

  18. 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

  19. Viral Macro Domains Reverse Protein ADP-Ribosylation.

    PubMed

    Li, Changqing; Debing, Yannick; Jankevicius, Gytis; Neyts, Johan; Ahel, Ivan; Coutard, Bruno; Canard, Bruno

    2016-10-01

    ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD(+) to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular integrity are demodified

  20. Viral Macro Domains Reverse Protein ADP-Ribosylation

    PubMed Central

    Li, Changqing; Debing, Yannick; Jankevicius, Gytis; Neyts, Johan; Ahel, Ivan

    2016-01-01

    ABSTRACT ADP-ribosylation is a posttranslational protein modification in which ADP-ribose is transferred from NAD+ to specific acceptors to regulate a wide variety of cellular processes. The macro domain is an ancient and highly evolutionarily conserved protein domain widely distributed throughout all kingdoms of life, including viruses. The human TARG1/C6orf130, MacroD1, and MacroD2 proteins can reverse ADP-ribosylation by acting on ADP-ribosylated substrates through the hydrolytic activity of their macro domains. Here, we report that the macro domain from hepatitis E virus (HEV) serves as an ADP-ribose-protein hydrolase for mono-ADP-ribose (MAR) and poly(ADP-ribose) (PAR) chain removal (de-MARylation and de-PARylation, respectively) from mono- and poly(ADP)-ribosylated proteins, respectively. The presence of the HEV helicase in cis dramatically increases the binding of the macro domain to poly(ADP-ribose) and stimulates the de-PARylation activity. Abrogation of the latter dramatically decreases replication of an HEV subgenomic replicon. The de-MARylation activity is present in all three pathogenic positive-sense, single-stranded RNA [(+)ssRNA] virus families which carry a macro domain: Coronaviridae (severe acute respiratory syndrome coronavirus and human coronavirus 229E), Togaviridae (Venezuelan equine encephalitis virus), and Hepeviridae (HEV), indicating that it might be a significant tropism and/or pathogenic determinant. IMPORTANCE Protein ADP-ribosylation is a covalent posttranslational modification regulating cellular protein activities in a dynamic fashion to modulate and coordinate a variety of cellular processes. Three viral families, Coronaviridae, Togaviridae, and Hepeviridae, possess macro domains embedded in their polyproteins. Here, we show that viral macro domains reverse cellular ADP-ribosylation, potentially cutting the signal of a viral infection in the cell. Various poly(ADP-ribose) polymerases which are notorious guardians of cellular

  1. ChemR23 Dampens Lung Inflammation and Enhances Anti-viral Immunity in a Mouse Model of Acute Viral Pneumonia

    PubMed Central

    de Nadai, Patricia; Glineur, Stéphanie; De Henau, Olivier; Luangsay, Souphalone; Van Gool, Frédéric; Communi, David; De Vuyst, Paul; Desmecht, Daniel; Parmentier, Marc

    2011-01-01

    Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23−/− mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23−/− mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies. PMID:22072972

  2. ChemR23 dampens lung inflammation and enhances anti-viral immunity in a mouse model of acute viral pneumonia.

    PubMed

    Bondue, Benjamin; Vosters, Olivier; de Nadai, Patricia; Glineur, Stéphanie; De Henau, Olivier; Luangsay, Souphalone; Van Gool, Frédéric; Communi, David; De Vuyst, Paul; Desmecht, Daniel; Parmentier, Marc

    2011-11-01

    Viral diseases of the respiratory tract, which include influenza pandemic, children acute bronchiolitis, and viral pneumonia of the elderly, represent major health problems. Plasmacytoid dendritic cells play an important role in anti-viral immunity, and these cells were recently shown to express ChemR23, the receptor for the chemoattractant protein chemerin, which is expressed by epithelial cells in the lung. Our aim was to determine the role played by the chemerin/ChemR23 system in the physiopathology of viral pneumonia, using the pneumonia virus of mice (PVM) as a model. Wild-type and ChemR23 knock-out mice were infected by PVM and followed for functional and inflammatory parameters. ChemR23(-/-) mice displayed higher mortality/morbidity, alteration of lung function, delayed viral clearance and increased neutrophilic infiltration. We demonstrated in these mice a lower recruitment of plasmacytoid dendritic cells and a reduction in type I interferon production. The role of plasmacytoid dendritic cells was further addressed by performing depletion and adoptive transfer experiments as well as by the generation of chimeric mice, demonstrating two opposite effects of the chemerin/ChemR23 system. First, the ChemR23-dependent recruitment of plasmacytoid dendritic cells contributes to adaptive immune responses and viral clearance, but also enhances the inflammatory response. Second, increased morbidity/mortality in ChemR23(-/-) mice is not due to defective plasmacytoid dendritic cells recruitment, but rather to the loss of an anti-inflammatory pathway involving ChemR23 expressed by non-leukocytic cells. The chemerin/ChemR23 system plays important roles in the physiopathology of viral pneumonia, and might therefore be considered as a therapeutic target for anti-viral and anti-inflammatory therapies.

  3. Assembly of multilayer films incorporating a viral protein cage architecture.

    PubMed

    Suci, Peter A; Klem, Michael T; Arce, Fernando T; Douglas, Trevor; Young, Mark

    2006-10-10

    Protein cage architectures such as viral capsids, heat shock proteins, ferritins, and DNA-binding proteins are nanoscale modular subunits that can be used to expand the structural and functional range of composite materials. Here, layer-by-layer (LbL) assembly was used to incorporate cowpea chlorotic mottle virus (CCMV) into multilayer films. Three types of multilayer films were prepared. In the first type, ionic interactions were employed to assemble CCMV into triple layers. In the second type, complementary biological interactions (streptavidin/biotin) were used for this purpose. In a third variation of LbL assembly, complementary biological interactions were employed to produce nanotextured films that exhibit in-plane order over a micron scale without the need to adsorb onto a prepatterned template.

  4. Understanding Viral Transmission Behavior via Protein Intrinsic Disorder Prediction: Coronaviruses

    PubMed Central

    Goh, Gerard Kian-Meng; Dunker, A. Keith; Uversky, Vladimir N.

    2012-01-01

    Besides being a common threat to farm animals and poultry, coronavirus (CoV) was responsible for the human severe acute respiratory syndrome (SARS) epidemic in 2002–4. However, many aspects of CoV behavior, including modes of its transmission, are yet to be fully understood. We show that the amount and the peculiarities of distribution of the protein intrinsic disorder in the viral shell can be used for the efficient analysis of the behavior and transmission modes of CoV. The proposed model allows categorization of the various CoVs by the peculiarities of disorder distribution in their membrane (M) and nucleocapsid (N). This categorization enables quick identification of viruses with similar behaviors in transmission, regardless of genetic proximity. Based on this analysis, an empirical model for predicting the viral transmission behavior is developed. This model is able to explain some behavioral aspects of important coronaviruses that previously were not fully understood. The new predictor can be a useful tool for better epidemiological, clinical, and structural understanding of behavior of both newly emerging viruses and viruses that have been known for a long time. A potentially new vaccine strategy could involve searches for viral strains that are characterized by the evolutionary misfit between the peculiarities of the disorder distribution in their shells and their behavior. PMID:23097708

  5. 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

  6. A temporal gate for viral enhancers to co-opt Toll-like-receptor transcriptional activation pathways upon acute infection.

    PubMed

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

    2015-04-01

    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

  7. The Pacific Ocean Virome (POV): A Marine Viral Metagenomic Dataset and Associated Protein Clusters for Quantitative Viral Ecology

    PubMed Central

    Hurwitz, Bonnie L.; Sullivan, Matthew B.

    2013-01-01

    Bacteria and their viruses (phage) are fundamental drivers of many ecosystem processes including global biogeochemistry and horizontal gene transfer. While databases and resources for studying function in uncultured bacterial communities are relatively advanced, many fewer exist for their viral counterparts. The issue is largely technical in that the majority (often 90%) of viral sequences are functionally ‘unknown’ making viruses a virtually untapped resource of functional and physiological information. Here, we provide a community resource that organizes this unknown sequence space into 27 K high confidence protein clusters using 32 viral metagenomes from four biogeographic regions in the Pacific Ocean that vary by season, depth, and proximity to land, and include some of the first deep pelagic ocean viral metagenomes. These protein clusters more than double currently available viral protein clusters, including those from environmental datasets. Further, a protein cluster guided analysis of functional diversity revealed that richness decreased (i) from deep to surface waters, (ii) from winter to summer, (iii) and with distance from shore in surface waters only. These data provide a framework from which to draw on for future metadata-enabled functional inquiries of the vast viral unknown. PMID:23468974

  8. The Pacific Ocean virome (POV): a marine viral metagenomic dataset and associated protein clusters for quantitative viral ecology.

    PubMed

    Hurwitz, Bonnie L; Sullivan, Matthew B

    2013-01-01

    Bacteria and their viruses (phage) are fundamental drivers of many ecosystem processes including global biogeochemistry and horizontal gene transfer. While databases and resources for studying function in uncultured bacterial communities are relatively advanced, many fewer exist for their viral counterparts. The issue is largely technical in that the majority (often 90%) of viral sequences are functionally 'unknown' making viruses a virtually untapped resource of functional and physiological information. Here, we provide a community resource that organizes this unknown sequence space into 27 K high confidence protein clusters using 32 viral metagenomes from four biogeographic regions in the Pacific Ocean that vary by season, depth, and proximity to land, and include some of the first deep pelagic ocean viral metagenomes. These protein clusters more than double currently available viral protein clusters, including those from environmental datasets. Further, a protein cluster guided analysis of functional diversity revealed that richness decreased (i) from deep to surface waters, (ii) from winter to summer, (iii) and with distance from shore in surface waters only. These data provide a framework from which to draw on for future metadata-enabled functional inquiries of the vast viral unknown.

  9. The anti-viral factor APOBEC3G enhances natural killer cell recognition of HIV-infected primary T cells

    PubMed Central

    Norman, Jason M.; Mashiba, Michael; McNamara, Lucy A; Onafuwa-Nuga, Adewunmi; Chiari-Fort, Estelle; Shen, Wenwen; Collins, Kathleen L.

    2011-01-01

    APOBEC3G (A3G) is an intrinsic antiviral factor that inhibits HIV replication by deaminating cytidine residues to uridine. This causes G-to-A hypermutation in the opposite strand and results in viral inactivation. HIV counteracts A3G through the activity of viral infectivity factor (Vif), which promotes A3G degradation. We report that viral protein R (Vpr), which interacts with a uracil glycosylase, also counteracts A3G by reducing uridine incorporation. However, this process results in activation of the DNA damage response pathway and expression of NK cell activating ligands. Our results reveal that pathogen-induced cytidine deamination and the DNA damage response to viral-mediated repair of uridine incorporation enhance recognition of HIV-infected cells by NK cells. PMID:21874023

  10. A Novel Role of the Potyviral Helper Component Proteinase Contributes To Enhance the Yield of Viral Particles

    PubMed Central

    Gallo, Araíz; Calvo, María; Pérez, José de Jesús

    2014-01-01

    ABSTRACT The helper component proteinase (HCPro) is an indispensable, multifunctional protein of members of the genus Potyvirus and other viruses of the family Potyviridae. This viral factor is directly involved in diverse steps of viral infection, such as aphid transmission, polyprotein processing, and suppression of host antiviral RNA silencing. In this paper, we show that although a chimeric virus based on the potyvirus Plum pox virus lacking HCPro, which was replaced by a heterologous silencing suppressor, caused an efficient infection in Nicotiana benthamiana plants, its viral progeny had very reduced infectivity. Making use of different approaches, here, we provide direct evidence of a previously unknown function of HCPro in which the viral factor enhances the stability of its cognate capsid protein (CP), positively affecting the yield of virions and consequently improving the infectivity of the viral progeny. Site-directed mutagenesis revealed that the ability of HCPro to stabilize CP and enhance the yield of infectious viral particles is not linked to any of its previously known activities and helped us to delimit the region of HCPro involved in this function in the central region of the protein. Moreover, the function is highly specific and cannot be fulfilled by the HCPro of a heterologous potyvirus. The importance of this novel requirement in regulating the sorting of the viral genome to be subjected to replication, translation, and encapsidation, thus contributing to the synchronization of these viral processes, is discussed. IMPORTANCE Potyviruses form one of the most numerous groups of plant viruses and are a major cause of crop loss worldwide. It is well known that these pathogens make use of virus-derived multitasking proteins, as well as dedicated host factors, to successfully infect their hosts. Here, we describe a novel requirement for the proper yield and infectivity of potyviral progeny. In this case, such a function is performed by the

  11. Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins.

    PubMed

    Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann; Becher, Paul

    2017-04-01

    RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5' terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  12. Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

    PubMed Central

    Kleine Büning, Maximiliane; Meyer, Denise; Austermann-Busch, Sophia; Roman-Sosa, Gleyder; Rümenapf, Tillmann

    2017-01-01

    RNA recombination is a major driving force for the evolution of RNA viruses and is significantly implicated in the adaptation of viruses to new hosts, changes of virulence, as well as in the emergence of new viruses including drug-resistant and escape mutants. However, the molecular details of recombination in animal RNA viruses are only poorly understood. In order to determine whether viral RNA recombination depends on translation of viral proteins, a nonreplicative recombination system was established which is based on cotransfection of cells with synthetic bovine viral diarrhea virus (family Flaviviridae) RNA genome fragments either lacking the internal ribosome entry site required for cap-independent translation or lacking almost the complete polyprotein coding region. The emergence of a number of recombinant viruses demonstrated that IRES-mediated translation of viral proteins is dispensable for efficient recombination and suggests that RNA recombination can occur in the absence of viral proteins. Analyses of 58 independently emerged viruses led to the detection of recombinant genomes with duplications, deletions and insertions in the 5′ terminal region of the open reading frame, leading to enlarged core fusion proteins detectable by Western blot analysis. This demonstrates a remarkable flexibility of the pestivirus core protein. Further experiments with capped and uncapped genome fragments containing a luciferase gene for monitoring the level of protein translation revealed that even a ∼1,000-fold enhancement of translation of viral proteins did not increase the frequency of RNA recombination. Taken together, this study highlights that nonreplicative RNA recombination does not require translation of viral proteins. PMID:28338950

  13. An evolutionary role for HIV latency in enhancing viral transmission.

    PubMed

    Rouzine, Igor M; Weinberger, Ariel D; Weinberger, Leor S

    2015-02-26

    HIV latency is the chief obstacle to eradicating HIV but is widely believed to be an evolutionary accident providing no lentiviral fitness advantage. However, findings of latency being "hardwired" into HIV's gene-regulatory circuitry appear inconsistent with latency being an evolutionary accident, given HIV's rapid mutation rate. Here, we propose that latency is an evolutionary "bet-hedging" strategy whose frequency has been optimized to maximize lentiviral transmission by reducing viral extinction during mucosal infections. The model quantitatively fits the available patient data, matches observations of high-frequency latency establishment in cell culture and primates, and generates two counterintuitive but testable predictions. The first prediction is that conventional CD8-depletion experiments in SIV-infected macaques increase latent cells more than viremia. The second prediction is that strains engineered to have higher replicative fitness—via reduced latency—will exhibit lower infectivity in animal-model mucosal inoculations. Therapeutically, the theory predicts treatment approaches that may substantially enhance "activate-and-kill" HIV-cure strategies. Copyright © 2015 Elsevier Inc. All rights reserved.

  14. Roles of serine and threonine residues of mumps virus P protein in viral transcription and replication.

    PubMed

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

    2014-04-01

    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. 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 novel strategies to

  15. 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

  16. Rodent Models of HAND and Drug Abuse: Exogenous Administration of Viral Protein(s) and Cocaine

    PubMed Central

    Yao, Honghong

    2015-01-01

    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

  17. 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

  18. Translational regulation of viral secretory proteins by the 5' coding regions and a viral RNA-binding protein.

    PubMed

    Nordholm, Johan; Petitou, Jeanne; Östbye, Henrik; da Silva, Diogo V; Dou, Dan; Wang, Hao; Daniels, Robert

    2017-08-07

    A primary function of 5' regions in many secretory protein mRNAs is to encode an endoplasmic reticulum (ER) targeting sequence. In this study, we show how the regions coding for the ER-targeting sequences of the influenza glycoproteins NA and HA also function as translational regulatory elements that are controlled by the viral RNA-binding protein (RBP) NS1. The translational increase depends on the nucleotide composition and 5' positioning of the ER-targeting sequence coding regions and is facilitated by the RNA-binding domain of NS1, which can associate with ER membranes. Inserting the ER-targeting sequence coding region of NA into different 5' UTRs confirmed that NS1 can promote the translation of secretory protein mRNAs based on the nucleotides within this region rather than the resulting amino acids. By analyzing human protein mRNA sequences, we found evidence that this mechanism of using 5' coding regions and particular RBPs to achieve gene-specific regulation may extend to human-secreted proteins. © 2017 Nordholm et al.

  19. Morphology-Dependent HIV-Enhancing Effect of Semen-Derived Enhancer of Viral Infection

    PubMed Central

    Qiao, Xin; Jeon, Jaekyun; Cole, Amy L.; Matos, Jason O.; Bautista, Stephany; Castillo, Justin; Hung, Ivan; Gan, Zhehong; Tatulian, Suren A.; Cole, Alexander M.; Chen, Bo

    2015-01-01

    PAP248–286 is a 39-residue fragment (residues 248 to 286) derived from protease cleavage of prostatic acidic phosphatase in semen. The amyloid fibrils formed in vitro by PAP248–286 can dramatically enhance human immunodeficiency virus (HIV) infection. To our knowledge, we present the first report that the HIV-enhancing potency of fibrils formed by PAP248–286 is morphology dependent. We identified pleomorphic fibrils by transmission electron microscopy in two buffer conditions. Our solid-state NMR data showed that these fibrils consist of molecules in distinct conformations. In agreement with NMR, fluorescence measurements confirmed that they are assembled along different pathways, with distinct molecular structures. Furthermore, our cell-based infectivity tests detected distinct HIV-enhancing potencies for fibrils in distinct morphologies. In addition, our transmission electron microscopy and NMR results showed that semen-derived enhancer of viral infection fibrils formed in sodium bicarbonate buffer remain stable over time, but semen-derived enhancer of viral infection fibrils formed in phosphate buffered saline keep evolving after the initial 7 days incubation period. Given time, most of the assemblies in phosphate buffered saline will turn into elongated thin fibrils. They have similar secondary structure but different packing than thin fibrils formed initially after 7 days incubation. PMID:25902442

  20. Tat acetylation modulates assembly of a viral-host RNA–protein transcription complex

    PubMed Central

    D'Orso, Iván; Frankel, Alan D.

    2009-01-01

    HIV-1 Tat enhances viral transcription elongation by forming a ribonucleoprotein complex with transactivating responsive (TAR) RNA and P-TEFb, an elongation factor composed of cyclin T1 (CycT1) and Cdk9 that phosphorylates the C-terminal domain of RNA polymerase II. Previous studies have shown that Lys-28 in the activation domain (AD) of Tat is essential for HIV-1 transcription and replication and is acetylated by p300/CBP-associated factor (PCAF), but the mechanistic basis of the Lys-28 requirement is unknown. Here, we show that Lys-28 acetylation modulates the affinity and stability of HIV-1 Tat–CycT1–TAR complexes by enhancing an interaction with the CycT1 Tat–TAR recognition motif. High-affinity assembly correlates strongly with stimulation of transcription elongation in vitro and Tat activation in vivo. In marked contrast, bovine lentiviral Tat proteins have evolved a high-affinity TAR interaction that does not require PCAF-mediated acetylation of the Tat AD or CycT1 for RNA binding, whereas HIV-2 Tat has evolved an intermediate mechanism that uses a duplicated TAR element and CycT1 to enhance RNA affinity and consequently transcription activation. The coevolution of Tat acetylation, CycT1 dependence, and TAR binding affinity is seen in viral replication assays using Tat proteins that rely on CycT1 for TAR binding but are acetylation deficient, where compensatory mutations rapidly accrue in TAR to generate high-affinity, CycT1-independent complexes reminiscent of the bovine viruses. Thus, lysine acetylation can be used to modulate and evolve the strength of a viral-host RNA–protein complex, thereby tuning the levels of transcription elongation. PMID:19223581

  1. 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

  2. Viral proteins that bridge unconnected proteins and components in the human PPI network.

    PubMed

    Rachita, H R; Nagarajaram, H A

    2014-07-29

    Viruses, despite having small genomes and few proteins, make an array of interactions with host proteins as they solely depend on host machinery for their replication and reproduction. Hence, analysis of the Human-Virus Protein-Protein Interaction Network (Hu-Vir PPI network) helps us to gain certain insights into the molecular mechanisms underlying the hijacking of host cell machinery by viruses for their perpetuation. Here we report an analysis of the Human-Virus Bridged PPI Networks that has led us to identify viral articulation points (VAPs) which connect unconnected components of the Human-PPI (Hu-PPI) network. VAPs cross-link peripheral nodes to the giant component of the Hu-PPI network. VAPs interact with a number of relatively lower topologically central human proteins and are conserved among related viruses. The linked nodes comprise of those that are mostly expressed during viral infection, as well as those that are found exclusively in some metabolic pathways, indicating that the novel viral mediation of certain human protein-protein interactions may form the basis for virus-specific tuning of the host machinery. The functional importance of VAPs and their interaction partners in virus replication make them potential drug targets against viral infection. Our investigations also led to the discovery of an example of a Human Endogenous Retrovirus (HERV) encoded protein, syncytin, as an Articulation Point (AP) in the Hu-PPI network, suggesting that VAPs may be retained in a genome if they result in any beneficial function in the host.

  3. 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.

  4. Effect of Acyclovir on Viral Protein Synthesis in Cells Infected with Herpes Simplex Virus Type 1

    PubMed Central

    Furman, Phillip A.; McGuirt, Paul V.

    1983-01-01

    The effect of the antiviral agent 9-(2-hydroxyethoxymethyl)guanine (acyclovir) on herpes simplex virus type 1 protein synthesis during virus replication was examined. Treatment of infected cells with acyclovir markedly affected the amounts of the four major glycosylated and certain non-glycosylated viral polypeptides synthesized; other viral polypeptides were made in normal amounts. The reduced amount of late protein synthesis was most likely due to the inhibition of progeny viral DNA synthesis by acyclovir. Images PMID:6301368

  5. 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

  6. Visualizing Viral Protein Structures in Cells Using Genetic Probes for Correlated Light and Electron Microscopy

    PubMed Central

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

    2015-01-01

    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

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

    PubMed

    Zhang, Hua; Song, Lei; Cong, Haolong; Tien, Po

    2015-10-01

    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. The nuclear protein Sam68 is found as an additional new host factor that interacts with the EV71 IRES during infection and could potentially

  8. 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

  9. A Family of Plasmodesmal Proteins with Receptor-Like Properties for Plant Viral Movement Proteins

    PubMed Central

    Schmitt-Keichinger, Corinne; Fernandez-Calvino, Lourdes; Didier, Pascal; Lerich, Alexander; Mutterer, Jérome; Thomas, Carole L.; Heinlein, Manfred; Mély, Yves; Maule, Andrew J.; Ritzenthaler, Christophe

    2010-01-01

    Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement. PMID:20886105

  10. A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

    PubMed

    Amari, Khalid; Boutant, Emmanuel; Hofmann, Christina; Schmitt-Keichinger, Corinne; Fernandez-Calvino, Lourdes; Didier, Pascal; Lerich, Alexander; Mutterer, Jérome; Thomas, Carole L; Heinlein, Manfred; Mély, Yves; Maule, Andrew J; Ritzenthaler, Christophe

    2010-09-23

    Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

  11. Targeted Killing of Virally Infected Cells by Radiolabeled Antibodies to Viral Proteins

    PubMed Central

    Dadachova, Ekaterina; Patel, Mahesh C; Toussi, Sima; Apostolidis, Christos; Morgenstern, Alfred; Brechbiel, Martin W; Gorny, Miroslaw K; Zolla-Pazner, Susan; Casadevall, Arturo; Goldstein, Harris

    2006-01-01

    Background The HIV epidemic is a major threat to health in the developing and western worlds. A modality that targets and kills HIV-1-infected cells could have a major impact on the treatment of acute exposure and the elimination of persistent reservoirs of infected cells. The aim of this proof-of-principle study was to demonstrate the efficacy of a therapeutic strategy of targeting and eliminating HIV-1-infected cells with radiolabeled antibodies specific to viral proteins in vitro and in vivo. Methods and Findings Antibodies to HIV-1 envelope glycoproteins gp120 and gp41 labeled with radioisotopes bismuth 213 (213Bi) and rhenium 188 (188Re) selectively killed chronically HIV-1-infected human T cells and acutely HIV-1-infected human peripheral blood mononuclear cells (hPBMCs) in vitro. Treatment of severe combined immunodeficiency (SCID) mice harboring HIV-1-infected hPBMCs in their spleens with a 213Bi- or 188Re-labeled monoclonal antibody (mAb) to gp41 resulted in a 57% injected dose per gram uptake of radiolabeled mAb in the infected spleens and in a greater than 99% elimination of HIV-1-infected cells in a dose-dependent manner. The number of HIV-1-infected thymocytes decreased 2.5-fold in the human thymic implant grafts of SCID mice treated with the 188Re-labeled antibody to gp41 compared with those treated with the 188Re-control mAb. The treatment did not cause acute hematologic toxicity in the treated mice. Conclusions The current study demonstrates the effectiveness of HIV-targeted radioimmunotherapy and may provide a novel treatment option in combination with highly active antiretroviral therapy for the eradication of HIV. PMID:17090209

  12. 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

  13. Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes.

    PubMed

    Casciano, Jessica C; Duchemin, Nicholas J; Lamontagne, R Jason; Steel, Laura F; Bouchard, Michael J

    2017-01-01

    Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases.

  14. Hepatitis B virus modulates store-operated calcium entry to enhance viral replication in primary hepatocytes

    PubMed Central

    Casciano, Jessica C.; Duchemin, Nicholas J.; Lamontagne, R. Jason; Steel, Laura F.; Bouchard, Michael J.

    2017-01-01

    Many viruses modulate calcium (Ca2+) signaling to create a cellular environment that is more permissive to viral replication, but for most viruses that regulate Ca2+ signaling, the mechanism underlying this regulation is not well understood. The hepatitis B virus (HBV) HBx protein modulates cytosolic Ca2+ levels to stimulate HBV replication in some liver cell lines. A chronic HBV infection is associated with life-threatening liver diseases, including hepatocellular carcinoma (HCC), and HBx modulation of cytosolic Ca2+ levels could have an important role in HBV pathogenesis. Whether HBx affects cytosolic Ca2+ in a normal hepatocyte, the natural site of an HBV infection, has not been addressed. Here, we report that HBx alters cytosolic Ca2+ signaling in cultured primary hepatocytes. We used single cell Ca2+ imaging of cultured primary rat hepatocytes to demonstrate that HBx elevates the cytosolic Ca2+ level in hepatocytes following an IP3-linked Ca2+ response; HBx effects were similar when expressed alone or in the context of replicating HBV. HBx elevation of the cytosolic Ca2+ level required extracellular Ca2+ influx and store-operated Ca2+ (SOC) entry and stimulated HBV replication in hepatocytes. We used both targeted RT-qPCR and transcriptome-wide RNAseq analyses to compare levels of SOC channel components and other Ca2+ signaling regulators in HBV-expressing and control hepatocytes and show that the transcript levels of these various proteins are not affected by HBV. We also show that HBx regulation of SOC-regulated Ca2+ accumulation is likely the consequence of HBV modulation of a SOC channel regulatory mechanism. In support of this, we link HBx enhancement of SOC-regulated Ca2+ accumulation to Ca2+ uptake by mitochondria and demonstrate that HBx stimulates mitochondrial Ca2+ uptake in primary hepatocytes. The results of our study may provide insights into viral mechanisms that affect Ca2+ signaling to regulate viral replication and virus-associated diseases

  15. Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation

    PubMed Central

    Rosa-Calatrava, Manuel; Puvion-Dutilleul, Francine; Lutz, Pierre; Dreyer, Dominique; De Thé, Hugues; Chatton, Bruno; Kedinger, Claude

    2003-01-01

    The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation. PMID:14528266

  16. Adenovirus protein IX sequesters host-cell promyelocytic leukaemia protein and contributes to efficient viral proliferation.

    PubMed

    Rosa-Calatrava, Manuel; Puvion-Dutilleul, Francine; Lutz, Pierre; Dreyer, Dominique; de Thé, Hugues; Chatton, Bruno; Kedinger, Claude

    2003-10-01

    The product of adenovirus type 5 (Ad5) gene IX, protein IX (pIX), is a multifunctional protein that stabilizes the viral capsid and has transcriptional activity. We show that pIX also contributes to the Ad5-induced reorganization of the host-cell nuclear ultrastructure: pIX induces the formation of specific and dynamic nuclear inclusions, and the host promyelocytic leukaemia (PML) protein, which is the main structural organizer of PML bodies, is stably relocated and confined within the pIX-induced inclusions late in infection. Our results suggest that Ad5 has evolved a unique strategy that leads to the sustained neutralization of PML bodies throughout infection, thereby ensuring optimal viral proliferation.

  17. Going against the Tide: Selective Cellular Protein Synthesis during Virally Induced Host Shutoff.

    PubMed

    Cao, Shuai; Dhungel, Pragyesh; Yang, Zhilong

    2017-09-01

    Many viral infections cause host shutoff, a state in which host protein synthesis is globally inhibited. Emerging evidence from vaccinia and influenza A virus infections indicates that subsets of cellular proteins are resistant to host shutoff and continue to be synthesized. Remarkably, the proteins of oxidative phosphorylation, the cellular-energy-generating machinery, are selectively synthesized in both cases. Identifying mechanisms that drive selective protein synthesis should facilitate understanding both viral replication and fundamental cell biology. Copyright © 2017 American Society for Microbiology.

  18. Graphical representation and mathematical characterization of protein sequences and applications to viral proteins.

    PubMed

    Ghosh, Ambarnil; Nandy, Ashesh

    2011-01-01

    Graphical representation and numerical characterization (GRANCH) of nucleotide and protein sequences is a new field that is showing a lot of promise in analysis of such sequences. While formulation and applications of GRANCH techniques for DNA/RNA sequences started just over a decade ago, analyses of protein sequences by these techniques are of more recent origin. The emphasis is still on developing the underlying technique, but significant results have been achieved in using these methods for protein phylogeny, mass spectral data of proteins and protein serum profiles in parasites, toxicoproteomics, determination of different indices for use in QSAR studies, among others. We briefly mention these in this chapter, with some details on protein phylogeny and viral diseases. In particular, we cover a systematic method developed in GRANCH to determine conserved surface exposed peptide segments in selected viral proteins that can be used for drug and vaccine targeting. The new GRANCH techniques and applications for DNAs and proteins are covered briefly to provide an overview to this nascent field.

  19. Recombinant protein-based viral disease diagnostics in veterinary medicine.

    PubMed

    Balamurugan, Vinayagamurthy; Venkatesan, Gnanavel; Sen, Arnab; Annamalai, Lakshmanan; Bhanuprakash, Veerakyathappa; Singh, Raj Kumar

    2010-09-01

    Identification of pathogens or antibody response to pathogens in human and animals modulates the treatment strategies for naive population and subsequent infections. Diseases can be controlled and even eradicated based on the epidemiology and effective prophylaxis, which often depends on development of efficient diagnostics. In addition, combating newly emerging diseases in human as well as animal healthcare is challenging and is dependent on developing safe and efficient diagnostics. Detection of antibodies directed against specific antigens has been the method of choice for documenting prior infection. Other than zoonosis, development of inexpensive vaccines and diagnostics is a unique problem in animal healthcare. The advent of recombinant DNA technology and its application in the biotechnology industry has revolutionized animal healthcare. The use of recombinant DNA technology in animal disease diagnosis has improved the rapidity, specificity and sensitivity of various diagnostic assays. This is because of the absence of host cellular proteins in the recombinant derived antigen preparations that dramatically decrease the rate of false-positive reactions. Various recombinant products are used for disease diagnosis in veterinary medicine and this article discusses recombinant-based viral disease diagnostics currently used for detection of pathogens in livestock and poultry.

  20. Chemotherapy targeting by DNA capture in viral protein particles

    PubMed Central

    Agadjanian, Hasmik; Chu, David; Hwang, Jae Youn; Wachsmann-Hogiu, Sebastian; Rentsendorj, Altan; Song, Lei; Valluripalli, Vinod; Lubow, Jay; Ma, Jun; Sharifi, Behrooz; Farkas, Daniel L; Medina-Kauwe, Lali K

    2012-01-01

    Aim This study tests the hypothesis that DNA intercalation and electrophilic interactions can be exploited to noncovalently assemble doxorubicin in a viral protein nanoparticle designed to target and penetrate tumor cells through ligand-directed delivery. We further test whether this new paradigm of doxorubicin targeting shows therapeutic efficacy and safety in vitro and in vivo. Materials & methods We tested serum stability, tumor targeting and therapeutic efficacy in vitro and in vivo using biochemical, microscopy and cytotoxicity assays. Results Self-assembly formed approximately 10-nm diameter serum-stable nanoparticles that can target and ablate HER2+ tumors at >10× lower dose compared with untargeted doxorubicin, while sparing the heart after intravenous delivery. The targeted nanoparticle tested here allows doxorubicin potency to remain unaltered during assembly, transport and release into target cells, while avoiding peripheral tissue damage and enabling lower, and thus safer, drug dose for tumor killing. Conclusion This nanoparticle may be an improved alternative to chemical conjugates and signal-blocking antibodies for tumor-targeted treatment. PMID:22385197

  1. 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

  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

    2016-07-07

    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.

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

    PubMed Central

    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.

    2016-01-01

    Viral proteins mimic host protein structure and function to redirect cellular processes and subvert innate defenses1. Small basic proteins compact and regulate both viral and cellular DNA genomes. Nucleosomes are the repeating units of cellular chromatin and play an important role in innate immune responses2. 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 histones3. Although protein VII binds viral DNA and is incorporated with viral genomes into virus particles4,5, it is unknown whether protein VII impacts cellular chromatin. Our observation that protein VII alters cellular chromatin led us to hypothesize that this impacts antiviral responses during adenovirus infection. We found 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 protein composition of host chromatin. We found that protein VII is necessary and sufficient for retention in 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 responses6,7. 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

  4. 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

  5. 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

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

    2015-12-30

    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. 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. 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. 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. 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. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Tat is a multifunctional viral protein that modulates cellular gene expression and functions.

    PubMed

    Clark, Evan; Nava, Brenda; Caputi, Massimo

    2017-02-07

    The human immunodeficiency virus type I (HIV-1) has developed several strategies to condition the host environment to promote viral replication and spread. Viral proteins have evolved to perform multiple functions, aiding in the replication of the viral genome and modulating the cellular response to the infection. Tat is a small, versatile, viral protein that controls transcription of the HIV genome, regulates cellular gene expression and generates a permissive environment for viral replication by altering the immune response and facilitating viral spread to multiple tissues. Studies carried out utilizing biochemical, cellular, and genomic approaches show that the expression and activity of hundreds of genes and multiple molecular networks are modulated by Tat via multiple mechanisms.

  7. 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

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

    PubMed

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

    2015-07-01

    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. 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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  9. Access of viral proteins to mitochondria via mitochondria-associated membranes.

    PubMed

    Williamson, Chad D; Colberg-Poley, Anamaris M

    2009-05-01

    By exploiting host cell machineries, viruses provide powerful tools for gaining insight into cellular pathways. Proteins from two unrelated viruses, human CMV (HCMV) and HCV, are documented to traffic sequentially from the ER into mitochondria, probably through the mitochondria-associated membrane (MAM) compartment. The MAM are sites of ER-mitochondrial contact enabling the direct transfer of membrane bound lipids and the generation of high calcium (Ca2+) microdomains for mitochondria signalling and responses to cellular stress. Both HCV core protein and HCMV UL37 proteins are associated with Ca2+ regulation and apoptotic signals. Trafficking of viral proteins to the MAM may allow viruses to manipulate a variety of fundamental cellular processes, which converge at the MAM, including Ca2+ signalling, lipid synthesis and transfer, bioenergetics, metabolic flow, and apoptosis. Because of their distinct topologies and targeted MAM sub-domains, mitochondrial trafficking (albeit it through the MAM) of the HCMV and HCV proteins predictably involves alternative pathways and, hence, distinct targeting signals. Indeed, we found that multiple cellular and viral proteins, which target the MAM, showed no apparent consensus primary targeting sequences. Nonetheless, these viral proteins provide us with valuable tools to access the poorly characterised MAM compartment, to define its cellular constituents and describe how virus infection alters these to its own end. Furthermore, because proper trafficking of viral proteins is necessary for their function, discovering the requirements for MAM to mitochondrial trafficking of essential viral proteins may provide novel targets for the rational design of anti-viral drugs.

  10. A trio of viral proteins tunes aphid-plant interactions in Arabidopsis thaliana.

    PubMed

    Westwood, Jack H; Groen, Simon C; 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

    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'). 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. 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 the effects of Fny-CMV on this plant and those seen previously in tobacco

  11. 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

  12. 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.

  13. Identification of a Novel Viral Protein Expressed from the PB2 Segment of Influenza A Virus

    PubMed Central

    Watanabe, Mariko; Goto, Hideo

    2015-01-01

    ABSTRACT Over the past 2 decades, several novel influenza virus proteins have been identified that modulate viral infections in vitro and/or in vivo. The PB2 segment, which is one of the longest influenza A virus segments, is known to encode only one viral protein, PB2. In the present study, we used reverse transcription-PCR (RT-PCR) targeting viral mRNAs transcribed from the PB2 segment to look for novel viral proteins encoded by spliced mRNAs. We identified a new viral protein, PB2-S1, encoded by a novel spliced mRNA in which the region corresponding to nucleotides 1513 to 1894 of the PB2 mRNA is deleted. PB2-S1 was detected in virus-infected cells and in cells transfected with a protein expression plasmid encoding PB2. PB2-S1 localized to mitochondria, inhibited the RIG-I-dependent interferon signaling pathway, and interfered with viral polymerase activity (dependent on its PB1-binding capability). The nucleotide sequences around the splicing donor and acceptor sites for PB2-S1 were highly conserved among pre-2009 human H1N1 viruses but not among human H1N1pdm and H3N2 viruses. PB2-S1-deficient viruses, however, showed growth kinetics in MDCK cells and virulence in mice similar to those of wild-type virus. The biological significance of PB2-S1 to the replication and pathogenicity of seasonal H1N1 influenza A viruses warrants further investigation. IMPORTANCE Transcriptome analysis of cells infected with influenza A virus has improved our understanding of the host response to viral infection, because such analysis yields considerable information about both in vitro and in vivo viral infections. However, little attention has been paid to transcriptomes derived from the viral genome. Here we focused on the splicing of mRNA expressed from the PB2 segment and identified a spliced viral mRNA encoding a novel viral protein. This result suggests that other, as yet unidentified viral proteins encoded by spliced mRNAs could be expressed in virus-infected cells. A viral

  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 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

  16. 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.

  17. Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance

    PubMed Central

    Ketscher, Lars; Hannß, Ronny; Morales, David J.; Basters, Anja; Guerra, Susana; Goldmann, Tobias; Hausmann, Annika; Prinz, Marco; Naumann, Ronald; Pekosz, Andrew; Utermöhlen, Olaf; Lenschow, Deborah J.; Knobeloch, Klaus-Peter

    2015-01-01

    Protein modification by the ubiquitin-like protein ISG15 is an interferon (IFN) effector system, which plays a major role in antiviral defense. ISG15 modification is counteracted by the isopeptidase USP18, a major negative regulator of IFN signaling, which was also shown to exert its regulatory function in an isopeptidase-independent manner. To dissect enzymatic and nonenzymatic functions of USP18 in vivo, we generated knock-in mice (USP18C61A/C61A) expressing enzymatically inactive USP18. USP18C61A/C61A mice displayed increased levels of ISG15 conjugates, validating that USP18 is a major ISG15 isopeptidase in vivo. Unlike USP18−/− mice, USP18C61A/C61A animals did not exhibit morphological abnormalities, fatal IFN hypersensitivity, or increased lethality, clearly showing that major USP18 functions are unrelated to its protease activity. Strikingly, elevated ISGylation in USP18C61A/C61A mice was accompanied by increased viral resistance against vaccinia virus and influenza B virus infections. Enhanced resistance upon influenza B infection in USP18C61A/C61A mice was completely reversed in USP18C61A/C61A mice, which additionally lack ISG15, providing evidence that the observed reduction in viral titers is ISG15 dependent. These results suggest that increasing ISGylation by specific inhibition of USP18 protease activity could constitute a promising antiviral strategy with only a minimal risk of severe adverse effects. PMID:25605921

  18. Selective inactivation of USP18 isopeptidase activity in vivo enhances ISG15 conjugation and viral resistance.

    PubMed

    Ketscher, Lars; Hannß, Ronny; Morales, David J; Basters, Anja; Guerra, Susana; Goldmann, Tobias; Hausmann, Annika; Prinz, Marco; Naumann, Ronald; Pekosz, Andrew; Utermöhlen, Olaf; Lenschow, Deborah J; Knobeloch, Klaus-Peter

    2015-02-03

    Protein modification by the ubiquitin-like protein ISG15 is an interferon (IFN) effector system, which plays a major role in antiviral defense. ISG15 modification is counteracted by the isopeptidase USP18, a major negative regulator of IFN signaling, which was also shown to exert its regulatory function in an isopeptidase-independent manner. To dissect enzymatic and nonenzymatic functions of USP18 in vivo, we generated knock-in mice (USP18(C61A/C61A)) expressing enzymatically inactive USP18. USP18(C61A/C61A) mice displayed increased levels of ISG15 conjugates, validating that USP18 is a major ISG15 isopeptidase in vivo. Unlike USP18(-/-) mice, USP18(C61A/C61A) animals did not exhibit morphological abnormalities, fatal IFN hypersensitivity, or increased lethality, clearly showing that major USP18 functions are unrelated to its protease activity. Strikingly, elevated ISGylation in USP18(C61A/C61A) mice was accompanied by increased viral resistance against vaccinia virus and influenza B virus infections. Enhanced resistance upon influenza B infection in USP18(C61A/C61A) mice was completely reversed in USP18(C61A/C61A) mice, which additionally lack ISG15, providing evidence that the observed reduction in viral titers is ISG15 dependent. These results suggest that increasing ISGylation by specific inhibition of USP18 protease activity could constitute a promising antiviral strategy with only a minimal risk of severe adverse effects.

  19. 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

  20. Critical role of human T-lymphotropic virus type 1 accessory proteins in viral replication and pathogenesis.

    PubMed

    Albrecht, Björn; Lairmore, Michael D

    2002-09-01

    Human T-cell lymphotropic virus type 1 (HTLV-1) infection is associated with a diverse range of lymphoproliferative and neurodegenerative diseases, yet pathogenic mechanisms induced by the virus remain obscure. This complex retrovirus contains typical structural and enzymatic genes but also unique regulatory and accessory genes in four open reading frames (ORFs) of the pX region of the viral genome (pX ORFs I to IV). The regulatory proteins encoded by pX ORFs III and IV, Tax and Rex, respectively, have been extensively characterized. In contrast the contribution of the four accessory proteins p12(I), p27(I), p13(II), and p30(II), encoded by pX ORFs I and II, to viral replication and pathogenesis remained unclear. 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. Emerging evidence indicates that the 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 p12(I) in lymphocyte activation. The endoplasmic reticulum and cis-Golgi localizing p12(I), encoded from pX ORF I, 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 p30(II) localizes to the nucleus and represses viral promoter activity, but may regulate cellular gene expression through p300/CBP or related coactivators of transcription. p13(II) targets mitochondrial proteins, where it alters the organelle morphology and may influence energy metabolism. Collectively, studies of the molecular functions of the HTLV-1 accessory proteins provide insight into strategies used by retroviruses that are associated with lymphoproliferative

  1. The nuclear export protein of H5N1 influenza A viruses recruits Matrix 1 (M1) protein to the viral ribonucleoprotein to mediate nuclear export.

    PubMed

    Brunotte, Linda; Flies, Joe; Bolte, Hardin; Reuther, Peter; Vreede, Frank; Schwemmle, Martin

    2014-07-18

    In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs.

  2. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures.

    PubMed

    Rao, Vasudev R; Eugenin, Eliseo A; Prasad, Vinayaka R

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood-brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes.

  3. Evaluating the Role of Viral Proteins in HIV-Mediated Neurotoxicity Using Primary Human Neuronal Cultures

    PubMed Central

    Rao, Vasudev R.; Eugenin, Eliseo A.; Prasad, Vinayaka R.

    2016-01-01

    Despite the inability of HIV-1 to infect neurons, over half of the HIV-1-infected population in the USA suffers from neurocognitive dysfunction. HIV-infected immune cells in the periphery enter the central nervous system by causing a breach in the blood–brain barrier. The damage to the neurons is mediated by viral and host toxic products released by activated and infected immune and glial cells. To evaluate the toxicity of any viral isolate, viral protein, or host inflammatory protein, we describe a protocol to assess the neuronal apoptosis and synaptic compromise in primary cultures of human neurons and astrocytes. PMID:26714725

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

    PubMed

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

    2012-09-01

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

  5. Viral Proteins Acquired from a Host Converge to Simplified Domain Architectures

    PubMed Central

    Rappoport, Nadav; Linial, Michal

    2012-01-01

    The infection cycle of viruses creates many opportunities for the exchange of genetic material with the host. Many viruses integrate their sequences into the genome of their host for replication. These processes may lead to the virus acquisition of host sequences. Such sequences are prone to accumulation of mutations and deletions. However, in rare instances, sequences acquired from a host become beneficial for the virus. We searched for unexpected sequence similarity among the 900,000 viral proteins and all proteins from cellular organisms. Here, we focus on viruses that infect metazoa. The high-conservation analysis yielded 187 instances of highly similar viral-host sequences. Only a small number of them represent viruses that hijacked host sequences. The low-conservation sequence analysis utilizes the Pfam family collection. About 5% of the 12,000 statistical models archived in Pfam are composed of viral-metazoan proteins. In about half of Pfam families, we provide indirect support for the directionality from the host to the virus. The other families are either wrongly annotated or reflect an extensive sequence exchange between the viruses and their hosts. In about 75% of cross-taxa Pfam families, the viral proteins are significantly shorter than their metazoan counterparts. The tendency for shorter viral proteins relative to their related host proteins accounts for the acquisition of only a fragment of the host gene, the elimination of an internal domain and shortening of the linkers between domains. We conclude that, along viral evolution, the host-originated sequences accommodate simplified domain compositions. We postulate that the trimmed proteins act by interfering with the fundamental function of the host including intracellular signaling, post-translational modification, protein-protein interaction networks and cellular trafficking. We compiled a collection of hijacked protein sequences. These sequences are attractive targets for manipulation of viral

  6. Dissociation of the CD4 downregulation and viral infectivity enhancement functions of human immunodeficiency virus type 1 Nef.

    PubMed Central

    Goldsmith, M A; Warmerdam, M T; Atchison, R E; Miller, M D; Greene, W C

    1995-01-01

    Recent evidence indicates that the nef gene of human immunodeficiency virus type 1 augments rather than inhibits viral replication in both cell culture and in vivo models. In addition, nef alters various normal cellular processes, including the display of CD4 on the cell surface. However, it remains unknown whether the enhancement of infectivity and the downregulation of CD4 represent linked or independent biologic properties of this single protein. In the present studies, mutational analyses were performed to define structure-function relationships within the Nef protein that mediate these effects. To assess the functional consequences of these mutations, sensitive and reliable assays were developed to quantitate the viral infectivity enhancement and CD4 downregulation functions of Nef. The results indicate that membrane-targeting sequences at the N terminus of Nef are important for both functions of Nef, while certain other conserved regions are dispensable for both functions. A conserved proline-X-X repeat segment in the central core of the protein, which is reminiscent of an SH3-binding domain, is critical for the enhancement of infectivity function but is dispensable for CD4 downregulation. However, the downregulation of CD4 by Nef appears to involve a two-step process requiring the initial dissociation of p56lck from CD4 to permit engagement of the endocytic apparatus by CD4. Together, these findings demonstrate that the infectivity enhancement and CD4 downregulation activities of human immunodeficiency virus type 1 Nef can be dissociated. Thus, these processes may be independent of one another in the viral replication cycle. PMID:7769669

  7. Self-assembly in the carboxysome: a viral capsid-like protein shell in bacterial cells.

    PubMed

    Yeates, T O; Tsai, Y; Tanaka, S; Sawaya, M R; Kerfeld, C A

    2007-06-01

    Many proteins self-assemble to form large supramolecular complexes. Numerous examples of these structures have been characterized, ranging from spherical viruses to tubular protein assemblies. Some new kinds of supramolecular structures are just coming to light, while it is likely there are others that have not yet been discovered. The carboxysome is a subcellular structure that has been known for more than 40 years, but whose structural and functional details are just now emerging. This giant polyhedral body is constructed as a closed shell assembled from several thousand protein subunits. Within this protein shell, the carboxysome encapsulates the CO(2)-fixing enzymes, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) and carbonic anhydrase; this arrangement enhances the efficiency of cellular CO(2) fixation. The carboxysome is present in many photosynthetic and chemoautotrophic bacteria, and so plays an important role in the global carbon cycle. It also serves as the prototypical member of what appears to be a large class of primitive protein-based organelles in bacteria. A series of crystal structures is beginning to reveal the secrets of how the carboxysome is assembled and how it enhances the efficiency of CO(2) fixation. Some of the assembly principles revealed in the carboxysome are reminiscent of those seen in icosahedral viral capsids. In addition, the shell appears to be perforated by pores for metabolite transport into and out of the carboxysome, suggesting comparisons to the pores through oligomeric transmembrane proteins, which serve to transport small molecules across the membrane bilayers of cells and eukaryotic organelles.

  8. 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.

  9. Evaluation of protein modification during anti-viral heat bioprocessing by electrospray ionization mass spectrometry.

    PubMed

    Smales, C M; Pepper, D S; James, D C

    2001-01-01

    During the preparation of therapeutic plasma and recombinant protein biopharmaceuticals heat-treatment is routinely applied as a means of viral inactivation. However, as most proteins denature and aggregate under heat stress, it is necessary to add thermostabilizing excipients to protein formulations destined for anti-viral heat-treatment in order to prevent protein damage. Anti-viral heat-treatment bioprocessing therefore requires that a balance be found between the bioprocessing conditions, virus kill and protein integrity. In this study we have utilized a simple model protein, beta-lactoglobulin, to investigate the relationship between virucidal heat-treatment conditions (protein formulation and temperature) and the type and extent of protein modification in the liquid state. A variety of industrially relevant heat-treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) we show here that protein modifications do occur with increasingly harsh heat-treatment. The predominant modification under these conditions was protein glycation by either glucose or fructose derived from hydrolyzed sucrose. Advanced glycation end products and additional unidentified products were also present in beta-lactoglobulin protein samples subjected to extended heat-treatment. These findings have implications for the improvement of anti-viral heat-treatment bioprocesses to ensure the safety and efficacy of protein biopharmaceuticals. CopyrightCopyright 2001 John Wiley & Sons, Ltd.

  10. Dual interaction of a geminivirus replication accessory factor with a viral replication protein and a plant cell cycle regulator.

    PubMed

    Settlage, S B; Miller, A B; Gruissem, W; Hanley-Bowdoin, L

    2001-01-20

    Geminiviruses replicate their small, single-stranded DNA genomes through double-stranded DNA intermediates in plant nuclei using host replication machinery. Like most dicot-infecting geminiviruses, tomato golden mosaic virus encodes a protein, AL3 or C3, that greatly enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that AL3 forms oligomers and interacts with the viral replication initiator AL1. Experiments reported here established that AL3 also interacts with a plant homolog of the mammalian tumor suppressor protein, retinoblastoma (pRb). Analysis of truncated AL3 proteins indicated that pRb and AL1 bind to similar regions of AL3, whereas AL3 oligomerization is dependent on a different region of the protein. Analysis of truncated AL1 proteins located the AL3-binding domain between AL1 amino acids 101 and 180 to a region that also includes the AL1 oligomerization domain and the catalytic site for initiation of viral DNA replication. Interestingly, the AL3-binding domain was fully contiguous with the domain that mediates AL1/pRb interactions. The potential significance of AL3/pRb binding and the coincidence of the domains responsible for AL3, AL1, and pRb interactions are discussed.

  11. The nucleolar protein GLTSCR2 is required for efficient viral replication

    PubMed Central

    Wang, Peng; Meng, Wen; Han, Shi-Chong; Li, Cui-Cui; Wang, Xiao-Jun; Wang, Xiao-Jia

    2016-01-01

    Glioma tumor suppressor candidate region gene 2 protein (GLTSCR2) is a nucleolar protein. In the investigation of the role of GLTSCR2 that played in the cellular innate immune response to viral infection, we found GLTSCR2 supported viral replication of rhabdovirus, paramyxovirus, and coronavirus in cells. Viral infection induced translocation of GLTSCR2 from nucleus to cytoplasm that enabled GLTSCR2 to attenuate type I interferon IFN-β and support viral replication. Cytoplasmic GLTSCR2 was able to interact with retinoic acid-inducible gene I (RIG-I) and the ubiquitin-specific protease 15 (USP15), and the triple interaction induced USP15 activity to remove K63-linked ubiquitination of RIG-I, leading to attenuation of RIG-I and IFN-β. Blocking cytoplasmic translocation of GLTSCR2, by deletion of its nuclear export sequence (NES), abrogated its ability to attenuate IFN-β and support viral replication. GLTSCR2-mediated attenuation of RIG-I and IFN-β led to alleviation of host cell innate immune response to viral infection. Our findings suggested that GLTSCR2 contributed to efficient viral replication, and GLTSCR2 should be considered as a potential target for therapeutic control of viral infection. PMID:27824081

  12. Separation and isolation of BTV dsRNA segments and viral proteins.

    PubMed

    Li, Joseph K-K; Huang, I-Jen; Hayama, Emiko

    2012-05-01

    Bluetongue virus (BTV) genome contains ten double-stranded RNA segments. The sequence of the plus strand of each of the BTV genomic double-stranded RNAs is the same as that of its mRNA, which encodes for a single viral protein, except the smallest S4 segment which can encode for two nonstructural proteins, primarily for the release assistance of the viral progeny. The separation and isolation of each BTV dsRNA segment and viral protein have provided extensive data related to its viral infection, pathology, suppression of host cellular functions, and eventual apoptosis of the infected host cells. This cytoplasmic virus is also an animal killer that costs the U.S. livestock industry at least $125 million yearly. However, this virus has no known effect on humans. Thus, it is very safe to carry out investigation with the virus, preferably in a BSL-2 laboratory.

  13. Influenza B virus non-structural protein 1 counteracts ISG15 antiviral activity by sequestering ISGylated viral proteins

    PubMed Central

    Zhao, Chen; Sridharan, Haripriya; Chen, Ran; Baker, Darren P.; Wang, Shanshan; Krug, Robert M.

    2016-01-01

    The ubiquitin-like protein ISG15 and its conjugation to proteins (ISGylation) are strongly induced by type I interferon. Influenza B virus encodes non-structural protein 1 (NS1B) that binds human ISG15 and provides an appropriate model for determining how ISGylation affects virus replication in human cells. Here using a recombinant virus encoding a NS1B protein defective in ISG15 binding, we show that NS1B counteracts ISGylation-mediated antiviral activity by binding and sequestering ISGylated viral proteins, primarily ISGylated viral nucleoprotein (NP), in infected cells. ISGylated NP that is not sequestered by mutant NS1B acts as a dominant-negative inhibitor of oligomerization of the more abundant unconjugated NP. Consequently formation of viral ribonucleoproteins that catalyse viral RNA synthesis is inhibited, causing decreased viral protein synthesis and virus replication. We verify that ISGylated NP is largely responsible for inhibition of viral RNA synthesis by generating recombinant viruses that lack known ISGylation sites in NP. PMID:27587337

  14. Marked Variability in the Extent of Protein Disorder within and between Viral Families

    PubMed Central

    Pushker, Ravindra; Mooney, Catherine; Davey, Norman E.; Jacqué, Jean-Marc; Shields, Denis C.

    2013-01-01

    Intrinsically disordered regions in eukaryotic proteomes contain key signaling and regulatory modules and mediate interactions with many proteins. Many viral proteomes encode disordered proteins and modulate host factors through the use of short linear motifs (SLiMs) embedded within disordered regions. However, the degree of viral protein disorder across different viruses is not well understood, so we set out to establish the constraints acting on viruses, in terms of their use of disordered protein regions. We surveyed predicted disorder across 2,278 available viral genomes in 41 families, and correlated the extent of disorder with genome size and other factors. Protein disorder varies strikingly between viral families (from 2.9% to 23.1% of residues), and also within families. However, this substantial variation did not follow the established trend among their hosts, with increasing disorder seen across eubacterial, archaebacterial, protists, and multicellular eukaryotes. For example, among large mammalian viruses, poxviruses and herpesviruses showed markedly differing disorder (5.6% and 17.9%, respectively). Viral families with smaller genome sizes have more disorder within each of five main viral types (ssDNA, dsDNA, ssRNA+, dsRNA, retroviruses), except for negative single-stranded RNA viruses, where disorder increased with genome size. However, surveying over all viruses, which compares tiny and enormous viruses over a much bigger range of genome sizes, there is no strong association of genome size with protein disorder. We conclude that there is extensive variation in the disorder content of viral proteomes. While a proportion of this may relate to base composition, to extent of gene overlap, and to genome size within viral types, there remain important additional family and virus-specific effects. Differing disorder strategies are likely to impact on how different viruses modulate host factors, and on how rapidly viruses can evolve novel instances of SLi

  15. Ultrasound enhances the transfection of plasmid DNA by non-viral vectors.

    PubMed

    Hosseinkhani, Hossein; Aoyama, Teruyoshi; Ogawa, Osamu; Tabata, Yasuhiko

    2003-04-01

    Increasing attention has been paid to technology used for the delivery of genetic materials into cells for gene therapy and the generation of genetically engineered cells. So far, viral vectors have been mainly used because of their inherently high transfection efficiency of gene. However, there are some problems to be resolved for the clinical applications, such as the pathogenicity and immunogenicity of viral vectors themselves. Therefore, many research trials with non-viral vectors have been performed to enhance their efficiency to a level comparable to the viral vector. Two directions of these trials exist: material improvement of non-viral vectors and their combination with various external physical stimuli. This paper reviews the latter research trials, with special attention paid to the enhancement of gene expression by ultrasound (US). The expression level of plasmid DNA by various cationized polymers and liposomes is promoted by US irradiation in vitro as well as in vivo. This US-enhanced expression of plasmid DNA will be discussed to emphasize the technical feasibility of US in gene therapy and biotechnology.

  16. Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells

    PubMed Central

    Haatveit, Hanne Merethe; Wessel, Øystein; Markussen, Turhan; Lund, Morten; Thiede, Bernd; Nyman, Ingvild Berg; Braaen, Stine; Dahle, Maria Krudtaa; Rimstad, Espen

    2017-01-01

    Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1–2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection. PMID:28335455

  17. Viral Protein Kinetics of Piscine Orthoreovirus Infection in Atlantic Salmon Blood Cells.

    PubMed

    Haatveit, Hanne Merethe; Wessel, Øystein; Markussen, Turhan; Lund, Morten; Thiede, Bernd; Nyman, Ingvild Berg; Braaen, Stine; Dahle, Maria Krudtaa; Rimstad, Espen

    2017-03-18

    Piscine orthoreovirus (PRV) is ubiquitous in farmed Atlantic salmon (Salmo salar) and the cause of heart and skeletal muscle inflammation. Erythrocytes are important target cells for PRV. We have investigated the kinetics of PRV infection in salmon blood cells. The findings indicate that PRV causes an acute infection of blood cells lasting 1-2 weeks, before it subsides into persistence. A high production of viral proteins occurred initially in the acute phase which significantly correlated with antiviral gene transcription. Globular viral factories organized by the non-structural protein µNS were also observed initially, but were not evident at later stages. Interactions between µNS and the PRV structural proteins λ1, µ1, σ1 and σ3 were demonstrated. Different size variants of µNS and the outer capsid protein µ1 appeared at specific time points during infection. Maximal viral protein load was observed five weeks post cohabitant challenge and was undetectable from seven weeks post challenge. In contrast, viral RNA at a high level could be detected throughout the eight-week trial. A proteolytic cleavage fragment of the µ1 protein was the only viral protein detectable after seven weeks post challenge, indicating that this µ1 fragment may be involved in the mechanisms of persistent infection.

  18. Live cell imaging reveals the relocation of dsRNA binding proteins upon viral infection.

    PubMed

    Barton, Deborah; Roovers, Elke; Gouil, Quentin; C da Fonseca, Guilherme; Reis, Rodrigo S; Jackson, Craig; Overall, Robyn; Fusaro, Adriana; Waterhouse, Peter

    2017-03-15

    Viral infection triggers a range of plant responses such as the activation of the RNA interference (RNAi) pathway. The double-stranded RNA binding (DRB) proteins, DRB3 and DRB4, are part of this pathway and aid in defending against DNA and RNA viruses, respectively. Using live cell imaging, we show that DRB2, DRB3 and DRB5 relocate from their uniform cytoplasmic distribution to concentrated accumulation in nascent viral replication complexes (VRCs) that develop following cell invasion by viral RNA. Inactivation of the DRB3 gene in Arabidopsis, by T-DNA insertion, rendered these plants less able to repress RNA viral replication. We propose a model for the early stages of virus defense in which DRB2, DRB3 and DRB5 are invasion sensors that relocate to nascent VRCs, where they bind to viral RNA and inhibit virus replication.

  19. 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

  20. Cigarette smoke selectively enhances viral PAMP- and virus-induced pulmonary innate immune and remodeling responses in mice.

    PubMed

    Kang, Min-Jong; Lee, Chun Geun; Lee, Jae-Young; Dela Cruz, Charles S; Chen, Zhijian J; Enelow, Richard; Elias, Jack A

    2008-08-01

    Viral infections have more severe consequences in patients who have been exposed to cigarette smoke (CS) than in those not exposed to CS. For example, in chronic obstructive pulmonary disease (COPD), viruses cause more severe disease exacerbation, heightened inflammation, and accelerated loss of lung function compared with other causes of disease exacerbation. Symptomatology and mortality in influenza-infected smokers is also enhanced. To test the hypothesis that these outcomes are caused by CS-induced alterations in innate immunity, we defined the effects of CS on pathogen-associated molecular pattern-induced (PAMP-induced) pulmonary inflammation and remodeling in mice. CS was found to enhance parenchymal and airway inflammation and apoptosis induced by the viral PAMP poly(I:C). CS and poly(I:C) also induced accelerated emphysema and airway fibrosis. The effects of a combination of CS and poly(I:C) were associated with early induction of type I IFN and IL-18, later induction of IL-12/IL-23 p40 and IFN-gamma, and the activation of double-stranded RNA-dependent protein kinase (PKR) and eukaryotic initiation factor-2alpha (eIF2alpha). Further analysis using mice lacking specific proteins indicated a role for TLR3-dependent and -independent pathways as well as a pathway or pathways that are dependent on mitochondrial antiviral signaling protein (MAVS), IL-18Ralpha, IFN-gamma, and PKR. Importantly, CS enhanced the effects of influenza but not other agonists of innate immunity in a similar fashion. These studies demonstrate that CS selectively augments the airway and alveolar inflammatory and remodeling responses induced in the murine lung by viral PAMPs and viruses.

  1. Hepatitis C viral protein translation: mechanisms and implications in developing antivirals.

    PubMed

    Hoffman, Brett; Liu, Qiang

    2011-11-01

    Hepatitis C viral protein translation occurs in a cap-independent manner through the use of an internal ribosomal entry site (IRES) present within the viral 5'-untranslated region. The IRES is composed of highly conserved structural domains that directly recruit the 40S ribosomal subunit to the viral genomic RNA. This frees the virus from relying on a large number of translation initiation factors that are required for cap-dependent translation, conferring a selective advantage to the virus especially in times when the availability of such factors is low. Although the mechanism of translation initiation on the Hepatitis C virus (HCV) IRES is well established, modulation of the HCV IRES activity by both cellular and viral factors is not well understood. As the IRES is essential in the HCV life cycle and as such remains well conserved in an otherwise highly heterogenic virus, the process of HCV protein translation represents an attractive target in the development of novel antivirals. This review will focus on the mechanisms of HCV protein translation and how this process is postulated to be modulated by cis-acting viral factors, as well as trans-acting viral and cellular factors. Numerous therapeutic approaches investigated in targeting HCV protein translation for the development of novel antivirals will also be discussed.

  2. Heat Shock Protein 90 Ensures Efficient Mumps Virus Replication by Assisting with Viral Polymerase Complex Formation.

    PubMed

    Katoh, Hiroshi; Kubota, Toru; Nakatsu, Yuichiro; Tahara, Maino; Kidokoro, Minoru; Takeda, Makoto

    2017-03-15

    Paramyxoviral RNAs are synthesized by a viral RNA-dependent RNA polymerase (RdRp) consisting of the large (L) protein and its cofactor phosphoprotein (P protein). The L protein is a multifunctional protein that catalyzes RNA synthesis, mRNA capping, and mRNA polyadenylation. Growing evidence shows that the stability of several paramyxovirus L proteins is regulated by heat shock protein 90 (Hsp90). In this study, we demonstrated that Hsp90 activity was important for mumps virus (MuV) replication. The Hsp90 activity was required for L-protein stability and activity because an Hsp90-specific inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), destabilized the MuV L protein and suppressed viral RNA synthesis. However, once the L protein formed a mature polymerase complex with the P protein, Hsp90 activity was no longer required for the stability and activity of the L protein. When the Hsp90 activity was inhibited, the MuV L protein was degraded through the CHIP (C terminus of Hsp70-interacting protein)-mediated proteasomal pathway. High concentrations of 17-AAG showed strong cytotoxicity to certain cell types, but combined use of an Hsp70 inhibitor, VER155008, potentiated degradation of the L protein, allowing a sufficient reduction of 17-AAG concentration to block MuV replication with minimum cytotoxicity. Regulation of the L protein by Hsp90 and Hsp70 chaperones was also demonstrated for another paramyxovirus, the measles virus. Collectively, our data show that the Hsp90/Hsp70 chaperone machinery assists in the maturation of the paramyxovirus L protein and thereby in the formation of a mature RdRp complex and efficient viral replication.IMPORTANCE Heat shock protein 90 (Hsp90) is nearly universally required for viral protein homeostasis. Here, we report that Hsp90 activity is required for efficient propagation of mumps virus (MuV). Hsp90 functions in the maintenance of the catalytic subunit of viral polymerase, the large (L) protein, prior to formation of a

  3. Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein.

    PubMed

    Cartier, Christine; Hemonnot, Bénédicte; Gay, Bernard; Bardy, Martine; Sanchiz, Céline; Devaux, Christian; Briant, Laurence

    2003-09-12

    Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.

  4. Common principles and intermediates of viral protein-mediated fusion: the HIV-1 paradigm

    PubMed Central

    Melikyan, Gregory B

    2008-01-01

    Enveloped viruses encode specialized fusion proteins which promote the merger of viral and cell membranes, permitting the cytosolic release of the viral cores. Understanding the molecular details of this process is essential for antiviral strategies. Recent structural studies revealed a stunning diversity of viral fusion proteins in their native state. In spite of this diversity, the post-fusion structures of these proteins share a common trimeric hairpin motif in which the amino- and carboxy-terminal hydrophobic domains are positioned at the same end of a rod-shaped molecule. The converging hairpin motif, along with biochemical and functional data, implies that disparate viral proteins promote membrane merger via a universal "cast-and-fold" mechanism. According to this model, fusion proteins first anchor themselves to the target membrane through their hydrophobic segments and then fold back, bringing the viral and cellular membranes together and forcing their merger. However, the pathways of protein refolding and the mechanism by which this refolding is coupled to membrane rearrangements are still not understood. The availability of specific inhibitors targeting distinct steps of HIV-1 entry permitted the identification of key conformational states of its envelope glycoprotein en route to fusion. These studies provided functional evidence for the direct engagement of the target membrane by HIV-1 envelope glycoprotein prior to fusion and revealed the role of partially folded pre-hairpin conformations in promoting the pore formation. PMID:19077194

  5. DNA methyltransferase DNMT3A associates with viral proteins and impacts HSV-1 infection.

    PubMed

    Rowles, Daniell L; Tsai, Yuan-Chin; Greco, Todd M; Lin, Aaron E; Li, Minghao; Yeh, Justin; Cristea, Ileana M

    2015-06-01

    Viral infections can alter the cellular epigenetic landscape, through modulation of either DNA methylation profiles or chromatin remodeling enzymes and histone modifications. These changes can act to promote viral replication or host defense. Herpes simplex virus type 1 (HSV-1) is a prominent human pathogen, which relies on interactions with host factors for efficient replication and spread. Nevertheless, the knowledge regarding its modulation of epigenetic factors remains limited. Here, we used fluorescently-labeled viruses in conjunction with immunoaffinity purification and MS to study virus-virus and virus-host protein interactions during HSV-1 infection in primary human fibroblasts. We identified interactions among viral capsid and tegument proteins, detecting phosphorylation of the capsid protein VP26 at sites within its UL37-binding domain, and an acetylation within the major capsid protein VP5. Interestingly, we found a nuclear association between viral capsid proteins and the de novo DNA methyltransferase DNA (cytosine-5)-methyltransferase 3A (DNMT3A), which we confirmed by reciprocal isolations and microscopy. We show that drug-induced inhibition of DNA methyltransferase activity, as well as siRNA- and shRNA-mediated DNMT3A knockdowns trigger reductions in virus titers. Altogether, our results highlight a functional association of viral proteins with the mammalian DNA methyltransferase machinery, pointing to DNMT3A as a host factor required for effective HSV-1 infection.

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

    PubMed Central

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

    2015-01-01

    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

  7. 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.

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

    PubMed Central

    Solvik, Tina

    2016-01-01

    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

  9. Detection of viral proteins in human cells lines by xeno-proteomics: elimination of the last valid excuse for not testing every cellular proteome dataset for viral proteins.

    PubMed

    Chernobrovkin, Alexey L; Zubarev, Roman A

    2014-01-01

    Cell cultures used routinely in proteomic experiments may contain proteins from other species because of infection, transfection or just contamination. Since infection or contamination may affect the results of a biological experiment, it is important to test the samples for the presence of "alien" proteins. Usually cells are tested only for the most common infections, and most of the existing tests are targeting specific contaminations. Here we describe a three-step procedure for reliable untargeted detection of viral proteins using proteomics data, and recommend this or similar procedure to be applied to every proteomics dataset submitted for publication.

  10. Post-translational modifications of hepatitis C viral proteins and their biological significance.

    PubMed

    Hundt, Jana; Li, Zhubing; Liu, Qiang

    2013-12-21

    Replication of hepatitis C virus (HCV) depends on the interaction of viral proteins with various host cellular proteins and signalling pathways. Similar to cellular proteins, post-translational modifications (PTMs) of HCV proteins are essential for proper protein function and regulation, thus, directly affecting viral life cycle and the generation of infectious virus particles. Cleavage of the HCV polyprotein by cellular and viral proteases into more than 10 proteins represents an early protein modification step after translation of the HCV positive-stranded RNA genome. The key modifications include the regulated intramembranous proteolytic cleavage of core protein, disulfide bond formation of core, glycosylation of HCV envelope proteins E1 and E2, methylation of nonstructural protein 3 (NS3), biotinylation of NS4A, ubiquitination of NS5B and phosphorylation of core and NS5B. Other modifications like ubiquitination of core and palmitoylation of core and NS4B proteins have been reported as well. For some modifications such as phosphorylation of NS3 and NS5A and acetylation of NS3, we have limited understanding of their effects on HCV replication and pathogenesis while the impact of other modifications is far from clear. In this review, we summarize the available information on PTMs of HCV proteins and discuss their relevance to HCV replication and pathogenesis.

  11. Temporal proteomic analysis and label-free quantification of viral proteins of an invertebrate iridovirus.

    PubMed

    İnce, İkbal Agah; Boeren, Sjef; van Oers, Monique M; Vlak, Just M

    2015-01-01

    Invertebrate iridescent virus 6 (IIV-6) is a nucleocytoplasmic virus with a ~212 kb linear dsDNA genome that encodes 215 putative ORFs. The IIV-6 virion-associated proteins consist of at least 54 virally encoded proteins. One of our previous findings showed that most of these proteins are encoded by genes from the early transcriptional class. This indicated that these structural proteins may not only function in the formation of the virion, but also in the initial stage of viral infection. In the current study, we followed the protein expression profile of IIV-6 over time in Drosophila S2 cells by label-free quantification using a proteomic approach. A total of 95 virally encoded proteins were detected in infected cells, of which 37 were virion proteins. The expressed IIV-6 virion proteins could be categorized into three main clusters based on their expression profiles: proteins with stably low expression levels during infection, proteins with exponentially increasing expression levels during infection and proteins that were initially highly abundant, but showed slightly reduced levels after 48 h post-infection. We thus provided novel information on the kinetics of virion and infected cell-specific protein levels that assists in our understanding of gene regulation in this lesser-known DNA virus model.

  12. 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.

  13. Lipids as modulators of membrane fusion mediated by viral fusion proteins.

    PubMed

    Teissier, Elodie; Pécheur, Eve-Isabelle

    2007-11-01

    Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called "rafts", or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. The Role of F-Box Proteins during Viral Infection

    PubMed Central

    Correa, Régis Lopes; Bruckner, Fernanda Prieto; de Souza Cascardo, Renan; Alfenas-Zerbini, Poliane

    2013-01-01

    The F-box domain is a protein structural motif of about 50 amino acids that mediates protein–protein interactions. The F-box protein is one of the four components of the SCF (SKp1, Cullin, F-box protein) complex, which mediates ubiquitination of proteins targeted for degradation by the proteasome, playing an essential role in many cellular processes. Several discoveries have been made on the use of the ubiquitin–proteasome system by viruses of several families to complete their infection cycle. On the other hand, F-box proteins can be used in the defense response by the host. This review describes the role of F-box proteins and the use of the ubiquitin–proteasome system in virus–host interactions. PMID:23429191

  19. Prediction of Interactions between Viral and Host Proteins Using Supervised Machine Learning Methods

    PubMed Central

    Barman, Ranjan Kumar; Saha, Sudipto; Das, Santasabuj

    2014-01-01

    Background Viral-host protein-protein interaction plays a vital role in pathogenesis, since it defines viral infection of the host and regulation of the host proteins. Identification of key viral-host protein-protein interactions (PPIs) has great implication for therapeutics. Methods In this study, a systematic attempt has been made to predict viral-host PPIs by integrating different features, including domain-domain association, network topology and sequence information using viral-host PPIs from VirusMINT. The three well-known supervised machine learning methods, such as SVM, Naïve Bayes and Random Forest, which are commonly used in the prediction of PPIs, were employed to evaluate the performance measure based on five-fold cross validation techniques. Results Out of 44 descriptors, best features were found to be domain-domain association and methionine, serine and valine amino acid composition of viral proteins. In this study, SVM-based method achieved better sensitivity of 67% over Naïve Bayes (37.49%) and Random Forest (55.66%). However the specificity of Naïve Bayes was the highest (99.52%) as compared with SVM (74%) and Random Forest (89.08%). Overall, the SVM and Random Forest achieved accuracy of 71% and 72.41%, respectively. The proposed SVM-based method was evaluated on blind dataset and attained a sensitivity of 64%, specificity of 83%, and accuracy of 74%. In addition, unknown potential targets of hepatitis B virus-human and hepatitis E virus-human PPIs have been predicted through proposed SVM model and validated by gene ontology enrichment analysis. Our proposed model shows that, hepatitis B virus “C protein” binds to membrane docking protein, while “X protein” and “P protein” interacts with cell-killing and metabolic process proteins, respectively. Conclusion The proposed method can predict large scale interspecies viral-human PPIs. The nature and function of unknown viral proteins (HBV and HEV), interacting partners of host protein

  20. Capsid, membrane and NS3 are the major viral proteins involved in autophagy induced by Japanese encephalitis virus.

    PubMed

    Wang, Xiujin; Hou, Lei; Du, Jige; Zhou, Lei; Ge, Xinna; Guo, Xin; Yang, Hanchun

    2015-08-05

    Japanese encephalitis virus (JEV) is an important zoonotic pathogen causing viral encephalitis in human and reproductive failure in pigs. In the present study, we first examined the autophagy induced by JEV infection in host cells, and then analyzed the JEV proteins involving in autophagy induction, and further investigated the relationship between viral protein and immunity-related GTPases M (IRGM). Our results showed that JEV infection could induce autophagy in host cells and autophagy promoted the replication of JEV in vitro; the cells transfected with individual plasmid that was expressing C, M and NS3 had a significantly higher conversion of LC3-I/II, and enhanced LC3 signals with the fluorescence punctuates accumulation which was completely co-localized with LC3 and increased number of autophagosomes-like vesicles, suggesting that C, M and NS3 are the major viral proteins involving in autophagy induction upon JEV infection; the virus titer in the cells treated by the siRNA specific for IRGM had a significant decrease, and the NS3 signals in the cells transfected with the plasmid that was expressing NS3 were completely co-localized with the IRGM signals, suggesting that the NS3 of JEV could target IRGM which may play a role in the replication of JEV. Our findings help to understand the role of autophagy in JEV and other flaviviruses infections.

  1. Analysis of astrovirus serotype 1 RNA, identification of the viral RNA-dependent RNA polymerase motif, and expression of a viral structural protein.

    PubMed Central

    Lewis, T L; Greenberg, H B; Herrmann, J E; Smith, L S; Matsui, S M

    1994-01-01

    We report the results from sequence analysis and expression studies of the gastroenteritis agent astrovirus serotype 1. We have cloned and sequenced 5,944 nucleotides (nt) of the estimated 7.2-kb RNA genome and have identified three open reading frames (ORFs). ORF-3, at the 3' end, is 2,361 nt in length and is fully encoded in both the genomic and subgenomic viral RNAs. Expression of ORF-3 in vitro yields an 87-kDa protein that is immunoprecipitated with a monoclonal antibody specific for viral capsids. This protein comigrates with an authentic 87-kDa astrovirus protein immunoprecipitated from infected cells, indicating that this region encodes a viral structural protein. The adjacent upstream ORF (ORF-2) is 1,557 nt in length and contains a viral RNA-dependent RNA polymerase motif. The viral RNA-dependent RNA polymerase motifs from four astrovirus serotypes are compared. Partial sequence (2,018 nt) of the most 5' ORF (ORF-1) reveals a 3C-like serine protease motif. The ORF-1 sequence is incomplete. These results indicate that the astrovirus genome is organized with nonstructural proteins encoded at the 5' end and structural proteins at the 3' end. ORF-2 has no start methionine and is in the -1 frame compared with ORF-1. We present sequence evidence for a ribosomal frameshift mechanism for expression of the viral polymerase. Images PMID:8254779

  2. The YXXL Sequences of a Transmembrane Protein of Bovine Leukemia Virus Are Required for Viral Entry and Incorporation of Viral Envelope Protein into Virions

    PubMed Central

    Inabe, Kazunori; Nishizawa, Masako; Tajima, Shigeru; Ikuta, Kazuyoshi; Aida, Yoko

    1999-01-01

    The cytoplasmic domain of an envelope transmembrane glycoprotein (gp30) of bovine leukemia virus (BLV) has two overlapping copies of the (YXXL)2 motif. The N-terminal motif has been implicated in in vitro signal transduction pathways from the external to the intracellular compartment and is also involved in infection and maintenance of high viral loads in sheep that have been experimentally infected with BLV. To determine the role of YXXL sequences in the replication of BLV in vitro, we changed the tyrosine or leucine residues of the N-terminal motif in an infectious molecular clone of BLV, pBLV-IF, to alanine to produce mutated proviruses designated Y487A, L490A, Y498A, L501A, and Y487/498A. Transient transfection of African green monkey kidney COS-1 cells with proviral DNAs that encoded wild-type and mutant sequences revealed that all of the mutated proviral DNAs synthesized mature envelope proteins and released virus particles into the growth medium. However, serial passages of fetal lamb kidney (FLK) cells, which are sensitive to infection with BLV, after transient transfection revealed that mutation of a second tyrosine residue in the N-terminal motif completely prevented the propagation of the virus. Similarly, Y498A and Y487/498A mutant BLV that was produced by the stably transfected COS-1 cells exhibited significantly reduced levels of cell-free virion-mediated transmission. Analysis of the protein compositions of mutant viruses demonstrated that lower levels of envelope protein were incorporated by two of the mutant virions than by wild-type and other mutant virions. Furthermore, a mutation of a second tyrosine residue decreased the specific binding of BLV particles to FLK cells and the capacity for viral penetration. Our data indicate that the YXXL sequences play critical roles in both viral entry and the incorporation of viral envelope protein into the virion during the life cycle of BLV. PMID:9882334

  3. HSC70 interactions with SV40 viral proteins differ between permissive and nonpermissive mammalian cells

    PubMed Central

    Sainis, Ioannis; Angelidis, Charalambos; Pagoulatos, Gerasimos N.; Lazaridis, Ioannis

    2000-01-01

    SV40 belongs to a group of DNA tumor viruses which induce the expression of the 70 Kd heat shock proteins, but the meaning of this induction remains unclear. Investigating the role of hsc70 in the SV40 life cycle, we found that the protein translocates to the nucleus late in infection of permissive CV1 cells, in contrast to infected nonpermissive BALB/3T3 and NIH/3T3 cells in which hsc70 remains cytoplasmic. Moreover, the pattern of hsc70 nuclear staining was diffused and clearly distinguishable from that observed after heat shock. In addition hsc70 late in infection coimmunoprecipitated with the viral capsid protein VP1, suggesting a role in the process of viral packaging. Interactions of hsc70 with the early viral oncoprotein T antigen were observed only in nonpermissive cells, indicating that the binding of the above proteins is specific to cells that do not support viral propagation. Finally, treatment of permissive CV1 cells with interferon γ, a known antiviral cytokine, resulted in hsc70 binding to T antigen. Our results suggest that the role of hsc70 in the process of SV40 infection is directly related to the ability of the host cells to support viral propagation and is clearly different between permissive and nonpermissive cell lines. PMID:11147964

  4. The anaphase promoting complex: a critical target for viral proteins and anti-cancer drugs.

    PubMed

    Heilman, Destin W; Green, Michael R; Teodoro, Jose G

    2005-04-01

    The study of animal viruses has provided extraordinary insights into cell cycle dynamics and tumor biology. The significance of the p53 and Rb tumor suppressor proteins, for example, was discovered due to their interactions with viral oncogenes. In the past several years, investigations with four viral proteins, human immunodeficiency virus type 1 (HIV-1) vpr, adenovirus E4orf4, chicken anemia virus (CAV) apoptin and human T lymphotropic virus type I (HTLV-I) Tax, have indicated that there are also critical viral targets involved in G2/M control. In particular, recent studies with E4orf4 and apoptin have shown that they induce G2/M arrest by targeting and inhibiting the anaphase-promoting complex/cyclosome (APC/C). Notably, these two viral proteins induce apoptosis selectively in transformed cells in a p53-independent manner; thus pathways affected by these proteins are of significant therapeutic interest. Further investigation of the underlying mechanism of G2/M arrest and subsequent apoptosis induced by viral APC/C inhibitors may shed light on the mechanisms of current cancer therapies and provide the foundation for developing novel therapeutic targets.

  5. Zinc finger antiviral protein inhibits coxsackievirus B3 virus replication and protects against viral myocarditis.

    PubMed

    Li, Min; Yan, Kepeng; Wei, Lin; Yang, Jie; Lu, Chenyu; Xiong, Fei; Zheng, Chunfu; Xu, Wei

    2015-11-01

    The host Zinc finger antiviral protein (ZAP) has been reported exhibiting antiviral activity against positive-stranded RNA viruses (Togaviridae), negative-stranded RNA viruses (Filoviridae) and retroviruses (Retroviridae). However, whether ZAP restricts the infection of enterovirus and the development of enterovirus mediated disease remains unknown. Here, we reported the antiviral properties of ZAP against coxsackievirus B3 (CVB3), a single-stranded RNA virus of the Enterovirus genus within the Picornaviridae as a major causative agent of viral myocarditis (VMC). We found that the expression of ZAP was significantly induced after CVB3 infection in heart tissues of VMC mice. ZAP potently inhibited CVB3 replication in cells after infection, while overexpression of ZAP in mice significantly increased the resistance to CVB3 replication and viral myocarditis by significantly reducing cardiac inflammatory cytokine production. The ZAP-responsive elements (ZREs) were mapped to the 3'UTR and 5'UTR of viral RNA. Taken together, ZAP confers resistance to CVB3 infection via directly targeting viral RNA and protects mice from acute myocarditis by suppressing viral replication and cardiac inflammatory cytokine production. Our finding further expands ZAP's range of viral targets, and suggests ZAP as a potential therapeutic target for viral myocarditis caused by CVB3.

  6. 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

  7. Sendai virus assembly: M protein binds to viral glycoproteins in transit through the secretory pathway.

    PubMed Central

    Sanderson, C M; McQueen, N L; Nayak, D P

    1993-01-01

    We have examined the relative ability of Sendai virus M (matrix) protein to associate with membranes containing viral glycoproteins at three distinct stages of the exocytic pathway prior to cell surface appearance. By the use of selective low-temperature incubations or the ionophore monensin, the transport of newly synthesized viral glycoproteins was restricted to either the pre-Golgi intermediate compartment (by incubation at 15 degrees C), the medial Golgi (in the presence of monensin), or the trans-Golgi network (by incubation at 20 degrees C). All three of these treatments resulted in a marked accumulation of the M protein on perinuclear Golgi-like membranes which in each case directly reflected the distribution of the viral F protein. Subsequent redistribution of the F protein to the plasma membrane by removal of the low-temperature (20 degrees C) block resulted in a concomitant redistribution of the M protein, thus implying association of the two components during intracellular transit. The extent of M protein-glycoprotein association was further examined by cell fractionation studies performed under each of the three restrictive conditions. Following equilibrium sedimentation of membranes derived from monensin-treated cells, approximately 40% of the recovered M protein was found to cofractionate with membranes containing the viral glycoproteins. Also, by flotation analyses, a comparable subpopulation of M protein was found to be membrane associated whether viral glycoproteins were restricted to the trans-Golgi network, the medial Golgi, or the pre-Golgi intermediate compartment. Additionally, transient expression of M protein alone from cloned cDNA showed that neither membrane association nor Golgi localization occurs in the absence of Sendai virus glycoproteins. Images PMID:8380460

  8. An integrated map of HIV-human protein complexes that facilitate viral infection.

    PubMed

    Emig-Agius, Dorothea; Olivieri, Kevin; Pache, Lars; Shih, Hsin Ling; Pustovalova, Olga; Bessarabova, Marina; Young, John A T; Chanda, Sumit K; Ideker, Trey

    2014-01-01

    Recent proteomic and genetic studies have aimed to identify a complete network of interactions between HIV and human proteins and genes. This HIV-human interaction network provides invaluable information as to how HIV exploits the host machinery and can be used as a starting point for further functional analyses. We integrated this network with complementary datasets of protein function and interaction to nominate human protein complexes with likely roles in viral infection. Based on our approach we identified a global map of 40 HIV-human protein complexes with putative roles in HIV infection, some of which are involved in DNA replication and repair, transcription, translation, and cytoskeletal regulation. Targeted RNAi screens were used to validate several proteins and complexes for functional impact on viral infection. Thus, our HIV-human protein complex map provides a significant resource of potential HIV-host interactions for further study.

  9. Molecular mechanisms deployed by virally encoded G protein-coupled receptors in human diseases.

    PubMed

    Montaner, Silvia; Kufareva, Irina; Abagyan, Ruben; Gutkind, J Silvio

    2013-01-01

    G protein-coupled receptors (GPCRs) represent the largest family of cell surface molecules involved in signal transduction. Surprisingly, open reading frames for multiple GPCRs were hijacked in the process of coevolution between Herpesviridae family viruses and their human and mammalian hosts. Virally encoded GPCRs (vGPCRs) evolved as parts of viral genomes, and this evolution allowed the power of host GPCR signaling circuitries to be harnessed in order to ensure viral replicative success. Phylogenetically, vGPCRs are distantly related to human chemokine receptors, although they feature several unique characteristics. Here, we describe the molecular mechanisms underlying vGPCR-mediated viral pathogenesis. These mechanisms include constitutive activity, aberrant coupling to human G proteins and β-arrestins, binding and activation by human chemokines, and dimerization with other GPCRs expressed in infected cells. The likely structural basis for these molecular events is described for the two closest viral homologs of human GPCRs. This information may aid in the development of novel targeted therapeutic strategies against viral diseases.

  10. 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

  11. 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

    2016-05-13

    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.

  12. A Narcissus mosaic viral vector system for protein expression and flavonoid production.

    PubMed

    Zhang, Huaibi; Wang, Lei; Hunter, Donald; Voogd, Charlotte; Joyce, Nigel; Davies, Kevin

    2013-07-13

    With the explosive numbers of sequences generated by next generation sequencing, the demand for high throughput screening to understand gene function has grown. Plant viral vectors have been widely used as tools in down-regulating plant gene expression. However, plant viral vectors can also express proteins in a very efficient manner and, therefore, can also serve as a valuable tool for characterizing proteins and their functions in metabolic pathways in planta. In this study, we have developed a Gateway®-based high throughput viral vector cloning system from Narcissus Mosaic Virus (NMV). Using the reporter genes of GFP and GUS, and the plant genes PAP1 (an R2R3 MYB which activates the anthocyanin pathway) and selenium-binding protein 1 (SeBP), we show that NMV vectors and the model plant Nicotiana benthamiana can be used for efficient protein expression, protein subcellular localization and secondary metabolite production. Our results suggest that not only can the plant viral vector system be employed for protein work but also can potentially be amenable to producing valuable secondary metabolites on a large scale, as the system does not require plant regeneration from seed or calli, which are stages where certain secondary metabolites can interfere with development.

  13. A Narcissus mosaic viral vector system for protein expression and flavonoid production

    PubMed Central

    2013-01-01

    Background With the explosive numbers of sequences generated by next generation sequencing, the demand for high throughput screening to understand gene function has grown. Plant viral vectors have been widely used as tools in down-regulating plant gene expression. However, plant viral vectors can also express proteins in a very efficient manner and, therefore, can also serve as a valuable tool for characterizing proteins and their functions in metabolic pathways in planta. Results In this study, we have developed a Gateway®-based high throughput viral vector cloning system from Narcissus Mosaic Virus (NMV). Using the reporter genes of GFP and GUS, and the plant genes PAP1 (an R2R3 MYB which activates the anthocyanin pathway) and selenium-binding protein 1 (SeBP), we show that NMV vectors and the model plant Nicotiana benthamiana can be used for efficient protein expression, protein subcellular localization and secondary metabolite production. Conclusions Our results suggest that not only can the plant viral vector system be employed for protein work but also can potentially be amenable to producing valuable secondary metabolites on a large scale, as the system does not require plant regeneration from seed or calli, which are stages where certain secondary metabolites can interfere with development. PMID:23849589

  14. Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

    PubMed Central

    Avey, Denis; Tepper, Sarah; Pifer, Benjamin; Bahga, Amritpal; Williams, Hunter; Gillen, Joseph; Li, Wenwei; Ogden, Sarah

    2016-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions. IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further

  15. Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36.

    PubMed

    Avey, Denis; Tepper, Sarah; Pifer, Benjamin; Bahga, Amritpal; Williams, Hunter; Gillen, Joseph; Li, Wenwei; Ogden, Sarah; Zhu, Fanxiu

    2016-07-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further characterized the consequences

  16. Presence of viral proteins in drinkable water--sufficient condition to consider water a vector of viral transmission?

    PubMed

    Gutiérrez, M F; Alvarado, M V; Martínez, E; Ajami, N J

    2007-01-01

    In order to determine the role of water as a possible vector for transmission of the most prevalent enteric viruses affecting infantile populations, 226 water samples were collected from Facatativa's (Colombian municipality located 30km away from Bogotá) water works in the years 2000, 2002, and 2005. The samples were clarified and virus was concentrated by filtering and ultrafiltering techniques. The presence of viral protein (VP) was assessed by enzyme immunoassay method (EIA) and viral RNA presence was detected by reverse trascriptase and polymerase chain reaction (RT-PCR). Using these techniques, one sample positive for Astrovirus (HAstV) was found in a sample collected from the river that supplies the aqueduct, two samples positive for Norovirus (NV) from fresh treated potable water and 13 samples positive for Rotavirus (RV), some in water from the plant during treatment and others from treated fresh water. RT-PCR inhibitors were also found in water samples obtained from the plant and in the fresh treated water. No inhibitors were found in the river water. VP, but no nucleic acid, was detected in the water samples at different stages of treatment, thus suggesting that the virus might have been complete and infectious at some stage prior to water purification.

  17. Protective Effect of Surfactant Protein D in Pulmonary Vaccinia Virus Infection: Implication of A27 Viral Protein

    PubMed Central

    Julien, Perino; Thielens, Nicole M.; Crouch, Erika; Spehner, Danièle; Crance, Jean-Marc; Favier, Anne-Laure

    2013-01-01

    Vaccinia virus (VACV) was used as a surrogate of variola virus (VARV) (genus Orthopoxvirus), the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D), constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/-) resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27. PMID:23518578

  18. 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

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

    PubMed

    Iijima, Kenta; Okudaira, Noriyuki; Tamura, Masato; Doi, Akihiro; Saito, Yoshikazu; Shimura, Mari; Goto, Motohito; Matsunaga, Akihiro; Kawamura, Yuki I; Otsubo, Takeshi; Dohi, Taeko; Hoshino, Shigeki; Kano, Shigeyuki; Hagiwara, Shotaro; Tanuma, Junko; Gatanaga, Hiroyuki; Baba, Masanori; Iguchi, Taku; Yanagita, Motoko; Oka, Shinichi; Okamura, Tadashi; Ishizaka, Yukihito

    2013-08-05

    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). 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. 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.

  20. 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

  1. Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system

    PubMed Central

    Maunder, H. E.; Wright, J.; Kolli, B. R.; Vieira, C. R.; Mkandawire, T. T.; Tatoris, S.; Kennedy, V.; Iqball, S.; Devarajan, G.; Ellis, S.; Lad, Y.; Clarkson, N. G.; Mitrophanous, K. A.; Farley, D. C.

    2017-01-01

    A key challenge in the field of therapeutic viral vector/vaccine manufacturing is maximizing production. For most vector platforms, the ‘benchmark' vector titres are achieved with inert reporter genes. However, expression of therapeutic transgenes can often adversely affect vector titres due to biological effects on cell metabolism and/or on the vector virion itself. Here, we exemplify the novel ‘Transgene Repression In vector Production' (TRiP) system for the production of both RNA- and DNA-based viral vectors. The TRiP system utilizes a translational block of one or more transgenes by employing the bacterial tryptophan RNA-binding attenuation protein (TRAP), which binds its target RNA sequence close to the transgene initiation codon. We report enhancement of titres of lentiviral vectors expressing Cyclo-oxygenase-2 by 600-fold, and adenoviral vectors expressing the pro-apoptotic gene Bax by >150,000-fold. The TRiP system is transgene-independent and will be a particularly useful platform in the clinical development of viral vectors expressing problematic transgenes. PMID:28345582

  2. Expression of the 1918 Influenza A Virus PB1-F2 Enhances the Pathogenesis of Viral and Secondary Bacterial Pneumonia

    PubMed Central

    McAuley, Julie L.; Hornung, Felicita; Boyd, Kelli L.; Smith, Amber M.; McKeon, Raelene; Bennink, Jack; Yewdell, Jonathan W.; McCullers, Jonathan A.

    2007-01-01

    Secondary bacterial pneumonia frequently claimed the lives of victims during the devastating 1918 influenza A virus pandemic. Little is known about the viral factors contributing to the lethality of the 1918 pandemic. Here we show that expression of the viral accessory protein PB1-F2 enhances inflammation during primary viral infection of mice and increases both the frequency and severity of secondary bacterial pneumonia. The priming effect of PB1-F2 on bacterial pneumonia could be recapitulated in mice by intranasal delivery of a synthetic peptide derived from the C-terminal portion of the PB1-F2. Relative to its isogenic parent, an influenza virus engineered to express a PB1-F2 with coding changes matching the 1918 pandemic strain was more virulent in mice, induced more pulmonary immunopathology, and led to more severe secondary bacterial pneumonia. These findings help explain both the unparalleled virulence of the 1918 strain and the high incidence of fatal pneumonia during the pandemic. PMID:18005742

  3. Oligomeric viral proteins: small in size, large in presence

    PubMed Central

    Jayaraman, Bhargavi; Smith, Amber M.; Fernandes, Jason D.; Frankel, Alan D.

    2016-01-01

    Viruses are obligate parasites that rely heavily on host cellular processes for replication. The small number of proteins typically encoded by a virus is faced with selection pressures that lead to the evolution of distinctive structural properties, allowing each protein to maintain its function under constraints such as small genome size, high mutation rate, and rapidly changing fitness conditions. One common strategy for this evolution is to utilize small building blocks to generate protein oligomers that assemble in multiple ways, thereby diversifying protein function and regulation. In this review, we discuss specific cases that illustrate how oligomerization is used to generate a single defined functional state, to modulate activity via different oligomeric states, or to generate multiple functional forms via different oligomeric states. PMID:27685368

  4. 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

  5. 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

  6. 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.

  7. Hydrophobic inactivation of influenza viruses confers preservation of viral structure with enhanced immunogenicity.

    PubMed

    Raviv, Yossef; Blumenthal, Robert; Tompkins, S Mark; Humberd, Jennifer; Hogan, Robert J; Viard, Mathias

    2008-05-01

    The use of inactivated influenza virus for the development of vaccines with broad heterosubtypic protection requires selective inactivation techniques that eliminate viral infectivity while preserving structural integrity. Here we tested if a hydrophobic inactivation approach reported for retroviruses could be applied to the influenza virus. By this approach, the transmembrane domains of viral envelope proteins are selectively targeted by the hydrophobic photoactivatable compound 1,5-iodonaphthyl-azide (INA). This probe partitions into the lipid bilayer of the viral envelope and upon far UV irradiation reacts selectively with membrane-embedded domains of proteins and lipids while the protein domains that localize outside the bilayer remain unaffected. INA treatment of influenza virus blocked infection in a dose-dependent manner without disrupting the virion or affecting neuraminidase activity. Moreover, the virus maintained the full activity in inducing pH-dependent lipid mixing, but pH-dependent redistribution of viral envelope proteins into the target cell membrane was completely blocked. These results indicate that INA selectively blocks fusion of the virus with the target cell membrane at the pore formation and expansion step. Using a murine model of influenza virus infection, INA-inactivated influenza virus induced potent anti-influenza virus serum antibody and T-cell responses, similar to live virus immunization, and protected against heterosubtypic challenge. INA treatment of influenza A virus produced a virus that is noninfectious, intact, and fully maintains the functional activity associated with the ectodomains of its two major envelope proteins, neuraminidase and hemagglutinin. When used as a vaccine given intranasally (i.n.), INA-inactivated influenza virus induced immune responses similar to live virus infection.

  8. The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins.

    PubMed Central

    O'Neill, R E; Talon, J; Palese, P

    1998-01-01

    Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex. PMID:9427762

  9. Host and viral proteins in the virion of Kaposi's sarcoma-associated herpesvirus.

    PubMed

    Bechtel, Jill T; Winant, Richard C; Ganem, Don

    2005-04-01

    Infection of cultured cells with Kaposi's sarcoma associated herpesvirus (KSHV) typically establishes a latent infection, in which only a few viral genes are expressed. Recently, it has been reported that a subset of lytic genes are transiently expressed very early after viral entry but that this burst of abortive lytic gene expression is terminated with the supervention of latency (H. H. Krishnan, P. P. Naranatt, M. S. Smith, L. Zeng, C. Bloomer, and B. Chandran, J. Virol. 78:3601-3620, 2004). To identify molecules imported into cells by KSHV that might influence this gene expression program, we have examined the protein composition of the KSHV particle. Immunoblotting of virus particles demonstrated that RTA, the lytic switch protein, and RAP, a viral protein that is a transcriptional and cell cycle modulator, were both incorporated into virus particles. In a second approach, polypeptides isolated from purified virions were identified by mass-spectrometric analysis of their constituent tryptic peptides. With this approach we were able to identify 18 major virion proteins, including structural, regulatory, and signaling proteins of both viral and cellular origin.

  10. 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.

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

    PubMed

    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.

  12. iLIR@viral: A web resource for LIR motif-containing proteins in viruses.

    PubMed

    Jacomin, Anne-Claire; Samavedam, Siva; Charles, Hannah; Nezis, Ioannis P

    2017-10-03

    Macroautophagy/autophagy has been shown to mediate the selective lysosomal degradation of pathogenic bacteria and viruses (xenophagy), and to contribute to the activation of innate and adaptative immune responses. Autophagy can serve as an antiviral defense mechanism but also as a proviral process during infection. Atg8-family proteins play a central role in the autophagy process due to their ability to interact with components of the autophagy machinery as well as selective autophagy receptors and adaptor proteins. Such interactions are usually mediated through LC3-interacting region (LIR) motifs. So far, only one viral protein has been experimentally shown to have a functional LIR motif, leaving open a vast field for investigation. Here, we have developed the iLIR@viral database ( http://ilir.uk/virus/ ) as a freely accessible web resource listing all the putative canonical LIR motifs identified in viral proteins. Additionally, we used a curated text-mining analysis of the literature to identify novel putative LIR motif-containing proteins (LIRCPs) in viruses. We anticipate that iLIR@viral will assist with elucidating the full complement of LIRCPs in viruses.

  13. Mechanisms of coronavirus cell entry mediated by the viral spike protein.

    PubMed

    Belouzard, Sandrine; Millet, Jean K; Licitra, Beth N; Whittaker, Gary R

    2012-06-01

    Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S) mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes-A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.

  14. Non-human Primate Schlafen11 Inhibits Production of Both Host and Viral Proteins.

    PubMed

    Stabell, Alex C; Hawkins, John; Li, Manqing; Gao, Xia; David, Michael; Press, William H; Sawyer, Sara L

    2016-12-01

    Schlafen11 (encoded by the SLFN11 gene) has been shown to inhibit the accumulation of HIV-1 proteins. We show that the SLFN11 gene is under positive selection in simian primates and is species-specific in its activity against HIV-1. The activity of human Schlafen11 is relatively weak compared to that of some other primate versions of this protein, with the versions encoded by chimpanzee, orangutan, gibbon, and marmoset being particularly potent inhibitors of HIV-1 protein production. Interestingly, we find that Schlafen11 is functional in the absence of infection and reduces protein production from certain non-viral (GFP) and even host (Vinculin and GAPDH) transcripts. This suggests that Schlafen11 may just generally block protein production from non-codon optimized transcripts. Because Schlafen11 is an interferon-stimulated gene with a broad ability to inhibit protein production from many host and viral transcripts, its role may be to create a general antiviral state in the cell. Interestingly, the strong inhibitors such as marmoset Schlafen11 consistently block protein production better than weak primate Schlafen11 proteins, regardless of the virus or host target being analyzed. Further, we show that the residues to which species-specific differences in Schlafen11 potency map are distinct from residues that have been targeted by positive selection. We speculate that the positive selection of SLFN11 could have been driven by a number of different factors, including interaction with one or more viral antagonists that have yet to be identified.

  15. 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.

  16. 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.

  17. Herpes simplex virus VP16, but not ICP0, is required to reduce histone occupancy and enhance histone acetylation on viral genomes in U2OS osteosarcoma cells.

    PubMed

    Hancock, Meaghan H; Cliffe, Anna R; Knipe, David M; Smiley, James R

    2010-02-01

    The herpes simplex virus (HSV) genome rapidly becomes associated with histones after injection into the host cell nucleus. The viral proteins ICP0 and VP16 are required for efficient viral gene expression and have been implicated in reducing the levels of underacetylated histones on the viral genome, raising the possibility that high levels of underacetylated histones inhibit viral gene expression. The U2OS osteosarcoma cell line is permissive for replication of ICP0 and VP16 mutants and appears to lack an innate antiviral repression mechanism present in other cell types. We therefore used chromatin immunoprecipitation to determine whether U2OS cells are competent to load histones onto HSV DNA and, if so, whether ICP0 and/or VP16 are required to reduce histone occupancy and enhance acetylation in this cell type. High levels of underacetylated histone H3 accumulated at several locations on the viral genome in the absence of VP16 activation function; in contrast, an ICP0 mutant displayed markedly reduced histone levels and enhanced acetylation, similar to wild-type HSV. These results demonstrate that U2OS cells are competent to load underacetylated histones onto HSV DNA and uncover an unexpected role for VP16 in modulating chromatin structure at viral early and late loci. One interpretation of these findings is that ICP0 and VP16 affect viral chromatin structure through separate pathways, and the pathway targeted by ICP0 is defective in U2OS cells. We also show that HSV infection results in decreased histone levels on some actively transcribed genes within the cellular genome, demonstrating that viral infection alters cellular chromatin structure.

  18. Structural biology of the Bcl-2 family and its mimicry by viral proteins

    PubMed Central

    Kvansakul, M; Hinds, M G

    2013-01-01

    Intrinsic apoptosis in mammals is regulated by protein–protein interactions among the B-cell lymphoma-2 (Bcl-2) family. The sequences, structures and binding specificity between pro-survival Bcl-2 proteins and their pro-apoptotic Bcl-2 homology 3 motif only (BH3-only) protein antagonists are now well understood. In contrast, our understanding of the mode of action of Bax and Bak, the two necessary proteins for apoptosis is incomplete. Bax and Bak are isostructural with pro-survival Bcl-2 proteins and also interact with BH3-only proteins, albeit weakly. Two sites have been identified; the in-groove interaction analogous to the pro-survival BH3-only interaction and a site on the opposite molecular face. Interaction of Bax or Bak with activator BH3-only proteins and mitochondrial membranes triggers a series of ill-defined conformational changes initiating their oligomerization and mitochondrial outer membrane permeabilization. Many actions of the mammalian pro-survival Bcl-2 family are mimicked by viruses. By expressing proteins mimicking mammalian pro-survival Bcl-2 family proteins, viruses neutralize death-inducing members of the Bcl-2 family and evade host cell apoptosis during replication. Remarkably, structural elements are preserved in viral Bcl-2 proteins even though there is in many cases little discernible sequence conservation with their mammalian counterparts. Some viral Bcl-2 proteins are dimeric, but they have distinct structures to those observed for mammalian Bcl-2 proteins. Furthermore, viral Bcl-2 proteins modulate innate immune responses regulated by NF-κB through an interface separate from the canonical BH3-binding groove. Our increasing structural understanding of the viral Bcl-2 proteins is leading to new insights in the cellular Bcl-2 network by exploring potential alternate functional modes in the cellular context. We compare the cellular and viral Bcl-2 proteins and discuss how alterations in their structure, sequence and binding specificity

  19. Effect of truncation of the N-terminal region of the viral hemorrhagic septicemia virus (VHSV) P protein on viral replication.

    PubMed

    Park, Ji Sun; Kim, Min Sun; Choi, Seung Hyuk; Kang, Yue Jai; Kim, Ki Hong

    2015-11-01

    The phosphoprotein (P) of viral hemorrhagic septicemia virus (VHSV) plays an essential role in viral replication by interconnecting the L protein and the N protein-RNA complex. In this study, to investigate the role of the N-terminal region of the P protein in viral replication, we mutated the first or the first and second or the first, second, and third ATG codon into TGA stop codons. The respective mutants were named P1, P2, and P3. Recombinant VHSVs containing each mutated P gene (rVHSV-P1, -P2, and -P3) were successfully generated by supplying the intact P protein in trans. The rVHSV-P2 and -P3 were not generated from cells expressing truncated P proteins (P1, P2 or P3 protein), but the rVHSV-P1 produced infectious viruses, even in cells without any P-protein-expressing plasmids. Nucleotide sequence analysis of the P gene of rVHSV-P1 showed that a mutation had occurred that resulted in the fourth amino acid (isoleucine, ATT) being changed to methionine (ATG) without a frameshift (P0.5), suggesting that strong selection pressure might facilitate mutations that are advantageous or essential for virus replication. Infectious rVHSV-P2 and -P3 were produced in cells expressing the P0.5 protein, suggesting that the first three amino acids of the P protein of VHSV are dispensable for viral replication. Furthermore, although the P1 protein was shorter than the P0.5 protein by only two amino acid residues, no viruses were produced when the P1 protein was supplied indicating that the fourth and the fifth amino acid residues are indispensable for normal P protein functions involved in viral replication.

  20. HIV-Enhancing and HIV-Inhibiting Properties of Cationic Peptides and Proteins.

    PubMed

    Cole, Alexander M; Cole, Amy L

    2017-05-15

    Cationic antimicrobial peptides and proteins have historically been ascribed roles in innate immunity that infer killing of microbial and viral pathogens and protection of the host. In the context of sexually transmitted HIV-1, we take an unconventional approach that questions this paradigm. It is becoming increasingly apparent that many of the cationic polypeptides present in the human genital or anorectal mucosa, or human semen, are capable of enhancing HIV-1 infection, often in addition to other reported roles as viral inhibitors. We explore how the in vivo environment may select for or against the HIV-enhancing aspects of these cationic polypeptides by focusing on biological relevance. We stress that the distinction between enhancing and inhibiting HIV-1 infection is not mutually exclusive to specific classes of cationic polypeptides. Understanding how virally enhancing peptides and proteins act to promote sexual transmission of HIV-1 would be important for the design of topical microbicides, mucosal vaccines, and other preventative measures.

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

    PubMed

    Hedil, Marcio; Kormelink, Richard

    2016-07-23

    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.

  2. 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

  3. The virally encoded killer proteins from Ustilago maydis

    USDA-ARS?s Scientific Manuscript database

    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 ...

  4. Arenavirus budding resulting from viral-protein-associated cell membrane curvature.

    PubMed

    Schley, David; Whittaker, Robert J; Neuman, Benjamin W

    2013-09-06

    Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations.

  5. Arenavirus budding resulting from viral-protein-associated cell membrane curvature

    PubMed Central

    Schley, David; Whittaker, Robert J.; Neuman, Benjamin W.

    2013-01-01

    Viral replication occurs within cells, with release (and onward infection) primarily achieved through two alternative mechanisms: lysis, in which virions emerge as the infected cell dies and bursts open; or budding, in which virions emerge gradually from a still living cell by appropriating a small part of the cell membrane. Virus budding is a poorly understood process that challenges current models of vesicle formation. Here, a plausible mechanism for arenavirus budding is presented, building on recent evidence that viral proteins embed in the inner lipid layer of the cell membrane. Experimental results confirm that viral protein is associated with increased membrane curvature, whereas a mathematical model is used to show that localized increases in curvature alone are sufficient to generate viral buds. The magnitude of the protein-induced curvature is calculated from the size of the amphipathic region hypothetically removed from the inner membrane as a result of translation, with a change in membrane stiffness estimated from observed differences in virion deformation as a result of protein depletion. Numerical results are based on experimental data and estimates for three arenaviruses, but the mechanisms described are more broadly applicable. The hypothesized mechanism is shown to be sufficient to generate spontaneous budding that matches well both qualitatively and quantitatively with experimental observations. PMID:23864502

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

    PubMed

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

    2015-06-24

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

  7. Structural framework for DNA translocation via the viral portal protein

    PubMed Central

    Lebedev, Andrey A; Krause, Margret H; Isidro, Anabela L; Vagin, Alexei A; Orlova, Elena V; Turner, Joanne; Dodson, Eleanor J; Tavares, Paulo; Antson, Alfred A

    2007-01-01

    Tailed bacteriophages and herpesviruses load their capsids with DNA through a tunnel formed by the portal protein assembly. Here we describe the X-ray structure of the bacteriophage SPP1 portal protein in its isolated 13-subunit form and the pseudoatomic structure of a 12-subunit assembly. The first defines the DNA-interacting segments (tunnel loops) that pack tightly against each other forming the most constricted part of the tunnel; the second shows that the functional dodecameric state must induce variability in the loop positions. Structural observations together with geometrical constraints dictate that in the portal–DNA complex, the loops form an undulating belt that fits and tightly embraces the helical DNA, suggesting that DNA translocation is accompanied by a ‘mexican wave' of positional and conformational changes propagating sequentially along this belt. PMID:17363899

  8. Human Cytomegalovirus Can Procure Deoxyribonucleotides for Viral DNA Replication in the Absence of Retinoblastoma Protein Phosphorylation

    PubMed Central

    Kuny, Chad V.

    2016-01-01

    ABSTRACT Viral DNA replication requires deoxyribonucleotide triphosphates (dNTPs). These molecules, which are found at low levels in noncycling cells, are generated either by salvage pathways or through de novo synthesis. Nucleotide synthesis utilizes the activity of a series of nucleotide-biosynthetic enzymes (NBEs) whose expression is repressed in noncycling cells by complexes between the E2F transcription factors and the retinoblastoma (Rb) tumor suppressor. Rb-E2F complexes are dissociated and NBE expression is activated during cell cycle transit by cyclin-dependent kinase (Cdk)-mediated Rb phosphorylation. The DNA virus human cytomegalovirus (HCMV) encodes a viral Cdk (v-Cdk) (the UL97 protein) that phosphorylates Rb, induces the expression of cellular NBEs, and is required for efficient viral DNA synthesis. A long-held hypothesis proposed that viral proteins with Rb-inactivating activities functionally similar to those of UL97 facilitated viral DNA replication in part by inducing the de novo production of dNTPs. However, we found that dNTPs were limiting even in cells infected with wild-type HCMV in which UL97 is expressed and Rb is phosphorylated. Furthermore, we revealed that both de novo and salvage pathway enzymes contribute to viral DNA replication during HCMV infection and that Rb phosphorylation by cellular Cdks does not correct the viral DNA replication defect observed in cells infected with a UL97-deficient virus. We conclude that HCMV can obtain dNTPs in the absence of Rb phosphorylation and that UL97 can contribute to the efficiency of DNA replication in an Rb phosphorylation-independent manner. IMPORTANCE Transforming viral oncoproteins, such as adenovirus E1A and papillomavirus E7, inactivate Rb. The standard hypothesis for how Rb inactivation facilitates infection with these viruses is that it is through an increase in the enzymes required for DNA synthesis, which include nucleotide-biosynthetic enzymes. However, HCMV UL97, which functionally

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

    PubMed

    Cui, Hongguang; Wang, Aiming

    2016-05-15

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

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

    PubMed Central

    Cui, Hongguang

    2016-01-01

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

  11. Multiple origins of viral capsid proteins from cellular ancestors

    PubMed Central

    Koonin, Eugene V.

    2017-01-01

    Viruses are the most abundant biological entities on earth and show remarkable diversity of genome sequences, replication and expression strategies, and virion structures. Evolutionary genomics of viruses revealed many unexpected connections but the general scenario(s) for the evolution of the virosphere remains a matter of intense debate among proponents of the cellular regression, escaped genes, and primordial virus world hypotheses. A comprehensive sequence and structure analysis of major virion proteins indicates that they evolved on about 20 independent occasions, and in some of these cases likely ancestors are identifiable among the proteins of cellular organisms. Virus genomes typically consist of distinct structural and replication modules that recombine frequently and can have different evolutionary trajectories. The present analysis suggests that, although the replication modules of at least some classes of viruses might descend from primordial selfish genetic elements, bona fide viruses evolved on multiple, independent occasions throughout the course of evolution by the recruitment of diverse host proteins that became major virion components. PMID:28265094

  12. Heat Shock Protein 27 Mediated Signaling in Viral Infection

    PubMed Central

    Rajaiya, Jaya; Yousuf, Mohammad A.; Singh, Gurdeep; Stanish, Heather; Chodosh, James

    2013-01-01

    Heat shock proteins (HSPs) play a critical role in many intracellular processes, including apoptosis and delivery of other proteins to intracellular compartments. Small HSPs have been shown previously to participate in many cellular functions, including IL-8 induction. Human adenovirus infection activates intracellular signaling, involving particularly the c-Src and mitogen-activated protein kinases [Natarajan, K., et al. (2003) J. Immunol. 170, 6234–6243]. HSP27 and MK2 are also phosphorylated, and c-Src, and its downstream targets, p38, ERK1/2, and c-Jun-terminal kinase (JNK), differentially mediate IL-8 and MCP-1 expression. Specifically, activation and translocation of transcription factor NFκB-p65 occurs in a p38-dependent fashion [Rajaiya, J., et al. (2009) Mol. Vision 15, 2879–2889]. Herein, we report a novel role for HSP27 in an association of p38 with NFκB-p65. Immunoprecipitation assays of virus-infected but not mock-infected cells revealed a signaling complex including p38 and NFκB-p65. Transfection with HSP27 short interfering RNA (siRNA) but not scrambled RNA disrupted this association and reduced the level of IL-8 expression. Transfection with HSP27 siRNA also reduced the level of nuclear localization of NFκB-p65 and p38. By use of tagged p38 mutants, we found that amino acids 279–347 of p38 are necessary for the association of p38 with NFκB-p65. These studies strongly suggest that HSP27, p38, and NFκB-p65 form a signalosome in virus-infected cells and influence downstream expression of pro-inflammatory mediators. PMID:22734719

  13. Proteasome-Dependent Degradation of Daxx by the Viral E1B-55K Protein in Human Adenovirus-Infected Cells ▿

    PubMed Central

    Schreiner, Sabrina; Wimmer, Peter; Sirma, Hüseyin; Everett, Roger D.; Blanchette, Paola; Groitl, Peter; Dobner, Thomas

    2010-01-01

    The death-associated protein Daxx found in PML (promyelocytic leukemia protein) nuclear bodies (PML-NBs) is involved in transcriptional regulation and cellular intrinsic antiviral resistence against incoming viruses. We found that knockdown of Daxx in a nontransformed human hepatocyte cell line using RNA interference (RNAi) techniques results in significantly increased adenoviral (Ad) replication, including enhanced viral mRNA synthesis and viral protein expression. This Daxx restriction imposed upon adenovirus growth is counteracted by early protein E1B-55K (early region 1B 55-kDa protein), a multifunctional regulator of cell-cycle-independent Ad5 replication. The viral protein binds to Daxx and induces its degradation through a proteasome-dependent pathway. We show that this process is independent of Ad E4orf6 (early region 4 open reading frame 6), known to promote the proteasomal degradation of cellular p53, Mre11, DNA ligase IV, and integrin α3 in combination with E1B-55K. These results illustrate the importance of the PML-NB-associated factor Daxx in virus growth restriction and suggest that E1B-55K antagonizes innate antiviral activities of Daxx and PML-NBs to stimulate viral replication at a posttranslational level. PMID:20484509

  14. Differential activities of cellular and viral macro domain proteins in binding of ADP-ribose metabolites.

    PubMed

    Neuvonen, Maarit; Ahola, Tero

    2009-01-09

    Macro domain is a highly conserved protein domain found in both eukaryotes and prokaryotes. Macro domains are also encoded by a set of positive-strand RNA viruses that replicate in the cytoplasm of animal cells, including coronaviruses and alphaviruses. The functions of the macro domain are poorly understood, but it has been suggested to be an ADP-ribose-binding module. We have here characterized three novel human macro domain proteins that were found to reside either in the cytoplasm and nucleus [macro domain protein 2 (MDO2) and ganglioside-induced differentiation-associated protein 2] or in mitochondria [macro domain protein 1 (MDO1)], and compared them with viral macro domains from Semliki Forest virus, hepatitis E virus, and severe acute respiratory syndrome coronavirus, and with a yeast macro protein, Poa1p. MDO2 specifically bound monomeric ADP-ribose with a high affinity (K(d)=0.15 microM), but did not bind poly(ADP-ribose) efficiently. MDO2 also hydrolyzed ADP-ribose-1'' phosphate, resembling Poa1p in all these properties. Ganglioside-induced differentiation-associated protein 2 did not show affinity for ADP-ribose or its derivatives, but instead bound poly(A). MDO1 was generally active in these reactions, including poly(A) binding. Individual point mutations in MDO1 abolished monomeric ADP-ribose binding, but not poly(ADP-ribose) binding; in poly(ADP-ribose) binding assays, the monomer did not compete against polymer binding. The viral macro proteins bound poly(ADP-ribose) and poly(A), but had a low affinity for monomeric ADP-ribose. Thus, the viral proteins do not closely resemble any of the human proteins in their biochemical functions. The differential activity profiles of the human proteins implicate them in different cellular pathways, some of which may involve RNA rather than ADP-ribose derivatives.

  15. Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner.

    PubMed

    Eckenfelder, Agathe; Ségéral, Emmanuel; Pinzón, Natalia; Ulveling, Damien; Amadori, Céline; Charpentier, Marine; Nidelet, Sabine; Concordet, Jean-Paul; Zagury, Jean-François; Paillart, Jean-Christophe; Berlioz-Torrent, Clarisse; Seitz, Hervé; Emiliani, Stéphane; Gallois-Montbrun, Sarah

    2016-12-20

    Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway.

  16. Argonaute proteins regulate HIV-1 multiply spliced RNA and viral production in a Dicer independent manner

    PubMed Central

    Eckenfelder, Agathe; Ségéral, Emmanuel; Pinzón, Natalia; Ulveling, Damien; Amadori, Céline; Charpentier, Marine; Nidelet, Sabine; Concordet, Jean-Paul; Zagury, Jean-François; Paillart, Jean-Christophe; Berlioz-Torrent, Clarisse; Seitz, Hervé

    2017-01-01

    Abstract Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway. PMID:28003477

  17. Glycosylation does not determine segregation of viral envelope proteins in the plasma membrane of epithelial cells

    PubMed Central

    1981-01-01

    Enveloped viruses are excellent tools for the study of the biogenesis of epithelial polarity, because they bud asymmetrically from confluent monolayers of epithelial cells and because polarized budding is preceded by the accumulation of envelope proteins exclusively in the plasma membrane regions from which the viruses bud. In this work, three different experimental approaches showed that the carbohydrate moieties do not determine the final surface localization of either influenza (WSN strain) or vesicular stomatitis virus (VSV) envelope proteins in infected Madin-Darby Canine Kidney (MDCK) cells, as determined by immunofluorescence and immunoelectron microscopy, using ferritin as a marker. Infected concanavalin A- and ricin 1-resistant mutants of MDCK cells, with alterations in glycosylation, exhibited surface distributions of viral glycoproteins identical to those of the parental cell line, i.e., influenza envelope proteins were exclusively found in the apical surface, whereas VSV G protein was localized only in the basolateral region. MDCK cells treated with tunicamycin, which abolishes the glycosylation of viral glycoproteins, exhibited the same distribution of envelope proteins as control cells, after infection with VSF or influenza. A temperature-sensitive mutant of influenza WSN, ts3, which, when grown at the nonpermissive temperature of 39.5 degrees C, retains the sialic acid residues in the envelope glycoproteins, showed, at both 32 degrees C (permissive temperature) and 39.5 degrees C, budding polarity and viral glycoprotein distribution identical to those of the parental WSN strain, when grown in MDCK cells. These results demonstrate that carbohydrate moieties are not components of the addressing signals that determine the polarized distribution of viral envelope proteins, and possibly of the intrinsic cellular plasma membrane proteins, in the surface of epithelial cells. PMID:6265461

  18. Rice grassy stunt virus nonstructural protein p5 serves as a viral suppressor of RNA silencing and interacts with nonstructural protein p3.

    PubMed

    Zhang, Chao; Liu, Xiao-juan; Wu, Kang-cheng; Zheng, Lu-Ping; Ding, Zuo-mei; Li, Fei; Zou, Peng; Yang, Liang; Wu, Jian-guo; Wu, Zu-jian

    2015-11-01

    Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes serious rice disease in Southeast Asian countries. In this study, a green fluorescent protein (GFP)-based transient expression assay was conducted to show that p5, encoded on RNA5 in the viral sense, is a viral suppressor of RNA silencing (VSR). Protein-protein interactions (PPIs) between p5 and all RGSV proteins except pC1 and pC2 were investigated using Gal4-based yeast two-hybrid (Y2H) experiments. The results demonstrated that p5 interacts with itself and with p3 encoded on RNA3 in the viral sense. p5-p5 and p5-p3 interactions were detected by bimolecular fluorescence complementation (BiFC) assay, and the p5-p3 interaction was confirmed by subcellular co-localization and co-immunoprecipitation (Co-IP) assays. Using the Y2H system, we demonstrated that the p5-p3 interaction requires both the N-terminal (amino acid residues 1 to 99) and C-terminal (amino acid residues 94 to 191) domains of p5. In addition, either p5 or p3 could enhance the pathogenicity of potato virus X (PVX) in Nicotiana benthamiana plants. A much more significant enhancement of PVX pathogenicity and accumulation was observed when p5 and p3 were expressed together. Our data also showed that RGSV p3 does not function as a VSR, and it had no effect on the VSR activity of p5 or the subcellular localization pattern of p5 in plant cells from Nicotiana benthamiana.

  19. Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

    PubMed

    Mounce, Bryan C; Tsan, Fei Chin; Droit, Lindsay; Kohler, Sarah; Reitsma, Justin M; Cirillo, Lisa A; Tarakanova, Vera L

    2011-11-25

    Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.

  20. Identification of Viral and Host Proteins That Interact with Murine Gammaherpesvirus 68 Latency-Associated Nuclear Antigen during Lytic Replication: a Role for Hsc70 in Viral Replication

    PubMed Central

    Salinas, Eduardo; Byrum, Stephanie D.; Moreland, Linley E.; Mackintosh, Samuel G.; Tackett, Alan J.

    2015-01-01

    ABSTRACT Latency-associated nuclear antigen (LANA) is a conserved, multifunctional protein encoded by members of the rhadinovirus subfamily of gammaherpesviruses, including Kaposi sarcoma-associated herpesvirus (KSHV) and murine gammaherpesvirus 68 (MHV68). We previously demonstrated that MHV68 LANA (mLANA) is required for efficient lytic replication. However, mechanisms by which mLANA facilitates viral replication, including interactions with cellular and viral proteins, are not known. Thus, we performed a mass spectrometry-based interaction screen that defined an mLANA protein-protein interaction network for lytic viral replication consisting of 15 viral proteins and 191 cellular proteins, including 19 interactions previously reported in KSHV LANA interaction studies. We also employed a stable-isotope labeling technique to illuminate high-priority mLANA-interacting host proteins. Among the top prioritized mLANA-binding proteins was a cellular chaperone, heat shock cognate protein 70 (Hsc70). We independently validated the mLANA-Hsc70 interaction through coimmunoprecipitation and in vitro glutathione S-transferase (GST) pulldown assays. Immunofluorescence and cellular fractionation analyses comparing wild-type (WT) to mLANA-null MHV68 infections demonstrated mLANA-dependent recruitment of Hsc70 to nuclei of productively infected cells. Pharmacologic inhibition and small hairpin RNA (shRNA)-mediated knockdown of Hsc70 impaired MHV68 lytic replication, which functionally correlated with impaired viral protein expression, reduced viral DNA replication, and failure to form viral replication complexes. Replication of mLANA-null MHV68 was less affected than that of WT virus by Hsc70 inhibition, which strongly suggests that Hsc70 function in MHV68 lytic replication is at least partially mediated by its interaction with mLANA. Together these experiments identify proteins engaged by mLANA during the MHV68 lytic replication cycle and define a previously unknown role for Hsc

  1. Actin-binding Protein Drebrin Regulates HIV-1-triggered Actin Polymerization and Viral Infection*

    PubMed Central

    Gordón-Alonso, Mónica; Rocha-Perugini, Vera; Álvarez, Susana; Ursa, Ángeles; Izquierdo-Useros, Nuria; Martinez-Picado, Javier; Muñoz-Fernández, María A.; Sánchez-Madrid, Francisco

    2013-01-01

    HIV-1 contact with target cells triggers F-actin rearrangements that are essential for several steps of the viral cycle. Successful HIV entry into CD4+ T cells requires actin reorganization induced by the interaction of the cellular receptor/co-receptor complex CD4/CXCR4 with the viral envelope complex gp120/gp41 (Env). In this report, we analyze the role of the actin modulator drebrin in HIV-1 viral infection and cell to cell fusion. We show that drebrin associates with CXCR4 before and during HIV infection. Drebrin is actively recruited toward cell-virus and Env-driven cell to cell contacts. After viral internalization, drebrin clustering is retained in a fraction of the internalized particles. Through a combination of RNAi-based inhibition of endogenous drebrin and GFP-tagged expression of wild-type and mutant forms, we establish drebrin as a negative regulator of HIV entry and HIV-mediated cell fusion. Down-regulation of drebrin expression promotes HIV-1 entry, decreases F-actin polymerization, and enhances profilin local accumulation in response to HIV-1. These data underscore the negative role of drebrin in HIV infection by modulating viral entry, mainly through the control of actin cytoskeleton polymerization in response to HIV-1. PMID:23926103

  2. Infectious Bursal Disease Virus-Host Interactions: Multifunctional Viral Proteins that Perform Multiple and Differing Jobs.

    PubMed

    Qin, Yao; Zheng, Shijun J

    2017-01-14

    Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive poultry disease caused by IBD virus (IBDV). The consequent immunosuppression increases susceptibility to other infectious diseases and the risk of subsequent vaccination failure as well. Since the genome of IBDV is relatively small, it has a limited number of proteins inhibiting the cellular antiviral responses and acting as destroyers to the host defense system. Thus, these virulence factors must be multifunctional in order to complete the viral replication cycle in a host cell. Insights into the roles of these viral proteins along with their multiple cellular targets in different pathways will give rise to a rational design for safer and effective vaccines. Here we summarize the recent findings that focus on the virus-cell interactions during IBDV infection at the protein level.

  3. Infectious Bursal Disease Virus-Host Interactions: Multifunctional Viral Proteins that Perform Multiple and Differing Jobs

    PubMed Central

    Qin, Yao; Zheng, Shijun J.

    2017-01-01

    Infectious bursal disease (IBD) is an acute, highly contagious and immunosuppressive poultry disease caused by IBD virus (IBDV). The consequent immunosuppression increases susceptibility to other infectious diseases and the risk of subsequent vaccination failure as well. Since the genome of IBDV is relatively small, it has a limited number of proteins inhibiting the cellular antiviral responses and acting as destroyers to the host defense system. Thus, these virulence factors must be multifunctional in order to complete the viral replication cycle in a host cell. Insights into the roles of these viral proteins along with their multiple cellular targets in different pathways will give rise to a rational design for safer and effective vaccines. Here we summarize the recent findings that focus on the virus–cell interactions during IBDV infection at the protein level. PMID:28098808

  4. A new bioinformatics approach to natural protein collections: permutation structure contrasts of viral and cellular systems.

    PubMed

    Graham, Daniel J

    2013-04-01

    Biological cells and viruses operate by different replication and symmetry paradigms. Cells are able to replicate independently and express little spatial symmetry; viruses require cells for replication while manifesting high symmetry. The author inquired whether different paradigms were reflected in the permutations of amino acid sequences. The hypothesis was that the permutation structure level and symmetry within viral protein collections exceed that of living cells. The rationale was that one symmetry aspect generally accompanies and promotes others in a system. The inquiry was readily answered given abundant sequence archives for proteins. The analysis of collections from diverse viral and cellular sources lends strong support. Additional insights into protein primary structure, the design of collections, and the role of information are provided as well.

  5. Toll-like receptor 2 senses hepatitis C virus core protein but not infectious viral particles

    PubMed Central

    Hoffmann, Marco; Zeisel, Mirjam B.; Jilg, Nikolaus; Paranhos-Baccalà, Glaucia; Stoll-Keller, Françoise; Wakita, Takaji; Hafkemeyer, Peter; Blum, Hubert E.; Barth, Heidi; Henneke, Philipp; Baumert, Thomas F.

    2009-01-01

    Toll-like receptors (TLRs) are pathogen recognition molecules activating the innate immune system. Cell surface expressed TLRs, such as TLR2 and TLR4 have been shown to play an important role in human host defenses against viruses through sensing of viral structural proteins. In this study, we aimed to elucidate whether TLR2 and TLR4 participate in inducing antiviral immunity against hepatitis C virus by sensing viral structural proteins. We studied TLR2 and TLR4 activation by cell-culture derived infectious virions (HCVcc) and serum-derived virions in comparison to purified recombinant HCV structural proteins and enveloped virus-like particles. Incubation of TLR2 or TLR4 transfected cell lines with recombinant core protein resulted in activation of TLR2-dependent signaling. In contrast, neither infectious virions nor enveloped HCV-like particles triggered TLR2 and TLR4 signaling. These findings suggest that monomeric HCV core protein but not intact infectious particles are sensed by TLR2. Impairment of core-TLR interaction in infectious viral particles may contribute to escape from innate antiviral immune responses. PMID:20375602

  6. 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

  7. 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.

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

    DOE PAGES

    Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; ...

    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

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

    ScienceCinema

    Sanchez, Savannah E. [San Diego State Univ., San Diego, CA (United States); Cuevas, Daniel A. [San Diego State Univ., San Diego, CA (United States); Rostron, Jason E. [San Diego State Univ., San Diego, CA (United States); Liang, Tiffany Y. [San Diego State Univ., San Diego, CA (United States); Pivaroff, Cullen G. [San Diego State Univ., San Diego, CA (United States); Haynes, Matthew R. [San Diego State Univ., San Diego, CA (United States); Nulton, Jim [San Diego State Univ., San Diego, CA (United States); Felts, Ben [San Diego State Univ., San Diego, CA (United States); Bailey, Barbara A. [San Diego State Univ., San Diego, CA (United States); Salamon, Peter [San Diego State Univ., San Diego, CA (United States); Edwards, Robert A. [San Diego State Univ., San Diego, CA (United States); Argonne National Lab. (ANL), Argonne, IL (United States); Burgin, Alex B. [Broad Institute, Cambridge, MA (United States); Segall, Anca M. [San Diego State Univ., San Diego, CA (United States); Rohwer, Forest [San Diego State Univ., San Diego, CA (United States)

    2016-07-12

    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.

  10. Zinc-finger antiviral protein inhibits HIV-1 infection by selectively targeting multiply spliced viral mRNAs for degradation

    PubMed Central

    Zhu, Yiping; Chen, Guifang; Lv, Fengxiang; Wang, Xinlu; Ji, Xin; Xu, Yihui; Sun, Jing; Wu, Li; Zheng, Yong-Tang; Gao, Guangxia

    2011-01-01

    The zinc-finger antiviral protein (ZAP) was originally identified as a host factor that inhibits the replication of Moloney murine leukemia virus. Here we report that ZAP inhibits HIV-1 infection by promoting the degradation of specific viral mRNAs. Overexpression of ZAP rendered cells resistant to HIV-1 infection in a ZAP expression level-dependent manner, whereas depletion of endogenous ZAP enhanced HIV-1 infection. Both human and rat ZAP inhibited the propagation of replication-competent HIV-1. ZAP specifically targeted the multiply spliced but not unspliced or singly spliced HIV-1 mRNAs for degradation. We provide evidence indicating that ZAP selectively recruits cellular poly(A)-specific ribonuclease (PARN) to shorten the poly(A) tail of target viral mRNA and recruits the RNA exosome to degrade the RNA body from the 3′ end. In addition, ZAP recruits cellular decapping complex through its cofactor RNA helicase p72 to initiate degradation of the target viral mRNA from the 5′ end. Depletion of each of these mRNA degradation enzymes reduced ZAP's activity. Our results indicate that ZAP inhibits HIV-1 by recruiting both the 5′ and 3′ mRNA degradation machinery to specifically promote the degradation of multiply spliced HIV-1 mRNAs. PMID:21876179

  11. 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

  12. Production of Uninfectious Human Immunodeficiency Virus Type 1 Containing Viral Protein R Fused to a Single-Chain Antibody against Viral Integrase

    PubMed Central

    Okui, Nobuo; Kobayashi, Noriko; Kitamura, Yoshihiro

    1998-01-01

    A single-chain antibody (scAb) against human immunodeficiency virus type 1 (HIV-1) integrase was expressed as a fusion protein of scAb and HIV-1 viral protein R (Vpr), together with the HIV-1 genome, in human 293T cells. The expression did not affect virion production much but markedly reduced the infectivity of progeny virions. The fusion protein was found to be incorporated into the virions. The incorporation appears to account for the reduced infectivity. PMID:9658154

  13. 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

  14. Disclosing the in vivo organization of a viral histone-like protein in Bacillus subtilis mediated by its capacity to recognize the viral genome

    PubMed Central

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

    2012-01-01

    Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage ϕ29 protein p6 has been described as a histone-like protein that compacts the viral genome by forming a nucleoprotein complex and plays a key role in the initiation of protein-primed DNA replication. In this work, we analyze the subcellular localization of protein p6 by immunofluorescence microscopy and show that, at early infection stages, it localizes in a peripheral helix-like configuration. Later, at middle infection stages, protein p6 is recruited to the bacterial nucleoid. This migrating process is shown to depend on the synthesis of components of the ϕ29 DNA replication machinery (i.e., terminal protein and DNA polymerase) needed for the replication of viral DNA, which is required to recruit the bulk of protein p6. Importantly, the double-stranded DNA-binding capacity of protein p6 is essential for its relocalization at the nucleoid. Altogether, the results disclose the in vivo organization of a viral histone-like protein in bacteria. PMID:22451942

  15. 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.

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

    PubMed

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

    2013-09-10

    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.

  17. Role of the C terminus of Lassa virus L protein in viral mRNA synthesis.

    PubMed

    Lehmann, Maria; Pahlmann, Meike; Jérôme, Hanna; Busch, Carola; Lelke, Michaela; Günther, Stephan

    2014-08-01

    The N terminus of arenavirus L protein contains an endonuclease presumably involved in "cap snatching." Here, we employed the Lassa virus replicon system to map other L protein sites that might be involved in this mechanism. Residues Phe-1979, Arg-2018, Phe-2071, Asp-2106, Trp-2173, Tyr-2179, Arg-2200, and Arg-2204 were important for viral mRNA synthesis but dispensable for genome replication. Thus, the C terminus of L protein is involved in the mRNA synthesis process, potentially by mediating cap binding.

  18. Transcriptional enhancer from milk protein genes

    DOEpatents

    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.

  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. High Serum Lipopolysaccharide-Binding Protein Level in Chronic Hepatitis C Viral Infection Is Reduced by Anti-Viral Treatments

    PubMed Central

    Nien, Hsiao-Ching; Hsu, Shih-Jer; Su, Tung-Hung; Yang, Po-Jen; Sheu, Jin-Chuan; Wang, Jin-Town; Chow, Lu-Ping; Chen, Chi-Ling

    2017-01-01

    Background Lipopolysaccharide-binding protein (LBP) has been reported to associate with metabolic diseases, such as obesity, diabetes, and non-alcoholic fatty liver disease. Since chronic hepatitis C virus (HCV) infection is associated with metabolic derangements, the relationship between LBP and HCV deserves additional studies. This study aimed to determine the serum LBP level in subjects with or without HCV infection and investigate the change of its level after anti-viral treatments with or without interferon. Methods and Findings We recruited 120 non-HCV subjects, 42 and 17 HCV-infected subjects respectively treated with peginterferon α-2a/ribavirin and direct-acting antiviral drugs. Basic information, clinical data, serum LBP level and abdominal ultrasonography were collected. All the subjects provided written informed consent before being enrolled approved by the Research Ethics Committee of the National Taiwan University Hospital. Serum LBP level was significantly higher in HCV-infected subjects than non-HCV subjects (31.0 ± 8.8 versus 20.0 ± 6.4 μg/mL; p-value < 0.001). After multivariate analyses, LBP at baseline was independently associated with body mass index, hemoglobin A1c, alanine aminotransferase (ALT) and HCV infection. Moreover, the baseline LBP was only significantly positively associated with ALT and inversely with fatty liver in HCV-infected subjects. The LBP level significantly decreased at sustained virologic response (27.4 ± 6.6 versus 34.6 ± 7.3 μg/mL, p-value < 0.001; 15.9 ± 4.4 versus 22.2 ± 5.7 μg/mL, p-value = 0.001), regardless of interferon-based or -free therapy. Conclusions LBP, an endotoxemia associated protein might be used as an inflammatory biomarker of both infectious and non-infectious origins in HCV-infected subjects. PMID:28107471

  1. Expression of IMP1 enhances production of murine leukemia virus vector by facilitating viral genomic RNA packaging.

    PubMed

    Mai, Yun; Gao, Guangxia

    2010-12-29

    Murine leukemia virus (MLV)-based retroviral vector is widely used for gene transfer. Efficient packaging of the genomic RNA is critical for production of high-titer virus. Here, we report that expression of the insulin-like growth factor II mRNA binding protein 1 (IMP1) enhanced the production of infectious MLV vector. Overexpression of IMP1 increased the stability of viral genomic RNA in virus producer cells and packaging of the RNA into progeny virus in a dose-dependent manner. Downregulation of IMP1 in virus producer cells resulted in reduced production of the retroviral vector. These results indicate that IMP1 plays a role in regulating the packaging of MLV genomic RNA and can be used for improving production of retroviral vectors.

  2. The interaction between claudin-1 and dengue viral prM/M protein for its entry.

    PubMed

    Che, Pulin; Tang, Hengli; Li, Qianjun

    2013-11-01

    Dengue disease is becoming a huge public health concern around the world as more than one-third of the world's population living in areas at risk of infection. In an effort to assess host factors interacting with dengue virus, we identified claudin-1, a major tight junction component, as an essential cell surface protein for dengue virus entry. When claudin-1 was knocked down in Huh 7.5 cells via shRNA, the amount of dengue virus entering host cells was reduced. Consequently, the progeny virus productions were decreased and dengue virus-induced CPE was prevented. Furthermore, restoring the expression of claudin-1 in the knockdown cells facilitated dengue virus entry. The interaction between claudin-1 and dengue viral prM protein was further demonstrated using the pull-down assay. Deletion of the extracellular loop 1 (ECL1) of claudin-1 abolished such interaction, so did point mutations C54A, C64A and I32M on ECL1. These results suggest that the interaction between viral protein prM and host protein claudin-1 was essential for dengue entry. Since host and viral factors involved in virus entry are promising therapeutic targets, determining the essential role of claudin-1 could lead to the discovery of entry inhibitors with attractive therapeutic potential against dengue disease.

  3. Human T-cell leukemia virus type 2 antisense viral protein 2 is dispensable for in vitro immortalization but functions to repress early virus replication in vivo.

    PubMed

    Yin, Han; Kannian, Priya; Dissinger, Nathan; Haines, Robyn; Niewiesk, Stefan; Green, Patrick L

    2012-08-01

    Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are closely related but pathogenically distinct human retroviruses. The antisense strand of the HTLV-1 genome encodes HTLV-1 basic leucine zipper (b-ZIP) protein (HBZ), a protein that inhibits Tax-mediated viral transcription, enhances T-cell proliferation, and promotes viral persistence. Recently, an HTLV-2 antisense viral protein (APH-2) was identified. Despite its lack of a typical b-ZIP domain, APH-2, like HBZ, interacts with cyclic AMP response element binding protein (CREB) and downregulates Tax-mediated viral transcription. Here, we provide evidence that the APH-2 C-terminal LXXLL motif is important for CREB binding and Tax repression. In order to investigate the functional role of APH-2 in the HTLV-2-mediated immortalization of primary T lymphocytes in vitro and in HTLV-2 infection in vivo, we generated APH-2 mutant viruses. In cell cultures, the immortalization capacities of APH-2 mutant viruses were indistinguishable from that of wild-type HTLV-2 (wtHTLV-2), indicating that, like HBZ, APH-2 is dispensable for viral infection and cellular transformation. In vivo, rabbits inoculated with either wtHTLV-2 or APH-2 mutant viruses established a persistent infection. However, the APH-2 knockout virus displayed an increased replication rate, as measured by an increased viral antibody response and a higher proviral load. In contrast to HTLV-1 HBZ, we show that APH-2 is dispensable for the establishment of an efficient infection and persistence in a rabbit animal model. Therefore, antisense proteins of HTLV-1 and HTLV-2 have evolved different functions in vivo, and further comparative studies will provide fundamental insights into the distinct pathobiologies of these two viruses.

  4. 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

  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. Quality by design approach for viral clearance by protein a chromatography

    PubMed Central

    Zhang, Min; Miesegaes, George R; Lee, Michael; Coleman, Daniel; Yang, Bin; Trexler-Schmidt, Melody; Norling, Lenore; Lester, Philip; Brorson, Kurt A; Chen, Qi

    2014-01-01

    Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus-like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X-MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design-of-experiment (DoE)—type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product. PMID:23860745

  7. Quality by design approach for viral clearance by protein a chromatography.

    PubMed

    Zhang, Min; Miesegaes, George R; Lee, Michael; Coleman, Daniel; Yang, Bin; Trexler-Schmidt, Melody; Norling, Lenore; Lester, Philip; Brorson, Kurt A; Chen, Qi

    2014-01-01

    Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus-like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X-MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design-of-experiment (DoE)-type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product.

  8. Non-human Primate Schlafen11 Inhibits Production of Both Host and Viral Proteins

    PubMed Central

    Stabell, Alex C.; Hawkins, John; Li, Manqing; Gao, Xia; David, Michael; Press, William H.; Sawyer, Sara L.

    2016-01-01

    Schlafen11 (encoded by the SLFN11 gene) has been shown to inhibit the accumulation of HIV-1 proteins. We show that the SLFN11 gene is under positive selection in simian primates and is species-specific in its activity against HIV-1. The activity of human Schlafen11 is relatively weak compared to that of some other primate versions of this protein, with the versions encoded by chimpanzee, orangutan, gibbon, and marmoset being particularly potent inhibitors of HIV-1 protein production. Interestingly, we find that Schlafen11 is functional in the absence of infection and reduces protein production from certain non-viral (GFP) and even host (Vinculin and GAPDH) transcripts. This suggests that Schlafen11 may just generally block protein production from non-codon optimized transcripts. Because Schlafen11 is an interferon-stimulated gene with a broad ability to inhibit protein production from many host and viral transcripts, its role may be to create a general antiviral state in the cell. Interestingly, the strong inhibitors such as marmoset Schlafen11 consistently block protein production better than weak primate Schlafen11 proteins, regardless of the virus or host target being analyzed. Further, we show that the residues to which species-specific differences in Schlafen11 potency map are distinct from residues that have been targeted by positive selection. We speculate that the positive selection of SLFN11 could have been driven by a number of different factors, including interaction with one or more viral antagonists that have yet to be identified. PMID:28027315

  9. 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

  10. Non-viral FoxM1 gene delivery to hepatocytes enhances liver repopulation.

    PubMed

    Xiang, D; Liu, C-C; Wang, M-J; Li, J-X; Chen, F; Yao, H; Yu, B; Lu, L; Borjigin, U; Chen, Y-X; Zhong, L; Wangensteen, K J; He, Z-Y; Wang, X; Hu, Y-P

    2014-05-22

    Hepatocyte transplantation as a substitute strategy of orthotopic liver transplantation is being studied for treating end-stage liver diseases. Several technical hurdles must be overcome in order to achieve the therapeutic liver repopulation, such as the problem of insufficient expansion of the transplanted hepatocytes in recipient livers. In this study, we analyzed the application of FoxM1, a cell-cycle regulator, to enhance the proliferation capacity of hepatocytes. The non-viral sleeping beauty (SB) transposon vector carrying FoxM1 gene was constructed for delivering FoxM1 into the hepatocytes. The proliferation capacities of hepatocytes with FoxM1 expression were examined both in vivo and in vitro. Results indicated that the hepatocytes with FoxM1 expression had a higher proliferation rate than wild-type (WT) hepatocytes in vitro. In comparison with WT hepatocytes, the hepatocytes with FoxM1 expression had an enhanced level of liver repopulation in the recipient livers at both sub-acute injury (fumaryl acetoacetate hydrolase (Fah)(-/-) mice model) and acute injury (2/3 partial hepatectomy mice model). Importantly, there was no increased risk of tumorigenicity with FoxM1 expression in recipients even after serial transplantation. In conclusion, expression of FoxM1 in hepatocytes enhanced the capacity of liver repopulation without inducing tumorigenesis. FoxM1 gene delivered by non-viral SB vector into hepatocytes may be a viable approach to promote therapeutic repopulation after hepatocyte transplantation.

  11. Viral expression cassette elements to enhance transgene target specificity and expression in gene therapy.

    PubMed

    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.

  12. A poliovirus 2A(pro) mutant unable to cleave 3CD shows inefficient viral protein synthesis and transactivation defects.

    PubMed Central

    Ventoso, I; Carrasco, L

    1995-01-01

    Four poliovirus mutants with modifications of tyrosine 88 in 2A(pro) were generated and introduced into the cloned poliovirus genome. Mutants Y88P and Y88L were nonviable, mutant Y88F showed a wild-type (WT) phenotype, and mutant Y88S showed a delayed cytopathic effect and formed small plaques in HeLa cells. Growth of Y88S in HeLa cells was restricted, giving rise to about 20% of the PFU production of the WT poliovirus. The 2A (Y88S) mutant synthesized significantly lower levels of viral proteins in HeLa cells than did the WT poliovirus, while the kinetics of p220 cleavage were identical for both viruses. Strikingly, the 2A (Y88S) mutant was unable to cleave 3CD, as shown by analysis of poliovirus proteins labeled with [35S]methionine or immunoblotted with a specific anti-3C serum. The ability of the Y88S mutant to form infectious virus and cleave 3CD can be complemented by the WT poliovirus. Synthesis of viral RNA was diminished in the Y88S mutant but less than the inhibition of translation of viral RNA. Experiments in which guanidine was used to inhibit poliovirus RNA synthesis suggest that the primary defect of the Y88S mutant virus is at the level of poliovirus RNA translation, while viral genome replication is much less affected. Transfection of HeLa cells infected with the WT poliovirus with a luciferase mRNA containing the poliovirus 5' untranslated sequence gives rise to a severalfold increase in luciferase activity. This enhanced translation of leader-luc mRNA was not observed when the transfected cells were infected with the 2A (Y88S) mutant. Moreover, cotransfection with mRNA encoding WT poliovirus 2A(pro) enhanced translation of leader-luc mRNA. This enhancement was much lower upon transfection with mRNA encoding 2A(Y88S), 2A(Y88L), or 2A(Y88P). These findings support the view that 2A(pro) itself, rather than the 3C' and/or 3D' products, is necessary for efficient translation of poliovirus RNA in HeLa cells. PMID:7666528

  13. Interleukin 12 Secretion Enhances Antitumor Efficacy of Oncolytic Herpes Simplex Viral Therapy for Colorectal Cancer

    PubMed Central

    Bennett, Joseph J.; Malhotra, Sandeep; Wong, Richard J.; Delman, Keith; Zager, Jonathan; St-Louis, Maryse; Johnson, Paul; Fong, Yuman

    2001-01-01

    Objective To assess the strategy of combining oncolytic herpes simplex virus (HSV) therapy with immunomodulatory therapy as treatment for experimental colon cancer. The oncolytic HSV recombinant NV1023 and the interleukin 12 (IL-12)-secreting oncolytic NV1042 virus were evaluated in vitro and in vivo with respect to antitumor efficacy. Summary Background Data Genetically engineered, replication-conditional, attenuated HSVs have shown oncolytic activity against a wide variety of solid malignancies. Other strategies for treating cancer have involved immunomodulation and cytokine gene transfer using viral vectors. This study has combined both of these strategies by inserting the murine IL-12 gene into a replication-competent HSV. This approach allows oncolytic therapy to replicate selectively within and lyse tumor cells while providing the host immune system with the cytokine stimulus necessary to recruit and activate inflammatory cells needed to enhance the antitumor effect. Methods NV1023 is a multimutant HSV based on the wild-type HSV-1 F strain. NV1042 was created by insertion of the mIL-12 gene into NV1023. Cytotoxicity and viral proliferation of both NV1023 and NV1042 within murine CT26 colorectal cancer cells were first shown. Cells infected with NV1042 were then shown to produce significant levels of IL-12. Using an experimental flank model of colon cancer, mice were treated with both high and low doses of NV1023 or NV1042 and were followed up for both cure and reduction in tumor burden. Results Both viruses could replicate within and kill CT26 cells in vitro, with 100% cytotoxicity achieved after infection by either virus. Only NV1042 could produce mIL-12. Therapy using high viral doses to treat animals in vivo showed equal efficacy between NV1023 and NV1042, with five of seven cures for each virus. When viral doses were lowered, only the cytokine-producing NV1042 virus could reduce tumor burden and cure animals of their disease. Conclusions Both NV1023 and

  14. 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

  15. A plant viral coat protein RNA binding consensus sequence contains a crucial arginine.

    PubMed Central

    Ansel-McKinney, P; Scott, S W; Swanson, M; Ge, X; Gehrke, L

    1996-01-01

    A defining feature of alfalfa mosaic virus (AMV) and ilarviruses [type virus: tobacco streak virus (TSV)] is that, in addition to genomic RNAs, viral coat protein is required to establish infection in plants. AMV and TSV coat proteins, which share little primary amino acid sequence identity, are functionally interchangeable in RNA binding and initiation of infection. The lysine-rich amino-terminal RNA binding domain of the AMV coat protein lacks previously identified RNA binding motifs. Here, the AMV coat protein RNA binding domain is shown to contain a single arginine whose specific side chain and position are crucial for RNA binding. In addition, the putative RNA binding domain of two ilarvirus coat proteins, TSV and citrus variegation virus, is identified and also shown to contain a crucial arginine. AMV and ilarvirus coat protein sequence alignment centering on the key arginine revealed a new RNA binding consensus sequence. This consensus may explain in part why heterologous viral RNA-coat protein mixtures are infectious. Images PMID:8890181

  16. 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.

  17. T Cell Inactivation by Poxviral B22 Family Proteins Increases Viral Virulence

    PubMed Central

    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

    2014-01-01

    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

  18. Infection Strategies of Bacterial and Viral Pathogens through Pathogen–Human Protein–Protein Interactions

    PubMed Central

    Durmuş Tekir, Saliha; Çakir, Tunahan; Ülgen, Kutlu Ö

    2012-01-01

    Since ancient times, even in today’s modern world, infectious diseases cause lots of people to die. Infectious organisms, pathogens, cause diseases by physical interactions with human proteins. A thorough analysis of these interspecies interactions is required to provide insights about infection strategies of pathogens. Here we analyzed the most comprehensive available pathogen–human protein interaction data including 23,435 interactions, targeting 5,210 human proteins. The data were obtained from the newly developed pathogen–host interaction search tool, PHISTO. This is the first comprehensive attempt to get a comparison between bacterial and viral infections. We investigated human proteins that are targeted by bacteria and viruses to provide an overview of common and special infection strategies used by these pathogen types. We observed that in the human protein interaction network the proteins targeted by pathogens have higher connectivity and betweenness centrality values than those proteins not interacting with pathogens. The preference of interacting with hub and bottleneck proteins is found to be a common infection strategy of all types of pathogens to manipulate essential mechanisms in human. Compared to bacteria, viruses tend to interact with human proteins of much higher connectivity and centrality values in the human network. Gene Ontology enrichment analysis of the human proteins targeted by pathogens indicates crucial clues about the infection mechanisms of bacteria and viruses. As the main infection strategy, bacteria interact with human proteins that function in immune response to disrupt human defense mechanisms. Indispensable viral strategy, on the other hand, is the manipulation of human cellular processes in order to use that transcriptional machinery for their own genetic material transcription. A novel observation about pathogen–human systems is that the human proteins targeted by both pathogens are enriched in the regulation of

  19. Differential Effects of Viral Vectors on Migratory Afferent Lymph Dendritic Cells In Vitro Predict Enhanced Immunogenicity In Vivo ▿

    PubMed Central

    Cubillos-Zapata, C.; Guzman, E.; Turner, A.; Gilbert, S. C.; Prentice, H.; Hope, J. C.; Charleston, B.

    2011-01-01

    Targeting dendritic cells (DC) is key to driving effective immune responses. Lymphatic cannulation provides access to the heterogeneous populations of DC draining peripheral sites in rodents and ruminants. Afferent lymph DEC-205+ CD11c+ SIRPα+ DC were preferentially infected ex vivo with three vaccine viral vectors: recombinant human replication-defective human adenovirus 5 (rhuAdV5), recombinant modified vaccinia virus Ankara (rMVA), and recombinant fowlpox virus (rFPV), all expressing green fluorescent protein (GFP). The rhuAdV5-infected cells remained viable, and peak GFP expression was observed 16 to 24 h posttransduction. Increasing the incubation period of DC with rhuAdV5 enhanced GFP expression. In contrast, DC infected with rMVA-GFP or rFPV-GFP became rapidly apoptotic and GFP expression peaked at 6 h postinfection. Delivery of foot-and-mouth disease virus (FMDV) A22 antigen to DC by rhuAdV5-FMDV-A22 ex vivo resulted in significantly greater CD4+ T cell proliferation than did delivery by rFPV-FMDV-A22. Delivery of rhuAdV5-GFP in oil adjuvant in vivo, to enhance DC-vector contact, resulted in increased GFP expression in migrating DC compared to that with vector alone. Similarly, CD4+ T cell responses were significantly enhanced when using rhuAdV5-FMDV-A22 in adjuvant. Therefore, the interaction between viral vectors and afferent lymph DC ex vivo can predict the outcome of in vivo immunization and provide a means of rapidly assessing the effects of vector modification. PMID:21752909

  20. Replacement of the human cytomegalovirus promoter with fish enhancer and core elements to control the expression of the G gene of viral haemorrhagic septicemia virus (VHSV).

    PubMed

    Martinez-Lopez, A; Chinchilla, B; Encinas, P; Gomez-Casado, E; Estepa, A; Coll, J M

    2012-12-15

    This work explores some of the possibilities to replace human cytomegalovirus (CMV) core and/or enhancer promoter control elements to create new expression vectors for use with fish. The work is relevant to fish vaccination, since DNA vaccines use eukaryotic expression plasmids controlled by the human cytomegalovirus (CMV) promoter to be effective against novirhabdoviruses, such as viral haemorrhagic septicemia virus (VHSV), one of the most devastating fish viral European diseases. To reduce possible homologous recombination with fish genome, core and enhancer sequences from fish origin, such as trout interferon-inducible myxovirus protein (Mx), zebrafish retrovirus long terminal repeat (LTR) and carp β-actin (AE6), were combined with those of CMV to design alternative hybrid promoters. The substitution of CMV core and/or enhancer with the corresponding elements of Mx or the LTR core maintained a similar in vitro protein G expression level than that obtained by using the CMV promoter. Vectors using the dsRNA-inducible Mx enhancer followed either by the LTR or the AE6 cores showed the highest in vitro protein G expression levels. Furthermore, synthetic constructs using the Mx enhancer maintained their polyI:C induction capabilities despite the core used. Some of these hybrid promoters might contribute to the development of all-fish-vectors for DNA vaccines while others might be useful for more basic studies.

  1. New Viral Vector for Superproduction of Epitopes of Vaccine Proteins in Plants

    PubMed Central

    Tyulkina, L.G.; Skurat, E.V.; Frolova, O.Yu.; Komarova, T.V.; Karger, E.M.; Atabekov, I.G.

    2011-01-01

    The novel viral vectors PVX-CP AltMV and PVXdt-CP AltMV are superexpressors of the capsid protein (CP). These viral vectors were constructed on the basis of the potato virus X (PVX) genome andAlternantheramosaic virus (AltMV) CP gene. The expression, based on the hybrid viral vectors, is genetically safe, since the systemic transport and formation of infective viral particles are blocked. CP AltMV can self-assemble into virus-like particles (VLPs) in the absence of genomic RNA. The vectors can be used for the presentation of foreign peptides (including epitopes of human pathogens) on the surface of the VLP. The N-terminal extracellular domain (M2e) of the influenza virus A M2 protein and its truncated variant (ΔM2e) were used as model heterologous peptides for the construction of the chimeric CP AltMV. Chimeric CP AltMV retains its ability to self-assemble into VLP. The epitopes of the M2 influenza virus protein were not eliminated during the process of accumulation, polymerization and purification of chimeric VLP AltMV, providing evidence of the stability of chimeric VLP with C-terminal heterologous epitopes. It appears that VLP produced by the vectors PVX-CP AltMV and PVXdt-CP AltMV can be used in the field of biotechnology for the presentation of the epitopes of vaccine proteins on their surfaces. The chimeric VLP AltMV with the presented foreign epitopes can be used as candidate vaccines. PMID:22649706

  2. 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.

  3. 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

  4. Neutralization of viral infectivity by zebrafish c-reactive protein isoforms.

    PubMed

    Bello-Perez, Melissa; Falco, Alberto; Medina-Gali, Regla; Pereiro, Patricia; Encinar, Jose Antonio; Novoa, Beatriz; Perez, Luis; Coll, Julio

    2017-09-12

    This work explores the unexpected in vivo and in vitro anti-viral functions of the seven c-reactive protein (crp1-7) genes of zebrafish (Danio rerio). First results showed heterogeneous crp1-7 transcript levels in healthy wild-type zebrafish tissues and organs and how those levels heterogeneously changed not only after bacterial but also after viral infections, including those in adaptive immunity-deficient rag1(-/-) mutants. As shown by microarray hybridization and proteomic techniques, crp2/CRP2 and crp5/CRP5 transcripts/proteins were among the most modulated during in vivo viral infection situations including the highest responses in the absence of adaptive immunity. In contrast crp1/CRP1/and crp7/CRP7 very often remained unmodulated. All evidences suggested that zebrafish crp2-6/CRP2-6 may have in vivo anti-viral activities in addition to their well known anti-bacterial and/or physiological functions in mammalians. Confirming those expectations, in vitro neutralization and in vivo protection against spring viremia carp virus (SVCV) infections were demonstrated by crp2-6/CRP2-6 using crp1-7 transfected and/or CRP1-7-enriched supernatant-treated fish cells and crp2-5-injected one-cell stage embryo eggs, respectively. All these findings discovered a crp1-7/CRP1-7 primitive anti-viral functional diversity.These findings may help to study similar functions on the one-gene-coded human CRP, which is widely used as a clinical biomarker for bacterial infections, tissue inflammation and coronary heart diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Cyclosporine inhibits flavivirus replication through blocking the interaction between host cyclophilins and viral NS5 protein.

    PubMed

    Qing, Min; Yang, Feng; Zhang, Bo; Zou, Gang; Robida, John M; Yuan, Zhiming; Tang, Hengli; Shi, Pei-Yong

    2009-08-01

    Although flaviviruses cause significant human diseases, no effective therapy is currently available. Host factors essential for viral replication are potential targets for antiviral development. Here we report that cyclophilins (CyPs), a family of cellular peptidyl-prolyl isomerases (PPIases), play a role in flavivirus replication. Huh-7.5 cells with knockdown of different isoforms of CyP were less efficient than parental cells in supporting flavivirus replication, including West Nile virus (WNV), dengue virus, and yellow fever virus. The low viral replication in CyP A (CyPA) knockdown cells could be rescued by trans supplying of a wild-type CyPA but not by trans supplying of a mutant CyPA (defective in the PPIase activity), indicating that the isomerase activity of CyPA is critical for viral replication. Immunoprecipitation and biochemical pulldown analyses showed that CyPA interacts with WNV genomic RNA and viral NS5 protein in the replication complex. Furthermore, antiviral experiments demonstrated that cyclosporine (Cs; an 11-amino-acid cyclic peptide known to block the PPIase activity of CyPA) inhibits flavivirus replication in cell culture at nontoxic concentrations. Time-of-addition and transient replicon results indicated that Cs inhibits flavivirus at the step of viral RNA synthesis. Biochemical analysis showed that Cs directly blocks the interaction between CyPA and WNV NS5 protein. Our results suggest that host CyPA is a component of flavivirus replication complex and could be targeted for potential antiviral development.

  6. 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.

  7. The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes.

    PubMed

    Zhang, Kun; Zhang, Yongliang; Yang, Meng; Liu, Songyu; Li, Zhenggang; Wang, Xianbing; Han, Chenggui; Yu, Jialin; Li, Dawei

    2017-04-07

    RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV) γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+) RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR) activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes.

  8. The Barley stripe mosaic virus γb protein promotes chloroplast-targeted replication by enhancing unwinding of RNA duplexes

    PubMed Central

    Yang, Meng; Liu, Songyu; Li, Zhenggang; Wang, Xianbing; Han, Chenggui; Yu, Jialin

    2017-01-01

    RNA viruses encode various RNA binding proteins that function in many steps of viral infection cycles. These proteins function as RNA helicases, methyltransferases, RNA-dependent RNA polymerases, RNA silencing suppressors, RNA chaperones, movement proteins, and so on. Although many of the proteins bind the viral RNA genome during different stages of infection, our knowledge about the coordination of their functions is limited. In this study, we describe a novel role for the Barley stripe mosaic virus (BSMV) γb as an enhancer of αa RNA helicase activity, and we show that the γb protein is recruited by the αa viral replication protein to chloroplast membrane sites of BSMV replication. Mutagenesis or deletion of γb from BSMV resulted in reduced positive strand (+) RNAα accumulation, but γb mutations abolishing viral suppressor of RNA silencing (VSR) activity did not completely eliminate genomic RNA replication. In addition, cis- or trans-expression of the Tomato bushy stunt virus p19 VSR protein failed to complement the γb replication functions, indicating that the direct involvement of γb in BSMV RNA replication is independent of VSR functions. These data support a model whereby two BSMV-encoded RNA-binding proteins act coordinately to regulate viral genome replication and provide new insights into strategies whereby double-stranded viral RNA unwinding is regulated, as well as formation of viral replication complexes. PMID:28388677

  9. Intraventricular injection of myxoma virus results in transient expression of viral protein in mouse brain ependymal and subventricular cells.

    PubMed

    France, Megan R; Thomas, Diana L; Liu, Jia; McFadden, Grant; MacNeill, Amy L; Roy, Edward J

    2011-01-01

    Oncolytic viruses that selectively infect and lyse cancer cells have potential as therapeutic agents. Myxoma virus, a poxvirus that is known to be pathogenic only in rabbits, has not been reported to infect normal tissues in humans or mice. We observed that when recombinant virus was injected directly into the lateral ventricle of the mouse brain, virally encoded red fluorescent protein was expressed in ependymal and subventricular cells. Cells were positive for nestin, a marker of neural stem cells. Rapamycin increased the number of cells expressing the virally encoded protein. However, protein expression was transient. Cells expressing the virally encoded protein did not undergo apoptosis and the ependymal lining remained intact. Myxoma virus appears to be safe when injected into the brain despite the transient expression of virally derived protein in a small population of periventricular cells.

  10. Prediction of antiviral peptides derived from viral fusion proteins potentially active against herpes simplex and influenza A viruses

    PubMed Central

    Jesús, Torres; Rogelio, López; Abraham, Cetina; Uriel, López; J- Daniel, García; Alfonso, Méndez-Tenorio; Lilia, Barrón Blanca

    2012-01-01

    There are very few antiviral drugs available to fight viral infections and the appearance of viral strains resistant to these antivirals is not a rare event. Hence, the design of new antiviral drugs is important. We describe the prediction of peptides with antiviral activity (AVP) derived from the viral glycoproteins involved in the entrance of herpes simplex (HSV) and influenza A viruses into their host cells. It is known, that during this event viral glycoproteins suffer several conformational changes due to protein-protein interactions, which lead to membrane fusion between the viral envelope and the cellular membrane. Our hypothesis is that AVPs can be derived from these viral glycoproteins, specifically from regions highly conserved in amino acid sequences, which at the same time have the physicochemical properties of being highly exposed (antigenic), hydrophilic, flexible, and charged, since these properties are important for protein-protein interactions. For that, we separately analyzed the HSV glycoprotein H and B, and influenza A viruses hemagglutinin (HA), using several bioinformatics tools. A set of multiple alignments was carried out, to find the most conserved regions in the amino acid sequences. Then, the physicochemical properties indicated above were analyzed. We predicted several peptides 12-20 amino acid length which by docking analysis were able to interact with the fusion viral glycoproteins and thus may prevent conformational changes in them, blocking the viral infection. Our strategy to design AVPs seems to be very promising since the peptides were synthetized and their antiviral activities have produced very encouraging results. PMID:23144542

  11. Superinfection Exclusion Is an Active Virus-Controlled Function That Requires a Specific Viral Protein

    PubMed Central

    2012-01-01

    Superinfection exclusion, a phenomenon in which a preexisting viral infection prevents a secondary infection with the same or a closely related virus, has been described for various viruses, including important pathogens of humans, animals, and plants. The phenomenon was initially used to test the relatedness of plant viruses. Subsequently, purposeful infection with a mild isolate has been implemented as a protective measure against virus isolates that cause severe disease. In the medical and veterinary fields, superinfection exclusion was found to interfere with repeated applications of virus-based vaccines to individuals with persistent infections and with the introduction of multicomponent vaccines. In spite of its significance, our understanding of this phenomenon is surprisingly incomplete. Recently, it was demonstrated that superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus, occurs only between isolates of the same strain, but not between isolates of different strains of the virus. In this study, I show that superinfection exclusion by CTV requires production of a specific viral protein, the p33 protein. Lack of the functional p33 protein completely eliminated the ability of the virus to exclude superinfection by the same or a closely related virus. Remarkably, the protein appeared to function only in a homology-dependent manner. A cognate protein from a heterologous strain failed to confer the exclusion, suggesting the existence of precise interactions of the p33 protein with other factors involved in this complex phenomenon. PMID:22398285

  12. Rabies viral mechanisms to escape the IFN system: the viral protein P interferes with IRF-3, Stat1, and PML nuclear bodies.

    PubMed

    Chelbi-Alix, Mounira K; Vidy, Aurore; El Bougrini, Jamila; Blondel, Danielle

    2006-05-01

    Interferons (IFNs) are a family of secreted proteins with antiviral, antiproliferative, and immunomodulatory activities. The different biologic actions of IFN are believed to be mediated by the products of specifically IFN-stimulated genes (ISG) in the target cells. The IFN response is the first line of defense against viral infections. Viruses, which require the cellular machinery for their replication, have evolved different ways to counteract the action of IFN by inhibiting IFN production or Jak-Stat signaling or by altering ISG products. This review focuses on the role of viral proteins from the RNA virus family, particularly rabies P protein. P protein mediates inhibition of the IFN system by different pathways: it inhibits IFN production by impairing IFN regulatory factor-3 (IRF-3) phosphorylation and IFN signaling by blocking nuclear transport of Stat1 and alters promyelocytic leukemia (PML) nuclear bodies by retaining PML in the cytoplasm.

  13. Polycarboxylates Enhance Beetle Antifreeze Protein Activity

    PubMed Central

    Amornwittawat, Natapol; Wang, Sen; Duman, John G.; Wen, Xin

    2008-01-01

    Summary Antifreeze proteins (AFPs) lower the noncolligative freezing point of water in the presence of ice below the ice melting point. The temperature difference between the melting point and the noncolligative freezing point is termed thermal hysteresis (TH). The magnitude of the TH depends on the specific activity and the concentration of AFP, and the concentration of enhancers in the solution. Known enhancers are certain low molecular mass molecules and proteins. Here, we investigated a series of polycarboxylates that enhance the TH activity of an AFP from the beetle Dendroides canadensis (DAFP) using differential scanning calorimetry (DSC). Triethylenetetramine-N,N,N′,N″,N‴,N‴-hexaacetate, the most efficient enhancer identified in this work, can increase the TH of DAFP by nearly 1.5 fold over than that of the published best enhancer, citrate. The Zn2+ coordinated carboxylate results in loss of the enhancement ability of the carboxylate on antifreeze activity. There is not an additional increase in TH when a weaker enhancer is added to a stronger enhancer solution. These observations suggest that the more carboxylate groups per enhancer molecule the better the efficiency of the enhancer and that the freedom of motion of these molecules is necessary for them to serve as enhancers for AFP. The hydroxyl groups in the enhancer molecules can also positively affect their TH enhancement efficiency, though not as strongly as carboxylate groups. Mechanisms are discussed. PMID:18620083

  14. A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells

    PubMed Central

    Campeau, Eric; Ruhl, Victoria E.; Rodier, Francis; Smith, Corey L.; Rahmberg, Brittany L.; Fuss, Jill O.; Campisi, Judith; Yaswen, Paul; Cooper, Priscilla K.; Kaufman, Paul D.

    2009-01-01

    The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we

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

    PubMed Central

    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 sigma 1 outer capsid protein. Amino acid sequence analysis predicts that the reovirus cell attachment protein sigma 1 contains an extended fiber domain (R. Bassel-Duby, A. Jayasuriya, D. Chatterjee, N. Sonenberg, J. V. Maizell, Jr., and B. N. Fields, Nature [London] 315:421-423, 1985). When sigma 1 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 sigma 1 has important implications for its function in cell attachment. Other evidence suggests that sigma 1 protein may occur in virions in both an extended and an unextended state. Images PMID:3275434

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

    SciTech Connect

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

    1990-03-01

    Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but 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.

  17. Expression of viral polymerase and phosphorylation of core protein determine core and capsid localization of the human hepatitis B virus.

    PubMed

    Deroubaix, Aurélie; Osseman, Quentin; Cassany, Aurélia; Bégu, Dominique; Ragues, Jessica; Kassab, Somar; Lainé, Sébastien; Kann, Michael

    2015-01-01

    Biopsies from patients show that hepadnaviral core proteins and capsids - collectively called core - are found in the nucleus and cytoplasm of infected hepatocytes. In the majority of studies, cytoplasmic core localization is related to low viraemia while nuclear core localization is associated with high viral loads. In order to better understand the molecular interactions leading to core localization, we analysed transfected hepatoma cells using immune fluorescence microscopy. We observed that expression of core protein in the absence of other viral proteins led to nuclear localization of core protein and capsids, while expression of core in the context of the other viral proteins resulted in a predominantly cytoplasmic localization. Analysis of which viral partner was responsible for cytoplasmic retention indicated that the HBx, surface proteins and HBeAg had no impact but that the viral polymerase was the major determinant. Further analysis revealed that ϵ, an RNA structure to which the viral polymerase binds, was essential for cytoplasmic retention. Furthermore, we showed that core protein phosphorylation at Ser 164 was essential for the cytoplasmic core localization phenotype, which is likely to explain differences observed between individual cells.

  18. Efficient SIVcpz replication in human lymphoid tissue requires viral matrix protein adaptation

    PubMed Central

    Bibollet-Ruche, Frederic; Heigele, Anke; Keele, Brandon F.; Easlick, Juliet L.; Decker, Julie M.; Takehisa, Jun; Learn, Gerald; Sharp, Paul M.; Hahn, Beatrice H.; Kirchhoff, Frank

    2012-01-01

    SIVs infecting wild-living apes in west central Africa have crossed the species barrier to humans on at least four different occasions, one of which spawned the AIDS pandemic. Although the chimpanzee precursor of pandemic HIV-1 strains must have been able to infect humans, the capacity of SIVcpz strains to replicate in human lymphoid tissues (HLTs) is not known. Here, we show that SIVcpz strains from two chimpanzee subspecies are capable of replicating in human tonsillary explant cultures, albeit only at low titers. However, SIVcpz replication in HLT was significantly improved after introduction of a previously identified human-specific adaptation at position 30 in the viral Gag matrix protein. An Arg or Lys at this position significantly increased SIVcpz replication in HLT, while the same mutation reduced viral replication in chimpanzee-derived CD4+ T cells. Thus, naturally occurring SIVcpz strains are capable of infecting HLTs, the major site of HIV-1 replication in vivo. However, efficient replication requires the acquisition of a host-specific adaptation in the viral matrix protein. These results identify Gag matrix as a major determinant of SIVcpz replication fitness in humans and suggest a critical role in the emergence of HIV/AIDS. PMID:22505456

  19. Protective effect of post-ischaemic viral delivery of heat shock proteins in vivo

    PubMed Central

    Badin, Romina A; Modo, Michael; Cheetham, Mike; Thomas, David L; Gadian, David G; Latchman, David S; Lythgoe, Mark F

    2009-01-01

    Heat shock proteins (HSPs) function as molecular chaperones involved in protein folding, transport and degradation and, in addition, they can promote cell survival both in vitro and in vivo after a range of stresses. Although some in vivo studies have suggested that HSP27 and HSP70 can be neuroprotective, current evidence is limited, particularly when HSPs have been delivered after an insult. The effect of overexpressing HSPs after transient occlusion of the middle cerebral artery in rats was investigated by delivering an attenuated herpes simplex viral vector (HSV-1) engineered to express HSP27 or HSP70 30 mins after tissue reperfusion. Magnetic resonance imaging scans were used to determine lesion size and cerebral blood flow at six different time points up to 1 month after stroke. Animals underwent two sensorimotor tests at the same time points to assess the relationship between lesion size and function. Results indicate that post-ischaemic viral delivery of HSP27, but not of HSP70, caused a statistically significant reduction in lesion size and induced a significant behavioural improvement compared with controls. This is the first evidence of effective post-ischaemic gene therapy with a viral vector expressing HSP27 in an experimental model of stroke. PMID:18781161

  20. Viral proteins of bovine papillomavirus type 4 during the development of alimentary canal tumours.

    PubMed

    Anderson, R A; Scobie, L; O'Neil, B W; Grindlay, G J; Campo, M S

    1997-07-01

    In cattle infection of the upper alimentary canal mucosa by bovine papillomavirus type 4 (BPV-4) results in the development of papillomas which can progress to cancer in animals fed on bracken fern. This paper describes a study of the cellular and subcellular distribution of a number of different BPV-4 products in experimentally-induced BPV-4 tumours. E8 and E4 proteins were detected solely as cytoplasmic antigens in the undifferentiated and differentiated layers of the papilloma, respectively; L2 was detected solely as a nuclear antigen in the differentiated layers, whereas E7 was present in either the nucleus or the cytoplasm depending on the differentiation stage of the keratinocyte. Replicative forms of viral DNA were detected from the spinous to the squamous layers. Viral antigens were not detected during papilloma regression or in carcinomas. E8 was most prominent in early developmental stages, while E4 and L2 were most abundant in mature papillomas. E7 was present in large amounts in both early and mature stages, declining at later stages. These results suggest a temporal and spatial requirement for the expression and function of the viral proteins.

  1. Interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission.

    PubMed

    Lorizate, Maier; Huarte, Nerea; Sáez-Cirión, Asier; Nieva, José L

    2008-01-01

    Membrane fusion and fission underlie two limiting steps of enveloped virus replication cycle: access to the interior of the host-cell (entry) and dissemination of viral progeny after replication (budding), respectively. These dynamic processes proceed mediated by specialized proteins that disrupt and bend the lipid bilayer organization transiently and locally. We introduced Wimley-White membrane-water partitioning free energies of the amino acids as an algorithm for predicting functional domains that may transmit protein conformational energy into membranes. It was found that many viral products possess unusually extended, aromatic-rich pre-transmembrane stretches predicted to stably reside at the membrane interface. Here, we review structure-function studies, as well as data reported on the interaction of representative peptides with model membranes, all of which sustain a functional role for these domains in viral fusion and fission. Since pre-transmembrane sequences also constitute antigenic determinants in a membrane-bound state, we also describe some recent results on their recognition and blocking at membrane interface by neutralizing antibodies.

  2. Genomic analysis of a Trichoplusia ni Betabaculovirus (TnGV) with three different viral enhancing factors and two unique genes.

    PubMed

    de Los Ángeles Bivian-Hernández, Ma; López-Tlacomulco, Juventino; Mares-Mares, Everardo; Ibarra, Jorge E; Del Rincón-Castro, María Cristina

    2017-08-30

    The complete genome of a Trichoplusia ni granulovirus (TnGV) is described and analyzed. The genome contains 175,360 bp (KU752557), becoming the third largest genome within the genus Betabaculovirus, smaller only than the Xestia c-nigrum GV (XecnGV) (178,733 pb) and the Pseudaletia unipuncta GV (PsunGV) (176,677 pb) genomes. The TnGV genome has a 39.81% C+G content and a total of 180 ORFs were identified, 96 of them in the granulin gene direction and 84 in the opposite direction. A total of 94.38% of the ORFs showed high identity with those of ClanGV, HaGV, and SlGV. Eight homologous regions (hrs) were identified as well as one apoptosis inhibitor (IAP-3). Interestingly, three viral enhancing factors (VEFs) were located in TnGV genome: VEF-1 (orf153), VEF-3 (orf155), and VEF-4 (orf164), additional to another metalloprotease (orf37). Two ORFs were unique to TnGV (orf100 and orf101) and another one was shared by only TnGV and AgseGV (orf2). Eleven of the deduced proteins showed high identity with proteins from nucleopolyhedroviruses, three with proteins from ascoviruses, and one with an entomopoxvirus protein. The largest deduced protein contains 1,213 amino acids (orf43) and the smallest deduced protein contains only 50 amino acids (orf143). Sequence identity and phylogenetic analyses showed that the closest related genomes to TnGV are, to date, those of PsunGV and XecnGV. This genome analysis may contribute to functional research on TnGV, and may form the bases for the utilization of this betabaculovirus as a pest control agent.

  3. The phiX174 protein J mediates DNA packaging and viral attachment to host cells.

    PubMed

    Bernal, Ricardo A; Hafenstein, Susan; Esmeralda, Raquel; Fane, Bentley A; Rossmann, Michael G

    2004-04-09

    Packaging of viral genomes into their respective capsids requires partial neutralization of the highly negatively charged RNA or DNA. Many viruses, including the Microviridae bacteriophages phiX174, G4, and alpha3, have solved this problem by coding for a highly positively charged nucleic acid-binding protein that is packaged along with the genome. The phiX174 DNA-binding protein, J, is 13 amino acid residues longer than the alpha3 and G4 J proteins by virtue of an additional nucleic acid-binding domain at the amino terminus. Chimeric phiX174 particles containing the smaller DNA-binding protein cannot be generated due to procapsid instability during DNA packaging. However, chimeric alpha3 and G4 phages, containing the phiX174 DNA-binding protein in place of the endogenous J protein, assemble and are infectious, but are less dense than the respective wild-type species. In addition, host cell attachment and native gel migration assays indicate surface variations of these viruses that are controlled by the nature of the J protein. The structure of alpha3 packaged with phiX174 J protein was determined to 3.5A resolution and compared with the previously determined structures of phiX174 and alpha3. The structures of the capsid and spike proteins in the chimeric particle remain unchanged within experimental error when compared to the wild-type alpha3 virion proteins. The amino-terminal region of the phiX174 J protein, which is missing from wild-type alpha3 virions, is mostly disordered in the alpha3 chimera. The differences observed between solution properties of wild-type phiX174, wild-type alpha3, and alpha3 chimera, including their ability to attach to host cells, correlates with the degree of order in the amino-terminal domain of the J protein. When ordered, this domain binds to the interior of the viral capsid and, thus, might control the flexibility of the capsid. In addition, the properties of the phiX174 J protein in the chimera and the results of mutational

  4. [HIV-1 p17 matrix protein is transported into the cell nucleus and binds with genomic viral RNA].

    PubMed

    Bukrinskaia, A G; Vorkunova, G K; Tentsov, Iu Iu

    1993-01-01

    We have shown that gag polyprotein p55 is cleaved in cytosol rapidly after its synthesis, during 2 h, and p17 enters the nuclei while p24 resides in cytosol. To determine whether the nascent p17 is associated with viral genomic RNA in the nuclei, the cells were fractionated, the viral complexes were immunoprecipitated by monoclonal antibodies against gag proteins, and RNA was extracted and analyzed by slot and blot hybridization. Monoclonal antibodies against p17 precipitated all the viral RNA from the nuclei including full-size genomic RNA and essential part from membranes while monoclonal antibodies against p24 did not precipitate any viral RNA from the nuclei. These data suggest that matrix protein is linked to genomic RNA in the nuclei and rise the possibility that p17 may transfer viral nucleocapsids from the nuclei to plasma membranes, the site of virus assembly.

  5. Regulatory Interaction between the Cellular Restriction Factor IFI16 and Viral pp65 (pUL83) Modulates Viral Gene Expression and IFI16 Protein Stability

    PubMed Central

    Pautasso, Sara; von Einem, Jens; Marschall, Manfred; Plachter, Bodo

    2016-01-01

    ABSTRACT A key player in the intrinsic resistance against human cytomegalovirus (HCMV) is the interferon-γ-inducible protein 16 (IFI16), which behaves as a viral DNA sensor in the first hours postinfection and as a repressor of viral gene transcription in the later stages. Previous studies on HCMV replication demonstrated that IFI16 binds to the viral protein kinase pUL97, undergoes phosphorylation, and relocalizes to the cytoplasm of infected cells. In this study, we demonstrate that the tegument protein pp65 (pUL83) recruits IFI16 to the promoter of the UL54 gene and downregulates viral replication, as shown by use of the HCMV mutant v65Stop, which lacks pp65 expression. Interestingly, at late time points of HCMV infection, IFI16 is stabilized by its interaction with pp65, which stood in contrast to IFI16 degradation, observed in herpes simplex virus 1 (HSV-1)-infected cells. Moreover, we found that its translocation to the cytoplasm, in addition to pUL97, strictly depends on pp65, as demonstrated with the HCMV mutant RV-VM1, which expresses a form of pp65 unable to translocate into the cytoplasm. Thus, these data reveal a dual role for pp65: during early infection, it modulates IFI16 activity at the promoter of immediate-early and early genes; subsequently, it delocalizes IFI16 from the nucleus into the cytoplasm, thereby stabilizing and protecting it from degradation. Overall, these data identify a novel activity of the pp65/IFI16 interactome involved in the regulation of UL54 gene expression and IFI16 stability during early and late phases of HCMV replication. IMPORTANCE The DNA sensor IFI16, a member of the PYHIN proteins, restricts HCMV replication by impairing viral DNA synthesis. Using a mutant virus lacking the tegument protein pp65 (v65Stop), we demonstrate that pp65 recruits IFI16 to the early UL54 gene promoter. As a putative counteraction to its restriction activity, pp65 supports the nucleocytoplasmic export of IFI16, which was demonstrated with the

  6. 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

  7. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. The structural protein ODV-EC27 of Autographa californica nucleopolyhedrovirus is a multifunctional viral cyclin.

    PubMed

    Belyavskyi, M; Braunagel, S C; Summers, M D

    1998-09-15

    Two major characteristics of baculovirus infection are arrest of the host cell at G2/M phase of the cell cycle with continuing viral DNA replication. We show that Autographa californica nucleopolyhedrovirus (AcMNPV) encodes for a multifunctional cyclin that may partially explain the molecular basis of these important characteristics of AcMNPV (baculovirus) infection. Amino acids 80-110 of the viral structural protein ODV-EC27 (-EC27) demonstrate 25-30% similarity with cellular cyclins within the cyclin box. Immunoprecipitation results using antibodies to -EC27 show that -EC27 can associate with either cdc2 or cdk6 resulting in active kinase complexes that can phosphorylate histone H1 and retinoblastoma protein in vitro. The cdk6-EC27 complex also associates with proliferating cell nuclear antigen (PCNA) and we demonstrate that PCNA is a structural protein of both the budded virus and the occlusion-derived virus. These results suggest that -EC27 can function as a multifunctional cyclin: when associated with cdc2, it exhibits cyclin B-like activity; when associated with cdk6, the complex possesses cyclin D-like activity and binds PCNA. The possible roles of such a multifunctional cyclin during the life cycle of baculovirus are discussed, along with potential implications relative to the expression of functionally authentic recombinant proteins by using baculovirus-infected cells.

  9. The unique IR2 protein of equine herpesvirus 1 negatively regulates viral gene expression.

    PubMed

    Kim, Seong K; Ahn, Byung C; Albrecht, Randy A; O'Callaghan, Dennis J

    2006-05-01

    The IR2 protein (IR2P) is a truncated form of the immediate-early protein (IEP) lacking the essential acidic transcriptional activation domain (TAD) and serine-rich tract and yet retaining binding domains for DNA and TFIIB and nuclear localization signal (NLS). Analysis of the IR2 promoter indicated that the IR2 promoter was upregulated by the EICP0P. The IR2P was first detected in the nucleus at 5 h postinfection in equine herpesvirus 1 (EHV-1)-infected HeLa and equine NBL6 cells. Transient-transfection assays revealed that (i) the IR2P by itself downregulated EHV-1 early promoters (EICP0, TK, EICP22, and EICP27) in a dose-dependent manner; (ii) the IR2P abrogated the IEP and the EICP27P (UL5) mediated transactivation of viral promoters in a dose-dependent manner; and (iii) the IR2P, like the IEP itself, also downregulated the IE promoter, indicating that the IEP TAD is not necessary to downregulate the IE promoter. In vitro interaction assays revealed that the IR2P interacts with TATA box-binding protein (TBP). The essential domain(s) of the IR2P that mediate negative regulation were mapped to amino acid residues 1 to 706, indicating that the DNA-binding domain and the NLS of the IR2P may be important for the downregulation. In transient-transfection and virus growth assays, the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector. Overall, these studies suggest that the IR2P downregulates viral gene expression by acting as a dominant-negative protein that blocks IEP-binding to viral promoters and/or squelching the limited supplies of TFIIB and TBP.

  10. The Unique IR2 Protein of Equine Herpesvirus 1 Negatively Regulates Viral Gene Expression

    PubMed Central

    Kim, Seong K.; Ahn, Byung C.; Albrecht, Randy A.; O'Callaghan, Dennis J.

    2006-01-01

    The IR2 protein (IR2P) is a truncated form of the immediate-early protein (IEP) lacking the essential acidic transcriptional activation domain (TAD) and serine-rich tract and yet retaining binding domains for DNA and TFIIB and nuclear localization signal (NLS). Analysis of the IR2 promoter indicated that the IR2 promoter was upregulated by the EICP0P. The IR2P was first detected in the nucleus at 5 h postinfection in equine herpesvirus 1 (EHV-1)-infected HeLa and equine NBL6 cells. Transient-transfection assays revealed that (i) the IR2P by itself downregulated EHV-1 early promoters (EICP0, TK, EICP22, and EICP27) in a dose-dependent manner; (ii) the IR2P abrogated the IEP and the EICP27P (UL5) mediated transactivation of viral promoters in a dose-dependent manner; and (iii) the IR2P, like the IEP itself, also downregulated the IE promoter, indicating that the IEP TAD is not necessary to downregulate the IE promoter. In vitro interaction assays revealed that the IR2P interacts with TATA box-binding protein (TBP). The essential domain(s) of the IR2P that mediate negative regulation were mapped to amino acid residues 1 to 706, indicating that the DNA-binding domain and the NLS of the IR2P may be important for the downregulation. In transient-transfection and virus growth assays, the IR2P reduced EHV-1 production by 23-fold compared to virus titers achieved in cells transfected with the empty vector. Overall, these studies suggest that the IR2P downregulates viral gene expression by acting as a dominant-negative protein that blocks IEP-binding to viral promoters and/or squelching the limited supplies of TFIIB and TBP. PMID:16641295

  11. Human papillomavirus major capsid protein L1 remains associated with the incoming viral genome throughout the entry process.

    PubMed

    DiGiuseppe, Stephen; Bienkowska-Haba, Malgorzata; Guion, Lucile G M; Keiffer, Timothy R; Sapp, Martin

    2017-05-31

    During infectious entry, acidification within the endosome triggers uncoating of the HPV capsid whereupon host cyclophilins facilitate the release of most of the major capsid protein, L1, from the minor capsid protein L2 and the viral genome. The L2/DNA complex traffics to the trans-Golgi network (TGN). Following the onset of mitosis, HPV-harboring transport vesicles bud from the TGN followed by association with mitotic chromosomes. During this time, the HPV genome remains in a vesicular compartment until the nucleus has completely reformed. Recent data suggests that while most of L1 protein dissociates and is degraded in the endosome, some L1 protein remains associated with the viral genome. The L1 protein has DNA binding activity and L2 protein has multiple domains capable of interacting with L1 capsomeres. In this study, we report that some L1 protein traffics with L2 and viral genome to the nucleus. The accompanying L1 protein is mostly full-length and retains conformation-dependent epitopes, which are recognized by neutralizing antibodies. Since more than one L1 molecule contributes to these epitopes and require assembly into capsomeres, we propose that L1 protein is present in form of pentamers. Furthermore, we provide evidence that L1 protein interacts directly with viral DNA within the capsid. Based on our findings, we propose that the L1 protein, likely arranged as capsomeres, stabilizes the viral genome within the subviral complex during intracellular trafficking.IMPORTANCE After internalization, the non-enveloped human papillomavirus virion uncoats in the endosome whereupon conformational changes result in a dissociation of a subset of the major capsid protein L1 from the minor capsid protein L2, which remains in complex with the viral DNA. Recent data suggests that some L1 protein may accompany the viral genome beyond the endosomal compartment. Herein, we demonstrate that conformationally intact L1 protein, likely still arranged as capsomeres, remains

  12. Human T-cell leukemia virus type 1 Tax requires direct access to DNA for recruitment of CREB binding protein to the viral promoter.

    PubMed

    Lenzmeier, B A; Giebler, H A; Nyborg, J K

    1998-02-01

    Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5'-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter.

  13. Human T-Cell Leukemia Virus Type 1 Tax Requires Direct Access to DNA for Recruitment of CREB Binding Protein to the Viral Promoter

    PubMed Central

    Lenzmeier, Brian A.; Giebler, Holli A.; Nyborg, Jennifer K.

    1998-01-01

    Efficient human T-cell leukemia virus type 1 (HTLV-1) replication and viral gene expression are dependent upon the virally encoded oncoprotein Tax. To activate HTLV-1 transcription, Tax interacts with the cellular DNA binding protein cyclic AMP-responsive element binding protein (CREB) and recruits the coactivator CREB binding protein (CBP), forming a nucleoprotein complex on the three viral cyclic AMP-responsive elements (CREs) in the HTLV-1 promoter. Short stretches of dG-dC-rich (GC-rich) DNA, immediately flanking each of the viral CREs, are essential for Tax recruitment of CBP in vitro and Tax transactivation in vivo. Although the importance of the viral CRE-flanking sequences is well established, several studies have failed to identify an interaction between Tax and the DNA. The mechanistic role of the viral CRE-flanking sequences has therefore remained enigmatic. In this study, we used high resolution methidiumpropyl-EDTA iron(II) footprinting to show that Tax extended the CREB footprint into the GC-rich DNA flanking sequences of the viral CRE. The Tax-CREB footprint was enhanced but not extended by the KIX domain of CBP, suggesting that the coactivator increased the stability of the nucleoprotein complex. Conversely, the footprint pattern of CREB on a cellular CRE lacking GC-rich flanking sequences did not change in the presence of Tax or Tax plus KIX. The minor-groove DNA binding drug chromomycin A3 bound to the GC-rich flanking sequences and inhibited the association of Tax and the Tax-CBP complex without affecting CREB binding. Tax specifically cross-linked to the viral CRE in the 5′-flanking sequence, and this cross-link was blocked by chromomycin A3. Together, these data support a model where Tax interacts directly with both CREB and the minor-groove viral CRE-flanking sequences to form a high-affinity binding site for the recruitment of CBP to the HTLV-1 promoter. PMID:9447968

  14. Enhanced CAR T-cell engineering using non-viral Sleeping Beauty transposition from minicircle vectors.

    PubMed

    Monjezi, R; Miskey, C; Gogishvili, T; Schleef, M; Schmeer, M; Einsele, H; Ivics, Z; Hudecek, M

    2017-01-01

    Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and broad clinical translation. Here, we demonstrate that CAR T cells can be engineered through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles (MCs). We analyzed genomic distribution of SB and LV integrations and show that a significantly higher proportion of MC-derived CAR transposons compared with LV integrants had occurred outside of highly expressed and cancer-related genes into genomic safe harbor loci that are not expected to cause mutagenesis or genotoxicity. CD19-CAR T cells engineered with our enhanced SB approach conferred potent reactivity in vitro and eradicated lymphoma in a xenograft model in vivo. Intriguingly, electroporation of SB MCs is substantially more effective and less toxic compared with conventional plasmids, and enables cost-effective rapid preparation of therapeutic CAR T-cell doses. This approach sets a new standard in advanced cellular and gene therapy and will accelerate and increase the availability of CAR T-cell therapy to treat hematologic malignancies.

  15. 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

  16. Antiviral agents targeted to interact with viral capsid proteins and a possible application to human immunodeficiency virus.

    PubMed Central

    Rossmann, M G

    1988-01-01

    The tertiary structure of most icosahedral viral capsid proteins consists of an eight-stranded antiparallel beta-barrel with a hydrophobic interior. In a group of picornaviruses, this hydrophobic pocket can be filled by suitable organic molecules, which thereby stop viral uncoating after attachment and penetration into the host cell. The antiviral activity of these agents is probably due to increased rigidity of the capsid protein, which inhibits disassembly. The hydrophobic pocket may be an essential functional component of the protein and, therefore, may have been conserved in the evolution of many viruses from a common precursor. Since eight-stranded anti-parallel beta-barrels, with a topology as in viral capsid proteins, are not generally found for other proteins involved in cell metabolism, this class of antiviral agents is likely to be more virus-specific and less cytotoxic. Furthermore, the greatest conservation of viral capsid proteins occurs within this pocket, whereas the least conserved part is the antigenic exterior. Thus, compounds that bind to such a pocket are likely to be effective against a broader group of serologically distinct viruses. Discovery of antiviral agents of this type will, therefore, depend on designing compounds that can enter and fit snugly into the hydrophobic pocket of a particular viral capsid protein. The major capsid protein, p24, of human immunodeficiency virus would be a likely suitable target. PMID:3133655

  17. Nuclear colocalization of cellular and viral myc proteins with HSP70 in myc-overexpressing cells.

    PubMed Central

    Koskinen, P J; Sistonen, L; Evan, G; Morimoto, R; Alitalo, K

    1991-01-01

    The c-myc oncogene and its viral counterpart v-myc encode phosphoproteins which have been located within cell nuclei, excluding nucleoli. We have expressed the c-myc gene under the simian virus 40 early promoter and studied the distribution of its protein product in transient expression assays in COS, HeLa, and 293 cells. We found three distinct patterns of c-myc immunofluorescence in the transfected cells: one-third of the c-myc-positive cells displayed a diffuse nuclear distribution, and in two-thirds of the cells the c-myc fluorescence was accumulated either in small amorphous or in large multilobed phase-dense nuclear structures. Unexpectedly, these structures also stained for the HSP70 heat shock protein in both heat-shocked and untreated cells. Our results indicate that both transient and stable overexpression of either the c-myc or v-myc protein induces translocation of the endogenous HSP70 protein from the cytoplasm to the nucleus, where it becomes sequestered in structures containing the myc protein. Interestingly, the closely related N-myc protein does not stimulate substantial nuclear expression of the HSP70 protein. Studies with chimeric myc proteins revealed that polypeptide sequences encoded by the second exon of c-myc are involved in colocalization with HSP70. Images PMID:1846202

  18. Differential effects of Sp cellular transcription factors on viral promoter activation by varicella-zoster virus (VZV) IE62 protein.

    PubMed

    Khalil, Mohamed I; Ruyechan, William T; Hay, John; Arvin, Ann

    2015-11-01

    The immediate early (IE) 62 protein is the major varicella-zoster virus (VZV) regulatory factor. Analysis of the VZV genome revealed 40 predicted GC-rich boxes within 36 promoters. We examined effects of ectopic expression of Sp1-Sp4 on IE62- mediated transactivation of three viral promoters. Ectopic expression of Sp3 and Sp4 enhanced IE62 activation of ORF3 and gI promoters while Sp3 reduced IE62 activation of ORF28/29 promoter and VZV DNA replication. Sp2 reduced IE62 transactivation of gI while Sp1 had no significant influence on IE62 activation with any of these viral promoters. Electrophoretic mobility shift assays (EMSA) confirmed binding of Sp1 and Sp3 but not Sp2 and Sp4 to the gI promoter. Sp1-4 bound to IE62 and amino acids 238-258 of IE62 were important for the interaction with Sp3 and Sp4 as well as Sp1. This work shows that Sp family members have differential effects on IE62-mediated transactivation in a promoter-dependent manner. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2016-01-01

    Hepatitis E virus (HEV), an important agent of viral hepatitis worldwide, can cause severe courses of infection in pregnant women and immunosuppressed patients. To date, HEV infections can only be treated with ribavirin (RBV). Major drawbacks of this therapy are that RBV is not approved for administration to pregnant women and that the virus can acquire mutations, which render the intra-host population less sensitive or even resistant to RBV. One of the proposed modes of action of RBV is a direct mutagenic effect on viral genomes, inducing mismatches and subsequent nucleotide substitutions. These transition events can drive the already error-prone viral replication beyond an error threshold, causing viral population extinction. In contrast, the expanded heterogeneous viral population can facilitate selection of mutant viruses with enhanced replication fitness. Emergence of these mutant viruses can lead to therapeutic failure. Consequently, the onset of RBV treatment in chronically HEV-infected individuals can result in two divergent outcomes: viral extinction versus selection of fitness-enhanced viruses. Following an overview of RNA viruses treated with RBV in clinics and a summary of the different antiviral modes of action of this drug, we focus on the mutagenic effect of RBV on HEV intrahost populations, and how HEV is able to overcome lethal mutagenesis. PMID:27754363

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

    PubMed

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

    2016-10-13

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

  1. Axin expression delays herpes simplex virus-induced autophagy and enhances viral replication in L929 cells.

    PubMed

    Choi, Eun-Jin; Kee, Sun-Ho

    2014-02-01

    Axin, a negative regulator of the Wnt signaling pathway, plays a critical role in various cellular events including cell proliferation and cell death. Axin-regulated cell death affects multiple processes, including viral replication. For example, axin expression suppresses herpes simplex virus (HSV)-induced necrotic cell death and enhances viral replication. Based on these observations, this study investigated the involvement of autophagy in regulation of HSV replication and found axin expression inhibits autophagy-mediated suppression of viral replication in L929 cells. HSV infection induced autophagy in a time- and viral dose-dependent manner in control L929 cells (L-EV), whereas virus-induced autophagy was delayed in axin-expressing L929 cells (L-axin). Subsequent analysis showed that induction of autophagy by rapamycin reduced HSV replication, and that inhibiting autophagy by 3-methyladenine (3MA) and beclin-1 knockdown facilitated viral replication in L-EV cells. In addition, preventing autophagy with 3MA suppressed virus-induced cytotoxicity in L-EV cells. In contrast, HSV replication in L-axin cells was resistant to changes in autophagy. These results suggest that axin expression may render L929 cells resistant to HSV-infection induced autophagy, leading to enhanced viral replication. © 2014 The Societies and Wiley Publishing Asia Pty Ltd.

  2. Enhanced Degradation of Misfolded Proteins Promotes Tumorigenesis.

    PubMed

    Chen, Liang; Brewer, Michael D; Guo, Lili; Wang, Ruoxing; Jiang, Peng; Yang, Xiaolu

    2017-03-28

    An adequate cellular capacity to degrade misfolded proteins is critical for cell survival and organismal health. A diminished capacity is associated with aging and neurodegenerative diseases; however, the consequences of an enhanced capacity remain undefined. Here, we report that the ability to clear misfolded proteins is increased during oncogenic transformation and is reduced upon tumor cell differentiation. The augmented capacity mitigates oxidative stress associated with oncogenic growth and is required for both the initiation and maintenance of malignant phenotypes. We show that tripartite motif-containing (TRIM) proteins select misfolded proteins for proteasomal degradation. The higher degradation power in tumor cells is attributed to the upregulation of the proteasome and especially TRIM proteins, both mediated by the antioxidant transcription factor Nrf2. These findings establish a critical role of TRIMs in protein quality control, connect the clearance of misfolded proteins to antioxidant defense, and suggest an intrinsic characteristic of tumor cells.

  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. [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. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2017-01-01

    SUMMARY 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] is unknown. Here, we demonstrate that [KIL-d] is a proteinaceous prion-like aggregate with non-Mendelian cytoplasmic transmission. Deep sequencing analyses revealed [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

  6. 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

  7. 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

  8. Viral infectivity and intracellular distribution of matrix (M) protein of canine distemper virus are affected by actin filaments.

    PubMed

    Klauschies, F; Gützkow, T; Hinkelmann, S; von Messling, V; Vaske, B; Herrler, G; Haas, L

    2010-09-01

    To investigate the role of cytoskeletal components in canine distemper virus (CDV) replication, various agents were used that interfere with turnover of actin filaments and microtubules. Only inhibition of actin filaments significantly reduced viral infectivity. Analysis of the intracellular localization of the viral matrix (M) protein revealed that it aligned along actin filaments. Treatment with actin filament-disrupting drugs led to a marked intracellular redistribution of M protein during infection as well as transfection. In contrast, the localization of the CDV fusion (F) protein was not significantly changed during transfection. Thus, a M protein-actin filament interaction appears to be important for generation of infectious CDV.

  9. The Adenovirus L4-33K Protein Regulates both Late Gene Expression Patterns and Viral DNA Packaging

    PubMed Central

    Wu, Kai; Guimet, Diana

    2013-01-01

    The adenovirus (Ad) L4-33K protein has been linked to disparate functions during infection. L4-33K is a virus-encoded alternative RNA splicing factor which activates splicing of viral late gene transcripts that contain weak 3′ splice sites. Additionally, L4-33K has been indicated to play a role in adenovirus assembly. We generated and characterized an Ad5 L4-33K mutant virus to further explore its function(s) during infection. Infectivity, viral genome replication, and most viral gene expression of the L4-33K mutant virus are comparable to those of the wild-type virus, except for a prominent decrease in the levels of the late proteins IIIa and pVI. The L4-33K mutant virus produces only empty capsids, indicating a defect in viral DNA packaging. We demonstrate that L4-33K does not preferentially bind to viral packaging sequences in vivo, and mutation of L4-33K does not interfere with the binding of the known viral packaging proteins IVa2, L4-22K, L1-52/55K, and IIIa to the packaging sequences in vivo. Collectively, these results demonstrate that the phenotype of an Ad5 L4-33K mutant virus is complex. The L4-33K protein regulates the accumulation of selective Ad late gene mRNAs and is involved in the proper transition of gene expression during the late phase of infection. The L4-33K protein also plays a role in adenovirus morphogenesis by promoting the packaging of the viral genome into the empty capsid. These results demonstrate the multifunctional nature of the L4-33K protein and its involvement in several different and critical aspects of viral infection. PMID:23552425

  10. Interaction of human plasma fibronectin with viral proteins of human immunodeficiency virus.

    PubMed

    Torre, D; Pugliese, A; Ferrario, G; Marietti, G; Forno, B; Zeroli, C

    1994-02-01

    Fibronectin (FN) is present in soluble and matrix forms in various body fluids and tissues, and has been shown to bind to several pathogens, including viruses. The interaction of FN with viral proteins of human immunodeficiency virus (HIV-1) was investigated by immunofluorescence technique using a cell line chronically infected with HIV-1 (H9-V). The results of this study showed that FN binds to HIV-1 infected cells, especially at FN concentration of 5 micrograms/ml. In addition, FN-pentapeptide has shown the ability to bind to HIV-1 infected cells. On the other hand, preincubation with antibodies against FN abolished the binding of FN to HIV-1 infected cells. Finally, FN has shown to bind to HIV-1 glycoproteins, including gp41 and gp120. In contrast, no binding to HIV-1 core proteins, including p15 and p24, was noted. We suggest that FN, in binding HIV-1 particles, may reduce viremia and thus may be involved in the clearance of viral proteins from the cells.

  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. 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. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. 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.

  14. Enhancement of antiviral capacity of transgenic silkworms against cytoplasmic polyhedrosis virus via knockdown of multiple viral genes.

    PubMed

    Jiang, Liang; Peng, Zhengwen; Guo, Huizhen; Sun, Jingchen; Sun, Qiang; Xia, Fei; Huang, Chunlin; Xu, Guowen; Xia, Qingyou

    2017-07-20

    Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a major pathogen of silkworms, causes serious economic losses in sericulture. The BmCPV genome contains 10 discrete dsRNA segments; among these, S1, S2, S3, S4, S6, and S7 encode virus structural proteins, whereas S5, S8, S9, and S10 encode nonstructural proteins. In an attempt to create an anti-BmCPV silkworm strain, we constructed transgenic RNAi vector pb-CNS for knockdown of S5, S8, S9, and S10, and pb-SNS targeting S1, S2, S4, S5, and S8. Transgenic silkworm line CNS and SNS were generated via microinjection of the practical diapause silkworm strain Furong. Following infection via the oral administration of a high dose of BmCPV, the mortality rates of the nontransgenic control, CNS, and SNS were 91%, 37%, and 41%, respectively. qPCR showed that the viral mRNA content in CNS and SNS was significantly lower than that in the nontransgenic line. The economic traits of CNS and SNS were not affected. These results suggest that the knockdown of multiple BmCPV genes significantly enhances the antiviral capacity of the silkworm. Copyright © 2017. Published by Elsevier Ltd.

  15. Enhanced neutralising antibody response to bovine viral diarrhoea virus (BVDV) induced by DNA vaccination in calves.

    PubMed

    R El-Attar, Laila M; Thomas, Carole; Luke, Jeremy; A Williams, James; Brownlie, Joe

    2015-07-31

    DNA vaccination is effective in inducing potent immunity in mice; however it appears to be less so in large animals. Increasing the dose of DNA plasmid to activate innate immunity has been shown to improve DNA vaccine adaptive immunity. Retinoic acid-inducible gene I (RIG-I) is a critical cytoplasmic double-stranded RNA pattern receptor required for innate immune activation in response to viral infection. RIG-I recognise viral RNA and trigger antiviral response, resulting in type I interferon (IFN) and inflammatory cytokine production. In an attempt to enhance the antibody response induced by BVDV DNA in cattle, we expressed BVDV truncated E2 (E2t) and NS3 codon optimised antigens from antibiotic free-plasmid vectors expressing a RIG-I agonist and designated either NTC E2t(co) and NTC NS3(co). To evaluate vaccine efficacy, groups of five BVDV-free calves were intramuscularly injected three times with NTC E2t(co) and NTC NS3(co) vaccine plasmids individually or in combination. Animals vaccinated with our (previously published) conventional DNA vaccines pSecTag/E2 and pTriExNS3 and plasmids expressing RIG-I agonist only presented both the positive and mock-vaccine groups. Our results showed that vaccines coexpressing E2t with a RIG-I agonist induced significantly higher E2 antigen specific antibody response (p<0.05). Additionally, E2t augmented the immune response to NS3 when the two vaccines were delivered in combination. Despite the lack of complete protection, on challenge day 4/5 calves vaccinated with NTC E2t(co) alone or NTC E2t(co) plus NTC NS3(co) had neutralising antibody titres exceeding 1/240 compared to 1/5 in the mock vaccine control group. Based on our results we conclude that co-expression of a RIG-I agonist with viral antigen could enhance DNA vaccine potency in cattle.

  16. Protein engineering strategies for the development of viral vaccines and immunotherapeutics.

    PubMed

    Koellhoffer, Jayne F; Higgins, Chelsea D; Lai, Jonathan R

    2014-01-21

    Vaccines that elicit a protective broadly neutralizing antibody (bNAb) response and monoclonal antibody therapies are critical for the treatment and prevention of viral infections. However, isolation of protective neutralizing antibodies has been challenging for some viruses, notably those with high antigenic diversity or those that do not elicit a bNAb response in the course of natural infection. Here, we discuss recent work that employs protein engineering strategies to design immunogens that elicit bNAbs or engineer novel bNAbs. We highlight the use of rational, computational, and combinatorial strategies and assess the potential of these approaches for the development of new vaccines and immunotherapeutics.

  17. Vectofusin-1, a New Viral Entry Enhancer, Strongly Promotes Lentiviral Transduction of Human Hematopoietic Stem Cells

    PubMed Central

    Fenard, David; Ingrao, Dina; Seye, Ababacar; Buisset, Julien; Genries, Sandrine; Martin, Samia; Kichler, Antoine; Galy, Anne

    2013-01-01

    Gene transfer into hCD34+ hematopoietic stem/progenitor cells (HSCs) using human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) has several promising therapeutic applications. Yet, efficiency, safety, and cost of LV gene therapy could be ameliorated by enhancing target cell transduction levels and reducing the amount of LV used on the cells. Several transduction enhancers already exist such as fibronectin fragments and cationic compounds, but all present limitations. In this study, we describe a new transduction enhancer called Vectofusin-1, which is a short cationic peptide, active on several LV pseudotypes. Vectofusin-1 is used as a soluble additive to safely increase the frequency of transduced HSCs and to augment the level of transduction to one or two copies of vector per cell in a vector dose-dependent manner. Vectofusin-1 acts at the entry step by promoting the adhesion and the fusion between viral and cellular membranes. Vectofusin-1 is therefore a promising additive that could significantly ameliorate hCD34+ cell-based gene therapy. PMID:23653154

  18. Basic residues within the ebolavirus VP35 protein are required for its viral polymerase cofactor function.

    PubMed

    Prins, Kathleen C; Binning, Jennifer M; Shabman, Reed S; Leung, Daisy W; Amarasinghe, Gaya K; Basler, Christopher F

    2010-10-01

    The ebolavirus (EBOV) VP35 protein binds to double-stranded RNA (dsRNA), inhibits host alpha/beta interferon (IFN-α/β) production, and is an essential component of the viral polymerase complex. Structural studies of the VP35 C-terminal IFN inhibitory domain (IID) identified specific structural features, including a central basic patch and a hydrophobic pocket, that are important for dsRNA binding and IFN inhibition. Several other conserved basic residues bordering the central basic patch and a separate cluster of basic residues, called the first basic patch, were also identified. Functional analysis of alanine substitution mutants indicates that basic residues outside the central basic patch are not required for dsRNA binding or for IFN inhibition. However, minigenome assays, which assess viral RNA polymerase complex function, identified these other basic residues to be critical for viral RNA synthesis. Of these, a subset located within the first basic patch is important for VP35-nucleoprotein (NP) interaction, as evidenced by the inability of alanine substitution mutants to coimmunoprecipitate with NP. Therefore, first basic patch residues are likely critical for replication complex formation through interactions with NP. Coimmunoprecipitation studies further demonstrate that the VP35 IID is sufficient to interact with NP and that dsRNA can modulate VP35 IID interactions with NP. Other basic residue mutations that disrupt the VP35 polymerase cofactor function do not affect interaction with NP or with the amino terminus of the viral polymerase. Collectively, these results highlight the importance of conserved basic residues from the EBOV VP35 C-terminal IID and validate the VP35 IID as a potential therapeutic target.

  19. Herpes simplex virus 1 VP22 regulates translocation of multiple viral and cellular proteins and promotes neurovirulence.

    PubMed

    Tanaka, Michiko; Kato, Akihisa; Satoh, Yuko; Ide, Takahiro; Sagou, Ken; Kimura, Kayo; Hasegawa, Hideki; Kawaguchi, Yasushi

    2012-05-01

    Herpes simplex virus 1 (HSV-1) protein VP22, encoded by the UL49 gene, is a major virion tegument protein. In the present study, we showed that VP22 was required for efficient redistribution of viral proteins VP16, VP26, ICP0, ICP4, and ICP27 and of cellular protein Hsc-70 to the cytoplasm of infected cells. We found that two dileucine motifs in VP22, at amino acids 235 and 236 and amino acids 251 and 252, were necessary for VP22 regulation of the proper cytoplasmic localization of these viral and cellular proteins. The dileucine motifs were also required for proper cytoplasmic localization of VP22 itself and for optimal expression of viral proteins VP16, VP22, ICP0, UL41, and glycoprotein B. Interestingly, a recombinant mutant virus with alanines substituted for the dileucines at amino acids 251 and 252 had a 50% lethal dose (LD(50)) for neurovirulence in mice following intracerebral inoculation about 10(3)-fold lower than the LD(50) of the repaired virus. Furthermore, the replication and spread of this mutant virus in the brains of mice following intracerebral inoculation were significantly impaired relative to those of the repaired virus. The ability of VP22 to regulate the localization and expression of various viral and cellular proteins, as shown in this study, was correlated with an increase in viral replication and neurovirulence in the experimental murine model. Thus, HSV-1 VP22 is a significant neurovirulence factor in vivo.

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

    PubMed

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

    2014-07-22

    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.

  1. Structural similarity enhances interaction propensity of proteins.

    PubMed

    Lukatsky, D B; Shakhnovich, B E; Mintseris, J; Shakhnovich, E I

    2007-02-02

    We study statistical properties of interacting protein-like surfaces and predict two strong, related effects: (i) statistically enhanced self-attraction of proteins; (ii) statistically enhanced attraction of proteins with similar structures. The effects originate in the fact that the probability to find a pattern self-match between two identical, even randomly organized interacting protein surfaces is always higher compared with the probability for a pattern match between two different, promiscuous protein surfaces. This theoretical finding explains statistical prevalence of homodimers in protein-protein interaction networks reported earlier. Further, our findings are confirmed by the analysis of curated database of protein complexes that showed highly statistically significant overrepresentation of dimers formed by structurally similar proteins with highly divergent sequences ("superfamily heterodimers"). We suggest that promiscuous homodimeric interactions pose strong competitive interactions for heterodimers evolved from homodimers. Such evolutionary bottleneck is overcome using the negative design evolutionary pressure applied against promiscuous homodimer formation. This is achieved through the formation of highly specific contacts formed by charged residues as demonstrated both in model and real superfamily heterodimers.

  2. Aquareovirus NS80 recruits viral proteins to its inclusions, and its C-terminal domain is the primary driving force for viral inclusion formation.

    PubMed

    Shao, Ling; Guo, Hong; Yan, Li-Ming; Liu, Huan; Fang, Qin

    2013-01-01

    Cytoplasmic inclusion bodies formed in reovirus-infected cells are the sites of viral replication and assembly. Previous studies have suggested that the NS80 protein of aquareovirus may be involved in the formation of viral inclusion bodies. However, it remains unknown whether other viral proteins are involved in the process, and what regions of NS80 may act coordinately in mediating inclusion formation. Here, we observed that globular cytoplasmic inclusions were formed in virus-infected cells and viral proteins NS80 and NS38 colocalized in the inclusions. During transfection, singly expressed NS80 could form cytoplasmic inclusions and recruit NS38 and GFP-tagged VP4 to these structures. Further treatment of cells with nocodazole, a microtubule inhibitor, did not disrupt the inclusion, suggesting that inclusion formation does not rely on microtubule network. Besides, we identified that the region 530-742 of NS80 was likely the minimal region required for inclusion formation, and the C-tail, coiled-coil region as well as the conserved linker region were essential for inclusion phenotype. Moreover, with series deletions from the N-terminus, a stepwise conversion occurred from large condensed cytoplasmic to small nuclear inclusions, then to a diffused intracellular distribution. Notablely, we found that the nuclear inclusions, formed by NS80 truncations (471 to 513-742), colocalized with cellular protein β-catenin. These data indicated that NS80 could be a major mediator in recruiting NS38 and VP4 into inclusion structures, and the C-terminus of NS80 is responsible for inclusion formation.

  3. The impact of viral RNA on the association free energies of capsid protein assembly: bacteriophage MS2 as a case study.

    PubMed

    ElSawy, Karim M

    2017-02-01

    A large number of single-stranded RNA viruses assemble their capsid and their genomic material simultaneously. The RNA viral genome plays multiple roles in this process that are currently only partly understood. In this work, we investigated the thermodynamic basis of the role of viral RNA on the assembly of capsid proteins. The viral capsid of bacteriophage MS2 was considered as a case study. The MS2 virus capsid is composed of 60 AB and 30 CC protein dimers. We investigated the effect of RNA stem loop (the translational repressor TR) binding to the capsid dimers on the dimer-dimer relative association free energies. We found that TR binding results in destabilization of AB self-association compared with AB and CC association. This indicates that the association of the AB and CC dimers is the most likely assembly pathway for the MS2 virus, which explains the experimental observation of alternating patterns of AB and CC dimers in dominant assembly intermediates of the MS2 virus. The presence of viral RNA, therefore, dramatically channels virus assembly to a limited number of pathways, thereby enhancing the efficiency of virus self-assembly process. Interestingly, Thr59Ser and Thr45Ala mutations of the dimers, in the absence of RNA stem loops, lead to stabilization of AB self-association compared with the AB and CC associations, thereby channelling virus assembly towards a fivefold (AB)5 pentamer intermediate, providing a testable hypothesis of our thermodynamic arguments.

  4. Adenovirus infection targets the cellular protein kinase CK2 and RNA-activated protein kinase (PKR) into viral inclusions of the cell nucleus.

    PubMed

    Souquere-Besse, Sylvie; Pichard, Evelyne; Filhol, Odile; Legrand, Valerie; Rosa-Calatrava, Manuel; Hovanessian, Ara G; Cochet, Claude; Puvion-Dutilleul, Francine

    2002-03-15

    The effects of the adenovirus infection on the distribution of the cellular protein kinase CK2 and double-stranded RNA-activated protein kinase (PKR) were examined at the ultrastructural level. Immunogold labeling revealed the redistribution of CK2 subunits and PKR to morphologically distinct structures of the cell nucleus. The electron-clear amorphous structures, designated pIX nuclear bodies in our previous work (Rosa-Calatrava et al., 2001), contained CK2 alpha and PKR. The protein crystals, which result from the regular assembly of hexon, penton base, and fiber proteins [Boulanger et al. (1970) J Gen Virol 6:329-332], contained CK2 beta and PKR. Both viral structures were devoid of viral RNA, including the PKR-inhibitor VA1 RNA generated by the RNA polymerase III. Instead, VA1 RNA accumulated in PKR-free viral compact rings in which the viral RNA generated by the RNA polymerase II was excluded.

  5. 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

  6. Similarities in intracellular transport of plant viral movement proteins BMB2 and TGB3.

    PubMed

    Lazareva, Ekaterina A; Lezzhov, Alexander A; Golyshev, Sergey A; Morozov, Sergey Y; Heinlein, Manfred; Solovyev, Andrey G

    2017-09-01

    The cell-to-cell transport of many plant viruses through plasmodesmata requires viral movement proteins (MPs) encoded by a 'triple gene block' (TGB) and termed TGB1, TGB2 and TGB3. TGB3 is a small integral membrane protein that contains subcellular targeting signals and directs both TGB2 and the helicase domain-containing TGB1 protein to plasmodesmata-associated structures. Recently, we described a 'binary movement block' (BMB) coding for two MPs, BMB1 and BMB2. The BMB2 protein associates with endoplasmic reticulum (ER) membranes, accumulates at plasmodesmata-associated membrane bodies and directs the BMB1 helicase to these structures. TGB3 transport to cell peripheral bodies was previously shown to bypass the secretory pathway and involve a non-conventional mechanism. Here, we provide evidence that the intracellular transport of both poa semilatent virus TGB3 and hibiscus green spot virus BMB2 to plasmodesmata-associated sites can occur via lateral translocation along the ER membranes. Agrobacterium-mediated transient co-expression in Nicotiana benthamiana leaves revealed that green fluorescent protein (GFP)-fused actin-binding domains of Arabidopsis fimbrin (ABD2-GFP) and mouse talin (TAL-GFP) inhibited the subcellular targeting of TGB3 and BMB2 to plasmodesmata-associated bodies, which resulted in TGB3 and BMB2 accumulation in the cytoplasm in association with aberrant ER structures. Inhibition of COPII budding complex formation by the expression of a dominant-negative mutant of the small GTPase Sar1 had no detectable effect on BMB2 subcellular targeting, which therefore could occur without exit from the ER in COPII transport vesicles. Collectively, the presented data support the current view that plant viral MPs exploit the ER:actin network for their intracellular transport.

  7. The BRCA-1 binding protein BRAP2 is a novel, negative regulator of nuclear import of viral proteins, dependent on phosphorylation flanking the nuclear localization signal.

    PubMed

    Fulcher, Alex J; Roth, Daniela M; Fatima, Shadma; Alvisi, Gualtiero; Jans, David A

    2010-05-01

    This study describes for the first time the ability of the novel BRCA1-binding protein 2 (BRAP2) to inhibit the nuclear import of specific viral proteins dependent on phosphorylation. Ectopic expression of BRAP2 in transfected African green monkey kidney COS-7 cells was found to significantly reduce nuclear localization signal (NLS)-dependent nuclear accumulation of either simian virus SV40 large-tumor antigen (T-ag) or human cytomegalovirus DNA polymerase processivity factor ppUL44; this was also observed in HL-60 human promyelocytic leukemia cells on induction of BRAP2 expression by vitamin D3 treatment. BRAP2 inhibition of nuclear accumulation was dependent on phosphorylation sites flanking the respective NLSs, where substitution of the cyclin-dependent kinase site T124 of T-ag with Ala or Asp prevented or enhanced BRAP2 inhibition of nuclear import, respectively. Substitution of T427 within the NLS of ppUL44 gave similar results, whereas no effect of BRAP2 was observed on nuclear targeting of other viral proteins, such as herpes simplex virus-1 pUL30, which lacks a phosphorylation site near its NLS, and the human immunodeficiency virus-1 Tat protein. Pulldowns/AlphaScreen assays indicated direct, high-affinity binding of BRAP2(442-592) to T-ag(111-135), strictly dependent on negative charge at T124 and the NLS. All results are consistent with BRAP2 being a novel, phosphorylation-regulated negative regulator of nuclear import, with potential as an antiviral agent.

  8. The ORF3 Protein of Porcine Circovirus Type 2 Is Involved in Viral Pathogenesis In Vivo

    PubMed Central

    Liu, Jue; Chen, Isabelle; Du, Qingyun; Chua, Huikheng; Kwang, Jimmy

    2006-01-01

    Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome. We previously showed that a novel identified protein, ORF3, was not essential for PCV2 replication in cultured PK15 cells and played a major role in virus-induced apoptosis. To evaluate the role of the ORF3 protein in viral pathogenesis in vivo, we inoculated 8-week-old BALB/c mice that have been developed for PCV2 replication with ORF3-deficient mutant PCV2 (mPCV2). By 42 days postinoculation, all of the mice inoculated with the mPCV2, as well as with wild-type PCV2 (wPCV2), had seroconverted to PCV2 capsid antibody, and the mutant induced levels of PCV2 antibodies that were higher than those of the wPCV2. The PCV2 genomic copy numbers in serum were significantly higher (P < 0.05) in the wPCV2-inoculated mice than in mice inoculated with the mPCV2. Also, the wPCV2 caused microscopic lesions characterized by lymphocyte depletion with histiocytic infiltration of lymphoid organs, but the mutant virus failed to induce any obvious pathological lesions. In situ hybridization and immunohistochemical analyses also showed that larger amounts of viral DNA and antigens were detected in the lymph nodes of the wPCV2-inoculated than mPCV2-inoculated mice. Furthermore, animals of the wPCV2-inoculated group showed significant downshifts of CD8+ T-cell subsets of peripheral blood lymphocytes compared to the control mice (P < 0.05) at various time points postinoculation. Also, the proportions of the CD4+ and CD4+ CD8+ cells were significantly reduced in wPCV2-inoculated mice at some time points postinoculation. In contrast, there are some reductions in the proportions of these subsets in the mutant virus-inoculated mice, but the proportions do not decrease significantly. Taken together, these results demonstrate that the ORF3 protein is also dispensable for viral replication in vivo and that it plays an important role in viral pathogenesis. PMID

  9. Neurotrophic factor expression after CNS viral injury produces enhanced sensitivity to psychostimulants: potential mechanism for addiction vulnerability.

    PubMed

    Solbrig, M V; Koob, G F; Parsons, L H; Kadota, T; Horscroft, N; Briese, T; Lipkin, W I

    2000-11-01

    Hypothesized risk factors for psychostimulant, amphetamine, and cocaine abuse include dopamine (DA) receptor polymorphisms, HIV infection, schizophrenia, drug-induced paranoias, and movement disorders; however, the molecular, cellular, and biochemical mechanisms that predispose to drug sensitivity or drive the development of addiction are incompletely understood. Using the Borna disease rat, an animal model of viral-induced encephalopathy wherein sensitivity to the locomotor and stereotypic behavioral effects of d-amphetamine and cocaine is enhanced (Solbrig et al., 1994, 1998), we identify a specific neurotrophin expression pattern triggered by striatal viral injury that increases tyrosine hydroxylase activity, an early step in DA synthesis, to produce a phenotype of enhanced amphetamine sensitivity. The reactive neurotrophin pattern provides a molecular framework for understanding how CNS viral injury, as well as other CNS adaptations producing similar growth factor activation profiles, may influence psychostimulant sensitivity.

  10. Rab18 is required for viral assembly of hepatitis C virus through trafficking of the core protein to lipid droplets.

    PubMed

    Dansako, Hiromichi; Hiramoto, Hiroki; Ikeda, Masanori; Wakita, Takaji; Kato, Nobuyuki

    2014-08-01

    During persistent infection of HCV, the HCV core protein (HCV-JFH-1 strain of genotype 2a) is recruited to lipid droplets (LDs) for viral assembly, but the mechanism of recruitment of the HCV core protein is uncertain. Here, we demonstrated that one of the Ras-related small GTPases, Rab18, was required for trafficking of the core protein around LDs. The knockdown of Rab18 reduced intracellular and extracellular viral infectivity, but not intracellular viral replication in HCV-JFH-1-infected RSc cells (an HuH-7-derived cell line). Exogenous expression of Rab18 increased extracellular viral infectivity almost two-fold. Furthermore, Rab18 was co-localized with the core protein in HCV-JFH-1-infected RSc cells, and the knockdown of Rab18 blocked recruitment of the HCV-JFH-1 core protein to LDs. These results suggest that Rab18 has an important role in viral assembly through the trafficking of the core protein to LDs.

  11. 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

  12. Enhanced protein production by engineered zinc finger proteins.

    PubMed

    Reik, Andreas; Zhou, Yuanyue; Collingwood, Trevor N; Warfe, Lyndon; Bartsevich, Victor; Kong, Yanhong; Henning, Karla A; Fallentine, Barrett K; Zhang, Lei; Zhong, Xiaohong; Jouvenot, Yann; Jamieson, Andrew C; Rebar, Edward J; Case, Casey C; Korman, Alan; Li, Xiao-Yong; Black, Amelia; King, David J; Gregory, Philip D

    2007-08-01

    Increasing the yield of therapeutic proteins from mammalian production cell lines reduces costs and decreases the time to market. To this end, we engineered a zinc finger protein transcription factor (ZFP TF) that binds a DNA sequence within the promoter driving transgene expression. This ZFP TF enabled >100% increase in protein yield from CHO cells in transient, stable, and fermentor production run settings. Expression vectors engineered to carry up to 10 ZFP binding sites further enhanced ZFP-mediated increases in protein production up to approximately 500%. The multimerized ZFP binding sites function independently of the promoter, and therefore across vector platforms. CHO cell lines stably expressing ZFP TFs demonstrated growth characteristics similar to parental cell lines. ZFP TF expression and gains in protein production were stable over >30 generations in the absence of antibiotic selection. Our results demonstrate that ZFP TFs can rapidly and stably increase protein production in mammalian cells.

  13. 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

  14. Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions.

    PubMed Central

    Lu, Y L; Spearman, P; Ratner, L

    1993-01-01

    The subcellular localization of human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) was examined by subcellular fractionation. In HIV-1-infected peripheral blood mononuclear cells, Vpr was found in the nuclear and membrane fractions as well as the conditioned medium. Expression of Vpr without other HIV-1 proteins, in two different eukaryotic expression systems, demonstrated a predominant localization of Vpr in the nuclear matrix and chromatin extract fractions. Deletion of the carboxyl-terminal 19-amino-acid arginine-rich sequence impaired Vpr nuclear localization. Indirect immunofluorescence confirmed the nuclear localization of Vpr and also indicated a perinuclear location. Expression of Vpr alone did not result in export of the protein from the cell, but when coexpressed with the Gag protein, Vpr was exported and found in virus-like particles. A truncated Gag protein, missing the p6 sequence and a portion of the p9 sequence, was incapable of exporting Vpr from the cell. Regulation of Vpr localization may be important in the influence of this protein on virus replication. Images PMID:8411357

  15. Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

    PubMed Central

    Gutiérrez, C; Freire, R; Salas, M; Hermoso, J M

    1994-01-01

    The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex. Images PMID:8306969

  16. Roles of the PVM M2-1, M2-2 and P gene ORF 2 (P-2) proteins in viral replication.

    PubMed

    Dibben, Oliver; Thorpe, Lindsay C; Easton, Andrew J

    2008-01-01

    A plasmid-based reverse genetics system for pneumonia virus of mice (PVM) using a synthetic minigenome is described. The system was used to investigate the functions of several viral proteins. The M2-1 protein of PVM was shown to enhance reporter gene expression when present at low levels, similar to the situation for the equivalent respiratory syncytial virus (RSV) M2-1 protein, but at high levels was shown to reduce gene expression from the minigenome activity, which differs significantly form the situation with RSV. Analysis of levels of nucleocapsid complex RNA showed that high levels of the PVM M2-1 protein inhibits RNA replication rather than transcription. In contrast, expression of the PVM M2-2 protein in conjunction with the polymerase proteins in a minigenome assay greatly reduced the levels of CAT reporter protein. This is similar to the situation with the RSV M2-2 protein although there is no significant sequence identity between the M2-2 proteins of the pneumoviruses. A significant difference between the genome organisations of RSV and PVM is that the P gene of PVM contains a second open reading frame, encoding the P-2 protein, which has no counterpart in the RSV P gene. Co-expression of the PVM P-2 protein with the minigenome inhibited virus gene expression. This resembles the situation seen with the accessory proteins expressed from alternate reading frames of the P gene of other paramyxoviruses. Analysis of levels of antigenome RNA and CAT mRNA produced by the minigenome in the presence of the P2 protein indicated that the protein inhibits viral transcription in a dose-dependent fashion.

  17. 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.

  18. Flos Farfarae Inhibits Enterovirus 71-Induced Cell Injury by Preventing Viral Replication and Structural Protein Expression.

    PubMed

    Chiang, Ya Wen; Yeh, Chia Feng; Yen, Ming Hong; Lu, Chi Yu; Chiang, Lien Chai; Shieh, Den En; Chang, Jung San

    2017-01-01

    Enterovirus 71 (EV71) infection can cause airway symptoms, brainstem encephalitis, neurogenic shock, and neurogenic pulmonary edema with high morbidity and mortality. There is no proven therapeutic modality. Flos Farfarae is the dried flower bud of Tussilago farfara L. that has been used to manage airway illnesses for thousands of years. It has neuro-protective activity and has been used to manage neuro-inflammatory diseases. However, it is unknown whether Flos Farfarae has activity against EV71-induced neuropathy. The current study used both human foreskin fibroblast (CCFS-1/KMC) and human rhabdomyosarcoma (RD) cell lines to test the hypothesis that a hot water extract of Flos Farfarae could effectively inhibit EV71 infection. The authenticity of Flos Farfarae was confirmed by HPLC-UV fingerprint. Through plaque reduction assays and flow cytometry, Flos Farfarae was found to inhibit EV71 infection ([Formula: see text]). Inhibition of viral replication and protein expression were further confirmed by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR), and western blot, respectively. The estimated IC[Formula: see text]s were 106.3[Formula: see text][Formula: see text]g/mL in CCFS-1/KMC, and 15.0[Formula: see text][Formula: see text]g/mL in RD cells. Therefore, Flos Farfarae could be beneficial to inhibit EV71 infection by preventing viral replication and structural protein expression.

  19. A Single Amino Acid Dictates Protein Kinase R Susceptibility to Unrelated Viral Antagonists

    PubMed Central

    Esparo, Nicolle M.; Child, Stephanie J.; Geballe, Adam P.

    2016-01-01

    During millions of years of coevolution with their hosts, cytomegaloviruses (CMVs) have succeeded in adapting to overcome host-specific immune defenses, including the protein kinase R (PKR) pathway. Consequently, these adaptations may also contribute to the inability of CMVs to cross species barriers. Here, we provide evidence that the evolutionary arms race between the antiviral factor PKR and its CMV antagonist TRS1 has led to extensive differences in the species-specificity of primate CMV TRS1 proteins. Moreover, we identify a single residue in human PKR that when mutated to the amino acid present in African green monkey (Agm) PKR (F489S) is sufficient to confer resistance to HCMVTRS1. Notably, this precise molecular determinant of PKR resistance has evolved under strong positive selection among primate PKR alleles and is positioned within the αG helix, which mediates the direct interaction of PKR with its substrate eIF2α. Remarkably, this same residue also impacts sensitivity to K3L, a poxvirus-encoded pseudosubstrate that structurally mimics eIF2α. Unlike K3L, TRS1 has no homology to eIF2α, suggesting that unrelated viral genes have convergently evolved to target this critical region of PKR. Despite its functional importance, the αG helix exhibits extraordinary plasticity, enabling adaptations that allow PKR to evade diverse viral antagonists while still maintaining its critical interaction with eIF2α. PMID:27780231

  20. Induction of Immune Tolerance to Foreign Protein via Adeno-Associated Viral Vector Gene Transfer in Mid-Gestation Fetal Sheep

    PubMed Central

    Davey, Marcus G.; Riley, John S.; Andrews, Abigail; Tyminski, Alec; Limberis, Maria; Pogoriler, Jennifer E.; Partridge, Emily; Olive, Aliza; Hedrick, Holly L.; Flake, Alan W.; Peranteau, William H.

    2017-01-01

    A major limitation to adeno-associated virus (AAV) gene therapy is the generation of host immune responses to viral vector antigens and the transgene product. The ability to induce immune tolerance to foreign protein has the potential to overcome this host immunity. Acquisition and maintenance of tolerance to viral vector antigens and transgene products may also permit repeat administration thereby enhancing therapeutic efficacy. In utero gene transfer (IUGT) takes advantage of the immunologic immaturity of the fetus to induce immune tolerance to foreign antigens. In this large animal study, in utero administration of AAV6.2, AAV8 and AAV9 expressing green fluorescent protein (GFP) to ~60 day fetal sheep (term: ~150 days) was performed. Transgene expression and postnatal immune tolerance to GFP and viral antigens were assessed. We demonstrate 1) hepatic expression of GFP 1 month following in utero administration of AAV6.2.GFP and AAV8.GFP, 2) in utero recipients of either AAV6.2.GFP or AAV8.GFP fail to mount an anti-GFP antibody response following postnatal GFP challenge and lack inflammatory cellular infiltrates at the intramuscular site of immunization, 3) a serotype specific anti-AAV neutralizing antibody response is elicited following postnatal challenge of in utero recipients of AAV6.2 or AAV8 with the corresponding AAV serotype, and 4) durable hepatic GFP expression was observed up to 6 months after birth in recipients of AAV8.GFP but expression was lost between 1 and 6 months of age in recipients of AAV6.2.GFP. The current study demonstrates, in a preclinical large animal model, the potential of IUGT to achieve host immune tolerance to the viral vector transgene product but also suggests that a single exposure to the vector capsid proteins at the time of IUGT is inadequate to induce tolerance to viral vector antigens. PMID:28141818

  1. Non-viral bone morphogenetic protein 2 transfection of rat dental pulp stem cells using calcium phosphate nanoparticles as carriers.

    PubMed

    Yang, Xuechao; Walboomers, X Frank; van den Dolder, Juliette; Yang, Fang; Bian, Zhuan; Fan, Mingwen; Jansen, John A

    2008-01-01

    Calcium phosphate nanoparticles have shown potential as non-viral vectors for gene delivery. The aim of this study was to induce bone morphogenetic protein (Bmp)2 transfection in rat dental pulp stem cells using calcium phosphate nanoparticles as a gene vector and then to evaluate the efficiency and bioactivity of the transfection. We also intended to investigate the behavior of transfected cells when seeded on 3-dimensional titanium fiber mesh scaffolds. Nanoparticles of calcium phosphate encapsulating plasmid deoxyribonucleic acid (DNA) (plasmid enhanced green fluorescent protein-BMP2) were prepared. Then, STRO-1-selected rat dental pulp stem cells were transfected using these nanoparticles. Transfection and bioactivity of the secreted BMP2 were examined. Thereafter, the transfected cells were cultured on a fibrous titanium mesh. The cultures were investigated using scanning electron microscipy and evaluated for cell proliferation, alkaline phosphatase activity and calcium content. Finally, real-time polymerase chain reaction was performed for odontogenesis-related gene expression. The results showed that the size of the DNA-loaded particles was approximately 100 nm in diameter. Nanoparticles could protect the DNA encapsulated inside from external DNase and release the loaded DNA in a low-acid environment (pH 3.0). In vitro, nanoparticle transfection was shown to be effective and to accelerate or promote the odontogenic differentiation of rat dental pulp stem cells when cultured in the 3-dimensional scaffolds. Based on our results, plasmid DNA-loaded calcium phosphate nanoparticles appear to be an effective non-viral vector for gene delivery and functioned well for odontogenic differentiation through Bmp2 transfection.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  3. A Single Amino Acid Residue Change in the P Protein of Parainfluenza Virus 5 Elevates Viral Gene Expression▿

    PubMed Central

    Timani, Khalid A.; Sun, Dengyun; Sun, Minghao; Keim, Celia; Lin, Yuan; Schmitt, Phuong Tieu; Schmitt, Anthony P.; He, Biao

    2008-01-01

    Parainfluenza virus 5 (PIV5) is a prototypical paramyxovirus. The V/P gene of PIV5 encodes two mRNA species through a process of pseudotemplated insertion of two G residues at a specific site during transcription, resulting in two viral proteins, V and P, whose N termini of 164 amino acid residues are identical. Previously it was reported that mutating six amino acid residues within this identical region results in a recombinant PIV5 (rPIV5-CPI−) that exhibits elevated viral protein expression and induces production of cytokines, such as beta interferon and interleukin 6. Because the six mutations correspond to the shared region of the V protein and the P protein, it is not clear whether the phenotypes associated with rPIV5-CPI− are due to mutations in the P protein and/or mutations in the V protein. To address this question, we used a minigenome system and recombinant viruses to study the effects of mutations on the functions of the P and V proteins. We found that the P protein with six amino acid residue changes (Pcpi−) was more efficient than wild-type P in facilitating replication of viral RNA, while the V protein with six amino acid residue changes (Vcpi−) still inhibits minigenome replication as does the wild-type V protein. These results indicate that elevated viral gene expression in rPIV5-CPI− virus-infected cells can be attributed to a P protein with an increased ability to facilitate viral RNA synthesis. Furthermore, we found that a single amino acid residue change at position 157 of the P protein from Ser (the residue in the wild-type P protein) to Phe (the residue in Pcpi−) is sufficient for elevated viral gene expression. Using mass spectrometry and 33P labeling, we found that residue S157 of the P protein is phosphorylated. Based on these results, we propose that phosphorylation of the P protein at residue 157 plays an important role in regulating viral RNA replication. PMID:18614634

  4. Interplay between SIRT1 and hepatitis B virus X protein in the activation of viral transcription.

    PubMed

    Deng, Jian-Jun; Kong, Ka-Yiu Edwin; Gao, Wei-Wei; Tang, Hei-Man Vincent; Chaudhary, Vidyanath; Cheng, Yun; Zhou, Jie; Chan, Chi-Ping; Wong, Danny Ka-Ho; Yuen, Man-Fung; Jin, Dong-Yan

    2017-04-01

    Hepatitis B virus (HBV) genome is organized into a minichromosome known as covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. SIRT1 is an NAD(+)-dependent protein deacetylase which activates HBV transcription by promoting the activity of cellular transcription factors and coactivators. How SIRT1 and viral transactivator X protein (HBx) might affect each other remains to be clarified. In this study we show synergy and mutual dependence between SIRT1 and HBx in the activation of HBV transcription. All human sirtuins SIRT1 through SIRT7 activated HBV gene expression. The steady-state levels of SIRT1 protein were elevated in HBV-infected liver tissues and HBV-replicating hepatoma cells. SIRT1 interacted with HBx and potentiated HBx transcriptional activity on precore promoter and covalently closed circular DNA (cccDNA) likely through a deacetylase-independent mechanism, leading to more robust production of cccDNA, pregenomic RNA and surface antigen. SIRT1 and HBx proteins were more abundant when both were expressed. SIRT1 promoted the recruitment of HBx as well as cellular transcriptional factors and coactivators such as PGC-1α and FXRα to cccDNA. Depletion of SIRT1 suppressed HBx recruitment. On the other hand, SIRT1 recruitment to cccDNA was compromised when HBx was deficient. Whereas pharmaceutical agonists of SIRT1 such as resveratrol activated HBV transcription, small-molecule inhibitors of SIRT1 including sirtinol and Ex527 exhibited anti-HBV activity. Taken together, our findings revealed not only the interplay between SIRT1 and HBx in the activation of HBV transcription but also new strategies and compounds for developing antivirals against HBV. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Integrated Computational Approach for Virtual Hit Identification against Ebola Viral Proteins VP35 and VP40

    PubMed Central

    Mirza, Muhammad Usman; Ikram, Nazia

    2016-01-01

    The Ebola virus (EBOV) has been recognised for nearly 40 years, with the most recent EBOV outbreak being in West Africa, where it created a humanitarian crisis. Mortalities reported up to 30 March 2016 totalled 11,307. However, up until now, EBOV drugs have been far from achieving regulatory (FDA) approval. It is therefore essential to identify parent compounds that have the potential to be developed into effective drugs. Studies on Ebola viral proteins have shown that some can elicit an immunological response in mice, and these are now considered essential components of a vaccine designed to protect against Ebola haemorrhagic fever. The current study focuses on chemoinformatic approaches to identify virtual hits against Ebola viral proteins (VP35 and VP40), including protein binding site prediction, drug-likeness, pharmacokinetic and pharmacodynamic properties, metabolic site prediction, and molecular docking. Retrospective validation was performed using a database of non-active compounds, and early enrichment of EBOV actives at different false positive rates was calculated. Homology modelling and subsequent superimposition of binding site residues on other strains of EBOV were carried out to check residual conformations, and hence to confirm the efficacy of potential compounds. As a mechanism for artefactual inhibition of proteins through non-specific compounds, virtual hits were assessed for their aggregator potential compared with previously reported aggregators. These systematic studies have indicated that a few compounds may be effective inhibitors of EBOV replication and therefore might have the potential to be developed as anti-EBOV drugs after subsequent testing and validation in experiments in vivo. PMID:27792169

  6. Integrated Computational Approach for Virtual Hit Identification against Ebola Viral Proteins VP35 and VP40.

    PubMed

    Mirza, Muhammad Usman; Ikram, Nazia

    2016-10-26

    The Ebola virus (EBOV) has been recognised for nearly 40 years, with the most recent EBOV outbreak being in West Africa, where it created a humanitarian crisis. Mortalities reported up to 30 March 2016 totalled 11,307. However, up until now, EBOV drugs have been far from achieving regulatory (FDA) approval. It is therefore essential to identify parent compounds that have the potential to be developed into effective drugs. Studies on Ebola viral proteins have shown that some can elicit an immunological response in mice, and these are now considered essential components of a vaccine designed to protect against Ebola haemorrhagic fever. The current study focuses on chemoinformatic approaches to identify virtual hits against Ebola viral proteins (VP35 and VP40), including protein binding site prediction, drug-likeness, pharmacokinetic and pharmacodynamic properties, metabolic site prediction, and molecular docking. Retrospective validation was performed using a database of non-active compounds, and early enrichment of EBOV actives at different false positive rates was calculated. Homology modelling and subsequent superimposition of binding site residues on other strains of EBOV were carried out to check residual conformations, and hence to confirm the efficacy of potential compounds. As a mechanism for artefactual inhibition of proteins through non-specific compounds, virtual hits were assessed for their aggregator potential compared with previously reported aggregators. These systematic studies have indicated that a few compounds may be effective inhibitors of EBOV replication and therefore might have the potential to be developed as anti-EBOV drugs after subsequent testing and validation in experiments in vivo.

  7. Cyclophilin A binds to the viral RNA and replication proteins, resulting in inhibition of tombusviral replicase assembly.

    PubMed

    Kovalev, Nikolay; Nagy, Peter D

    2013-12-01

    Replication of plus-stranded RNA viruses is greatly affected by numerous host-encoded proteins that act as restriction factors. Cyclophilins, which are a large family of cellular prolyl isomerases, have been found to inhibit Tomato bushy stunt tombusvirus (TBSV) replication in a Saccharomyces cerevisiae model based on genome-wide screens and global proteomics approaches. In this report, we further characterize single-domain cyclophilins, including the mammalian cyclophilin A and plant Roc1 and Roc2, which are orthologs of the yeast Cpr1p cyclophilin, a known inhibitor of TBSV replication in yeast. We found that recombinant CypA, Roc1, and Roc2 strongly inhibited TBSV replication in a cell-free replication assay. Additional in vitro studies revealed that CypA, Roc1, and Roc2 cyclophilins bound to the viral replication proteins, and CypA and Roc1 also bound to the viral RNA. These interactions led to inhibition of viral RNA recruitment, the assembly of the viral replicase complex, and viral RNA synthesis. A catalytically inactive mutant of CypA was also able to inhibit TBSV replication in vitro due to binding to the replication proteins and the viral RNA. Overexpression of CypA and its mutant in yeast or plant leaves led to inhibition of tombusvirus replication, confirming that CypA is a restriction factor for TBSV. Overall, the current work has revealed a regulatory role for the cytosolic single-domain Cpr1-like cyclophilins in RNA virus replication.

  8. Enhanced β2-adrenergic receptor (β2AR) signaling by adeno-associated viral (AAV)-mediated gene transfer

    PubMed Central

    Jones, Stacie M; Hiller, F Charles; Jacobi, Sandie E; Foreman, Susan K; Pittman, Laura M; Cornett, Lawrence E

    2003-01-01

    Background β2-Adrenergic receptors (β2AR) play important regulatory roles in a variety of cells and organ systems and are important therapeutic targets in the treatment of airway and cardiovascular disease. Prolonged use of β-agonists results in tolerance secondary to receptor down-regulation resulting in reduced therapeutic efficiency. The purpose of this work is to evaluate the signaling capabilities of the β2AR expressed by a recombinant adeno-associated viral (AAV) vector that also included an enhanced green fluorescent protein (EGFP) gene (AAV-β2AR/EGFP). Results By epifluorescence microscopy, ~40% of infected HEK 293 cells demonstrated EGFP expression. β2AR density measured with [3H]dihydroalprenolol ([3H]DHA) increased either 13- or 77-fold in infected cells compared to mock infected controls depending on the culture conditions used. The [3H]DHA binding was to a single receptor population with a dissociation constant of 0.42 nM, as would be expected for wild-type β2AR. Agonist competition assays with [3H]DHA showed the following rank order of potency: isoproterenol>epinephrine> norepinephrine, consistent with β2AR interaction. Isoproterenol-stimulated cyclic AMP levels were 5-fold higher in infected cells compared to controls (314 ± 43 vs. 63.4 ± 9.6 nmol/dish; n = 3). Receptor trafficking demonstrated surface expression of β2AR with vehicle treatment and internalization following isoproterenol treatment. Conclusions We conclude that HEK 293 cells infected with AAV-β2AR/EGFP effectively express β2AR and that increased expression of these receptors results in enhanced β2AR signaling. This method of gene transfer may provide an important means to enhance function in in vivo systems. PMID:14656380

  9. Molecular mechanisms of interferon resistance mediated by viral-directed inhibition of PKR, the interferon-induced protein kinase.

    PubMed

    Gale, M; Katze, M G

    1998-04-01

    The interferon (IFN)-induced cellular antiviral response is the first line of defense against viral infection within an animal host. In order to establish a productive infection, eukaryotic viruses must first overcome the IFN-induced blocks imposed on viral replication. The double-stranded RNA-activated protein kinase (PKR) is a key component mediating the antiviral actions of IFN. This IFN-induced protein kinase can restrict viral replication through its ability to phosphorylate the protein synthesis initiation factor eukaryotic initiation factor-2 alpha-subunit and reduce levels of viral protein synthesis. Viruses, therefore, must block the function of PKR in order to avoid these deleterious antiviral effects associated with PKR activity. Indeed, many viruses have developed effective measures to repress PKR activity during infection. This review will focus primarily on an overview of the different molecular mechanisms employed by these viruses to meet a common goal: the inhibition of PKR function, uncompromised viral protein synthesis, and unrestricted virus replication. The past few years have seen exciting new advances in this area. Rather unexpectedly, this area of research has benefited from the use of the yeast system to study PKR. Other recent advances include studies on PKR regulation by the herpes simplex viruses and data from our laboratory on the medically important hepatitis C viruses. We speculate that IFN is ineffective as a therapeutic agent against hepatitis C virus because the virus can effectively repress PKR function. Finally, we will discuss briefly the future directions of this PKR field.

  10. 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

  11. Ribosomal protein L4 interacts with viral protein VP3 and regulates the replication of infectious bursal disease virus.

    PubMed

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

    2016-01-04

    VP3 protein is a structural protein which plays important roles in the virus assembly and the inhibition of antiviral innate immunity of infectious bursal disease virus (IBDV). To explore the potential roles of VP3 in the interplay of IBDV with the host cell, an immunoprecipitation (IP)-coupled mass spectra (MS) screening was performed and the host cellular ribosomal protein L4 (RPL4) was identified as a putative interacting partner of VP3 protein. The interaction of RPL4 with VP3 was further confirmed by co-immunoprecipitation (co-IP) and their colocalization in DF1 cells were observed by confocal microscopy. In addition, knockdown of RPL4 in DF1 cells resulted in reductions of the viral protein pVP2 expression and the virus titers, which reveals a significant role of RPL4 in IBDV replication. Taken together, we indicated for the first time that ribosomal protein L4 (RPL4) was an interacting partner of VP3 and involved in the modulation of IBDV replication. The present study contributes to further understanding the pathogenic mechanism of IBDV. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Protein modification during anti-viral heat-treatment bioprocessing of factor VIII concentrates, factor IX concentrates, and model proteins in the presence of sucrose.

    PubMed

    Smales, C Mark; Pepper, Duncan S; James, David C

    2002-01-05

    To ensure the optimal safety of plasma derived and new generation recombinant proteins, heat treatment is customarily applied in the manufacturing of such biopharmaceuticals as a means of viral inactivation. In subjecting proteins to anti-viral heat-treatment it is necessary to use high concentrations of thermostabilizing excipients to prevent protein damage, and it is therefore imperative that the correct balance between bioprocessing conditions, maintenance of protein integrity and virus kill is found. In this study we have utilized model proteins (lysozyme, fetuin, and human serum albumin) and plasma-derived therapeutic proteins (factor VIII and factor IX) to investigate the protein modifications that occur during anti-viral heat treatment. Specifically, we investigated the relationship between bioprocessing conditions and the type and extent of protein modification under a variety of industrially relevant wet and lyophilized heat treatments using sucrose as a thermostabilizing agent. Heat treatment led to the formation of disulfide crosslinks and aggregates in proteins containing free cysteine residues. Terminal oligosaccharide sialic acid residues were hydrolyzed from the glycan moieties of glycoproteins during anti-viral heat treatment. Heat treatment promoted sucrose hydrolysis to yield glucose and fructose, leading, in turn, to the glycation of lysine amino groups in those proteins containing di-lysine motifs. During extended hear treatments, 1,2-dicarbonyl type advanced glycation end-products were also formed. Glycation-type modifications were more prevalent in wet heat-treated protein formulations.

  13. 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

  14. Newly identified phosphorylation site in the vesicular stomatitis virus P protein is required for viral RNA synthesis.

    PubMed

    Mondal, Arindam; Victor, Ken G; Pudupakam, R S; Lyons, Charles E; Wertz, Gail W

    2014-02-01

    The vesicular stomatitis virus (VSV) RNA-dependent RNA polymerase consists of two viral proteins; the large (L) protein is the main catalytic subunit, and the phosphoprotein (P) is an essential cofactor for polymerase function. The P protein interacts with the L protein and the N-RNA template, thus connecting the polymerase to the template. P protein also binds to free N protein to maintain it in a soluble, encapsidation-competent form. Previously, five sites of phosphorylation were identified on the P protein and these sites were reported to be differentially important for mRNA synthesis or genomic replication. The previous studies were carried out by biochemical analysis of portions of the authentic viral P protein or by analysis of bacterium-expressed, exogenously phosphorylated P protein by mutagenesis. However, there has been no systematic biochemical search for phosphorylation sites on authentic, virus-expressed P protein. In this study, we analyzed the P protein isolated from VSV-infected cells for sites of phosphorylation by mass spectrometry. We report the identification of Tyr14 as a previously unidentified phosphorylation site of VSV P and show that it is essential for viral transcription and replication. However, our mass spectral analysis failed to observe the phosphorylation of previously reported C-terminal residues Ser226 and Ser227 and mutagenic analyses did not demonstrate a role for these sites in RNA synthesis.

  15. 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

  16. Dengue virus NS1 protein interacts with the ribosomal protein RPL18: this interaction is required for viral translation and replication in Huh-7 cells.

    PubMed

    Cervantes-Salazar, Margot; Angel-Ambrocio, Antonio H; Soto-Acosta, Ruben; Bautista-Carbajal, Patricia; Hurtado-Monzon, Arianna M; Alcaraz-Estrada, Sofia L; Ludert, Juan E; Del Angel, Rosa M

    2015-10-01

    Given dengue virus (DENV) genome austerity, it uses cellular molecules and structures for virion entry, translation and replication of the genome. NS1 is a multifunctional protein key to viral replication and pathogenesis. Identification of cellular proteins that interact with NS1 may help in further understanding the functions of NS1. In this paper we isolated a total of 64 proteins from DENV infected human hepatic cells (Huh-7) that interact with NS1 by affinity chromatography and immunoprecipitation assays. The subcellular location and expression levels during infection of the ribosomal proteins RPS3a, RPL7, RPL18, RPL18a plus GAPDH were determined. None of these proteins changed their expression levels during infection; however, RPL-18 was redistributed to the perinuclear region after 48hpi. Silencing of the RPL-18 does not affect cell translation efficiency or viability, but it reduces significantly viral translation, replication and viral yield, suggesting that the RPL-18 is required during DENV replicative cycle.

  17. 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

  18. 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).

  19. Expression of Major Capsid Protein VP-1 in the Absence of Viral Particles in Thymomas Induced by Murine Polyomavirus

    PubMed Central

    Sanjuan, Norberto; Porrás, Analía; Otero, Javier; Perazzo, Sofía

    2001-01-01

    Thymomas induced by polyomavirus strain PTA in mice are known to express the major capsid protein VP-1. Since the expression of a late structural protein such as VP-1 is considered a sign of virus replication, the present work attempted to clarify the implication of the presence of this protein in tumor cells. Electron microscopy of tumors showed a striking absence of viral particles in the vast majority of the cells. However, immunoelectron microscopy of the same samples demonstrated intranuclear VP-1 in most cells despite the absence of viral particles. Very little infectious virus was recovered from tumors. A change in the electrophoretic mobility of VP-1 from thymomas was detected compared with VP-1 from productively infected cells. The data presented in this work prove that the expression of VP-1 in polyomavirus-induced tumors is not synonymous with the presence of infectious virus, suggesting a possible defect in viral encapsidation. PMID:11222714

  20. Isolation and immunizations with hepatitis A viral structural proteins: induction of antiprotein, antiviral, and neutralizing responses.

    PubMed

    Hughes, J V; Stanton, L W

    1985-08-01

    An immune affinity purification procedure for hepatitis A virus (HAV) was designed which yielded milligram quantities of the virus with greater than 95% purity. The major structural proteins VP-1, VP-2, and VP-3 were isolated from the purified virus by electroelution from sodium dodecyl sulfate-polyacrylamide gels and used to immunize Lewis rats (three to four doses, 10 to 15 micrograms per dose). The two Lewis rats immunized with VP-1 developed a strong antibody response to VP-1, as determined by Western blot analysis and immune precipitation of the denatured protein. These animals also developed a good antibody response to the whole virus, as demonstrated by a positive response in a competitive radioimmunoassay (HAV antibody test) and by precipitation of the whole virus. In addition, both animals developed a low titer neutralizing antibody to HAV, as demonstrated by an in vitro cell culture assay. While the two rats receiving VP-2 developed only minimal responses to the protein and to the virus by the same assays described above, one of the two developed a significant neutralizing antibody to HAV. The immunization of one Lewis rat with VP-3 induced a good antibody response to both denatured protein and the whole virus. This serum sample was also demonstrated to neutralize the viral infectivity. Finally, two rabbits that had received inoculations of sodium dodecyl sulfate and heat-disrupted HAV (containing 20 to 30 micrograms of each protein per dose) developed good antiprotein responses to all of the proteins and good antiviral responses, including a consistently significant neutralizing activity. The neutralizing antibody responses suggest that the structural proteins of HAV, or a portion of them, could provide the basis for a subunit vaccine for HAV.

  1. 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.

  2. Regulation of Multiple Stages of Hepadnavirus Replication by the Carboxyl-Terminal Domain of Viral Core Protein in trans

    PubMed Central

    Liu, Kuancheng; Ludgate, Laurie; Yuan, Zhenghong

    2014-01-01

    ABSTRACT Mutational analyses have indicated that the carboxyl-terminal domain (CTD) of hepadnavirus core protein and its state of phosphorylation are critical for multiple steps in viral replication. Also, CTD interacts with host proteins in a phosphorylation state-dependent manner. To ascertain the role of CTD in viral replication without perturbing its sequence and the role of CTD-host interactions, CTD of the human hepatitis B virus (HBV) or duck hepatitis B virus (DHBV) core protein, either the wild type (WT) or with alanine or glutamic acid/aspartic acid substitutions at the phosphorylation sites, was expressed in cells replicating DHBV with the WT core protein. A dramatic decrease in phosphorylation of the DHBV core protein (DHBc) was observed when the WT and most HBV core protein CTD (HCTD) variants were coexpressed in trans, which was accompanied by a profound reduction of viral core DNA and, in particular, the double-stranded DNA. One HCTD variant that failed to change DHBc phosphorylation also had no effect on DHBV core DNA. All WT and variant HCTDs and DHBc CTDs (DCTDs) decreased the DHBV covalently closed circular (CCC) DNA. Identification of CTD-host interactions indicated that CDK2 binding by CTD may mediate its inhibitory effect on DHBc phosphorylation and reverse transcription via competition with DHBc for the host kinase, whereas importin α binding by CTD may contribute to inhibition of CCC DNA production by competitively blocking the nuclear import of viral nucleocapsids. These results suggest the possibility of blocking multiple steps of viral replication, especially CCC DNA formation, via inhibition of CTD functions. IMPORTANCE Mutational analyses have suggested that the carboxyl-terminal domain (CTD) of hepadnavirus core protein is critical for viral replication. However, results from mutational analyses are open to alternative interpretations. Also, how CTD affects virus replication remains unclear. In this study, we took an alternative

  3. Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens.

    PubMed

    Valansi, Clari; Moi, David; Leikina, Evgenia; Matveev, Elena; Graña, Martín; Chernomordik, Leonid V; Romero, Héctor; Aguilar, Pablo S; Podbilewicz, Benjamin

    2017-03-06

    Cell-cell fusion is inherent to sexual reproduction. Loss of HAPLESS 2/GENERATIVE CELL SPECIFIC 1 (HAP2/GCS1) proteins results in gamete fusion failure in diverse organisms, but their exact role is unclear. In this study, we show that Arabidopsis thaliana HAP2/GCS1 is sufficient to promote mammalian cell-cell fusion. Hemifusion and complete fusion depend on HAP2/GCS1 presence in both fusing cells. Furthermore, expression of HAP2 on the surface of pseudotyped vesicular stomatitis virus results in homotypic virus-cell fusion. We demonstrate that the Caenorhabditis elegans Epithelial Fusion Failure 1 (EFF-1) somatic cell fusogen can replace HAP2/GCS1 in one of the fusing membranes, indicating that HAP2/GCS1 and EFF-1 share a similar fusion mechanism. Structural modeling of the HAP2/GCS1 protein family predicts that they are homologous to EFF-1 and viral class II fusion proteins (e.g., Zika virus). We name this superfamily Fusexins: fusion proteins essential for sexual reproduction and exoplasmic merger of plasma membranes. We suggest a common origin and evolution of sexual reproduction, enveloped virus entry into cells, and somatic cell fusion.

  4. Interplay of viral miRNAs and host mRNAs and proteins

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2011-10-01

    Recent experiments indicate that several viruses may encode microRNAs (miRNAs) in cells. Such RNAs may interfere with the host mRNAs and proteins. We present a kinetic analysis of this interplay. In our treatment, the viral miRNA is considered to be able to associate with the host mRNA with subsequent degradation. This process may result in a decline of the mRNA population and also in a decline of the population of the protein encoded by this mRNA. With these ingredients, we first show the types of the corresponding steady-state kinetics in the cases of positive and negative regulation of the miRNA synthesis by the protein. In addition, we scrutinize the situation when the protein regulates the virion replication or, in other words, provides a feedback for the replication. For the negative feedback, the replication rate is found to increase with increasing the intracellular virion population. For the positive feedback, the replication rate first increases and then drops. These features may determine the stability of steady states.

  5. Arabidopsis HAP2/GCS1 is a gamete fusion protein homologous to somatic and viral fusogens

    PubMed Central

    Valansi, Clari; Moi, David; Leikina, Evgenia; Matveev, Elena; Chernomordik, Leonid V.

    2017-01-01

    Cell–cell fusion is inherent to sexual reproduction. Loss of HAPLESS 2/GENERATIVE CELL SPECIFIC 1 (HAP2/GCS1) proteins results in gamete fusion failure in diverse organisms, but their exact role is unclear. In this study, we show that Arabidopsis thaliana HAP2/GCS1 is sufficient to promote mammalian cell–cell fusion. Hemifusion and complete fusion depend on HAP2/GCS1 presence in both fusing cells. Furthermore, expression of HAP2 on the surface of pseudotyped vesicular stomatitis virus results in homotypic virus–cell fusion. We demonstrate that the Caenorhabditis elegans Epithelial Fusion Failure 1 (EFF-1) somatic cell fusogen can replace HAP2/GCS1 in one of the fusing membranes, indicating that HAP2/GCS1 and EFF-1 share a similar fusion mechanism. Structural modeling of the HAP2/GCS1 protein family predicts that they are homologous to EFF-1 and viral class II fusion proteins (e.g., Zika virus). We name this superfamily Fusexins: fusion proteins essential for sexual reproduction and exoplasmic merger of plasma membranes. We suggest a common origin and evolution of sexual reproduction, enveloped virus entry into cells, and somatic cell fusion. PMID:28137780

  6. Toll-interacting protein inhibits HIV-1 infection and regulates viral latency.

    PubMed

    Li, Chuan; Kuang, Wen-Dong; Qu, Di; Wang, Jian-Hua

    2016-06-24

    HIV-1 latency is mainly characterized by a reversible silencing of long-terminal repeat (LTR)-driven transcription of provirus. The existing of repressive factors has been described to contribute to transcription silencing of HIV-1. Toll-interacting protein (Tollip) has been identified as a repressor of Toll like receptors (TLR)-mediated signaling. Our previous study has found that Tollip inhibited NF-κB-dependent HIV-1 promoter LTR-driven transcription, indicating the potential role of Tollip in governing viral latency. In this study, by using HIV-1 latently infected Jurkat T-cell and central memory CD4(+) T-cells, we demonstrate the role of Tollip in regulating HIV-1 latency, as the knock-down of Tollip promoted HIV-1 reactivation from both HIV-1 latently infected Jurkat CD4(+) T cells and primary central memory T cells (TCM). Moreover, we found that the activities of LTRs derived from multiple HIV-1 subtypes could be repressed by Tollip; Knock-down of Tollip promoted HIV-1 transcription and infection in CD4(+) T cells. Our data indicate a key role of Tollip in suppressing HIV-1 infection and regulating viral latency, which provides a potential host target for combating HIV-1 infection and latency.

  7. PJA2 ubiquitinates the HIV-1 Tat protein with atypical chain linkages to activate viral transcription

    PubMed Central

    Faust, Tyler B.; Li, Yang; Jang, Gwendolyn M.; Johnson, Jeffrey R.; Yang, Shumin; Weiss, Amit; Krogan, Nevan J.; Frankel, Alan D.

    2017-01-01

    Transcription complexes that assemble at the HIV-1 promoter efficiently initiate transcription but generate paused RNA polymerase II downstream from the start site. The virally encoded Tat protein hijacks positive transcription elongation factor b (P-TEFb) to phosphorylate and activate this paused polymerase. In addition, Tat undergoes a series of reversible post-translational modifications that regulate distinct steps of the transcription cycle. To identify additional functionally important Tat cofactors, we performed RNAi knockdowns of sixteen previously identified Tat interactors and found that a novel E3 ligase, PJA2, ubiquitinates Tat in a non-degradative manner and specifically regulates the step of HIV transcription elongation. Interestingly, several different lysine residues in Tat can function as ubiquitin acceptor sites, and variable combinations of these lysines support both full transcriptional activity and viral replication. Further, the polyubiquitin chain conjugated to Tat by PJA2 can itself be assembled through variable ubiquitin lysine linkages. Importantly, proper ubiquitin chain assembly by PJA2 requires that Tat first binds its P-TEFb cofactor. These results highlight that both the Tat substrate and ubiquitin modification have plastic site usage, and this plasticity is likely another way in which the virus exploits the host molecular machinery to expand its limited genetic repertoire. PMID:28345603

  8. SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release

    PubMed Central

    Raghava, Smita; Giorda, Kristina M.; Romano, Fabian B.; Heuck, Alejandro P.; Hebert, Daniel N.

    2014-01-01

    Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results found that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with bodipy-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of between 1 and 5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers. PMID:23651212

  9. Nuclear envelope breakdown induced by herpes simplex virus type 1 involves the activity of viral fusion proteins

    SciTech Connect

    Maric, Martina; Haugo, Alison C.; Dauer, William; Johnson, David; Roller, Richard J.

    2014-07-15

    Herpesvirus infection reorganizes components of the nuclear lamina usually without loss of integrity of the nuclear membranes. We report that wild-type HSV infection can cause dissolution of the nuclear envelope in transformed mouse embryonic fibroblasts that do not express torsinA. Nuclear envelope breakdown is accompanied by an eight-fold inhibition of virus replication. Breakdown of the membrane is much more limited during infection with viruses that lack the gB and gH genes, suggesting that breakdown involves factors that promote fusion at the nuclear membrane. Nuclear envelope breakdown is also inhibited during infection with virus that does not express UL34, but is enhanced when the US3 gene is deleted, suggesting that envelope breakdown may be enhanced by nuclear lamina disruption. Nuclear envelope breakdown cannot compensate for deletion of the UL34 gene suggesting that mixing of nuclear and cytoplasmic contents is insufficient to bypass loss of the normal nuclear egress pathway. - Highlights: • We show that wild-type HSV can induce breakdown of the nuclear envelope in a specific cell system. • The viral fusion proteins gB and gH are required for induction of nuclear envelope breakdown. • Nuclear envelope breakdown cannot compensate for deletion of the HSV UL34 gene.

  10. 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

    USDA-ARS?s Scientific Manuscript database

    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...

  11. 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.

  12. BFV activates the NF-kappaB pathway through its transactivator (BTas) to enhance viral transcription

    SciTech Connect

    Wang Jian; Tan Juan; Zhang Xihui; Guo Hongyan; Zhang Qicheng; Guo Tingting; Geng Yunqi; Qiao Wentao

    2010-05-10

    Multiple families of viruses have evolved sophisticated strategies to regulate nuclear factor-kappaB (NF-kappaB) signaling, which plays a pivotal role in diverse cellular events, including virus-host interactions. In this study, we report that bovine foamy virus (BFV) is able to activate the NF-kappaB pathway through the action of its transactivator, BTas. Both cellular IKKbeta and IkappaBalpha also participate in this activation. In addition, we demonstrate that BTas induces the processing of p100, which implies that BTas can activate NF-kappaB through a noncanonical pathway as well. Co-immunoprecipitation analysis shows that BTas interacts with IKK catalytic subunits (IKKalpha and IKKbeta), which may be responsible for regulation of IKK kinase activity and persistent NF-kappaB activation. Furthermore, our results indicate that the level of BTas-mediated LTR transcription correlates with the activity of cellular NF-kappaB. Together, this study suggests that BFV activates the NF-kappaB pathway through BTas to enhance viral transcription.

  13. Thixotropic solutions enhance viral-mediated gene transfer to airway epithelia.

    PubMed

    Seiler, Michael P; Luner, Paul; Moninger, Thomas O; Karp, Philip H; Keshavjee, Shaf; Zabner, Joseph

    2002-08-01

    Adenovirus-mediated gene transfer to airway epithelia is inefficient in part because its receptor is absent on the apical surface of the airways. Targeting adenovirus to other receptors, increasing the viral concentration, and even prolonging the incubation time with adenovirus vectors can partially overcome the lack of receptors and facilitate gene transfer. Unfortunately, mucociliary clearance would prevent prolonged incubation time in vivo. Thixotropic solutions (TS) are gels that upon a vigorous shearing force reversibly become liquid. We hypothesized that formulating recombinant adenoviruses in TS would decrease virus clearance and thus enhance gene transfer to the airway epithelia. We found that clearance of virus-sized fluorescent beads by human airway epithelia in vitro and by monkey trachea in vivo were markedly decreased when the beads were formulated in TS compared with phosphate-buffered saline (PBS). Adenovirus formulated in TS significantly increased adenovirus-mediated gene transfer of a reporter gene in human airway epithelia in vitro and in murine airway epithelia in vivo. Furthermore, an adenovirus encoding the cystic fibrosis transmembrane regulator (CFTR) gene (AdCFTR) formulated in TS was more efficient in correcting the chloride transport defect in cystic fibrosis airway epithelia than AdCFTR formulated in PBS. These data indicate a novel strategy to augment the efficiency of gene transfer to the airways that may be applicable to a number of different gene transfer vectors and could be of value in gene transfer to cystic fibrosis (CF) airway epithelia in vivo.

  14. Canine enteric coronaviruses: emerging viral pathogens with distinct recombinant spike proteins.

    PubMed

    Licitra, Beth N; Duhamel, Gerald E; Whittaker, Gary R

    2014-08-22

    Canine enteric coronavirus (CCoV) is an alphacoronavirus infecting dogs that is closely related to enteric coronaviruses of cats and pigs. While CCoV has traditionally caused mild gastro-intestinal clinical signs, there are increasing reports of lethal CCoV infections in dogs, with evidence of both gastrointestinal and systemic viral dissemination. Consequently, CCoV is now considered to be an emerging infectious disease of dogs. In addition to the two known serotypes of CCoV, novel recombinant variants of CCoV have been found containing spike protein N-terminal domains (NTDs) that are closely related to those of feline and porcine strains. The increase in disease severity in dogs and the emergence of novel CCoVs can be attributed to the high level of recombination within the spike gene that can occur during infection by more than one CCoV type in the same host.

  15. The ULb′ Region of the Human Cytomegalovirus Genome Confers an Increased Requirement for the Viral Protein Kinase UL97

    PubMed Central

    Wang, Depeng; Li, Gang; Schauflinger, Martin; Nguyen, Christopher C.; Hall, Ellie D.; Yurochko, Andrew D.; von Einem, Jens

    2013-01-01

    We report a requirement for the viral protein kinase UL97 in human cytomegalovirus (HCMV) replication that maps to the ULb′ region of the viral genome. A UL97-null (Δ97) mutant of strain TB40/E, which encodes a full-length ULb′ region, exhibited replication defects, particularly in production of cell-free virus, that were more severe than those seen with a Δ97 mutant of laboratory strain AD169, which harbors extensive deletions in its ULb′ region. These differences were recapitulated with additional HCMV strains by treatment with a UL97 kinase inhibitor, 1-(β-l-ribofuranosyl)-2-isopropylamino-5,6-dichlorobenzimidazole (maribavir). We observed lower levels of viral DNA synthesis and an increased requirement for UL97 in viral late gene expression in strains with full-length ULb′ regions. Analysis of UL97-deficient TB40/E infections by electron microscopy revealed fewer C-capsids in nuclei, unusual viral particles in the cytoplasmic assembly compartment, and defective viral nuclear egress. Partial inhibition of viral DNA synthesis caused defects in production of cell-free virus that were up to ∼100-fold greater than those seen with cell-associated virus in strains TB40/E and TR, suggesting that UL97-dependent defects in cell-free virus production in strains with full-length ULb′ regions were secondary to DNA synthesis defects. Accordingly, a chimeric virus in which the ULb′ region of TB40/E was replaced with that of AD169 showed reduced effects of UL97 inhibition on viral DNA synthesis, late gene expression, and production of cell-free virus compared to parental TB40/E. Together, these results argue that the ULb′ region encodes a factor(s) which invokes an increased requirement for UL97 during viral DNA synthesis. PMID:23536674

  16. Iridovirus Bcl-2 protein inhibits apoptosis in the early stage of viral infection.

    PubMed

    Lin, Pei-Wen; Huang, Yi-Jen; John, Joseph Abraham Christopher; Chang, Ya-Nan; Yuan, Chung-Hsiang; Chen, Wen-Ya; Yeh, Chiao-Hwa; Shen, San-Tai; Lin, Fu-Pang; Tsui, Wen-Huei; Chang, Chi-Yao

    2008-01-01

    The grouper iridovirus (GIV) belongs to the family Iridoviridae, whose genome contains an antiapoptotic B-cell lymphoma (Bcl)-2-like gene. This study was carried-out to understand whether GIV blocks apoptosis in its host. UV-irradiated grouper kidney (GK) cells underwent apoptosis. However, a DNA fragmentation assay of UV-exposed GK cells after GIV infection revealed an inhibition of apoptosis. The UV- or heat-inactivated GIV failed to inhibit apoptosis, implying that a gene or protein of the viral particle might contribute to an apoptosis inhibitory function. The DNA ladder assay for GIV-infected GK cells after UV irradiation confirmed that apoptosis inhibition was an early process which occurred as early as 5 min post-infection. A GIV-Bcl sequence comparison showed distant sequence similarities to that of human and four viruses; however, all possessed the putative Bcl-2 homology (BH) domains of BH1, BH2, BH3, and BH4, as well as a transmembrane domain. Northern blot hybridization showed that GIV-Bcl transcription began at 2 h post-infection, and the mRNA level significantly increased in the presence of cycloheximide or aphidicolin, indicating that this GIV-Bcl is an immediate-early gene. This was consistent with the Western blot results, which also revealed that the virion carries the Bcl protein. We observed the localization of GIV-Bcl on the mitochondrial membrane and other defined intracellular areas. By immunostaining, it was proven that GIV-Bcl-expressing cells effectively inhibited apoptosis. Taken together, these results demonstrate that GIV inhibits the promotion of apoptosis by GK cells, which is mediated by the immediate early expressed viral Bcl gene.

  17. DNA vaccines encoding viral glycoproteins induce nonspecific immunity and Mx protein synthesis in fish.

    PubMed

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

    2000-08-01

    Protective immunity by vaccination with plasmid DNA encoding a viral glycoprotein (G) has long been assumed to result from the induction of a specific immune response. We report here that the initial protection may be due to the induction of alpha/beta interferon, with long-term protection due to a specific response to the encoded viral G. DNA vaccines encoding the Gs of three serologically unrelated fish rhabdoviruses were used to vaccinate rainbow trout against a lethal challenge with infectious hematopoietic necrosis virus (IHNV). All three vaccines, each encoding the G gene of either IHNV (IHNV-G), snakehead rhabdovirus (SHRV) (SHRV-G), or spring viremia of carp virus (SVCV) (SVCV-G), elicited protective immunity against IHNV. Vaccinated fish were challenged at 30 or 70 days postvaccination with lethal doses of IHNV. At 30 days postvaccination, only 5% of fish that had received any of the G vaccines died, whereas more than 50% of the control fish succumbed to virus challenge. When fish were vaccinated and challenged at 70 days postvaccination, only 12% of the IHNV-G-vaccinated fish died compared to 68% for the SHRV-G- and 76% for the SVCV-G-vaccinated fish. Assays for trout Mx protein, an indicator of alpha/beta interferon induction, showed that only fish vaccinated with a G-containing plasmid produced high levels of Mx protein in the kidneys and liver. Interestingly, at day 7 after virus challenge, all of the fish vaccinated with the IHNV-G plasmid were negative for Mx, but the SHRV-G- and SVCV-G-vaccinated fish still showed detectable levels of Mx. These results suggest that DNA vaccines in fish induce an early, nonspecific antiviral protection mediated by an alpha/beta interferon and, later, a specific immune response.

  18. Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation.

    PubMed

    Lehr, Elizabeth E; Qadadri, Brahim; Brown, Calla R; Brown, Darron R

    2003-09-30

    Human papillomavirus type 59 (HPV 59) is an oncogenic type related to HPV 18. HPV 59 was recently propagated in the athymic mouse xenograft system. A continuous keratinocyte cell line infected with HPV 59 was created from a foreskin xenograft grown in an athymic mouse. Cells were cultured beyond passage 50. The cells were highly pleomorphic, containing numerous abnormally shaped nuclei and mitotic figures. HPV 59 sequences were detected in the cells by DNA in situ hybridization in a diffuse nuclear distribution. Southern blots were consistent with an episomal state of HPV 59 DNA at approximately 50 copies per cell. Analysis of the cells using a PCR/reverse blot strip assay, which amplifies a portion of the L1 open reading frame, was strongly positive. Differentiation of cells in monolayers was induced by growth in F medium containing 2 mM calcium chloride for 10 days. Cells were harvested as a single tissue-like sheet, and histologic analysis revealed a four-to-six cell-thick layer. Transcripts encoding involucrin, a cornified envelope protein, and the E1/E4 and E1/E4/L1 viral transcripts were detected after several days of growth in F medium containing 2 mM calcium chloride. The E1/E4 and L1 proteins were detected by immunohistochemical analysis, and virus particles were seen in electron micrographs in a subset of differentiated cells. An extract of differentiated cells was prepared by vigorous sonication and was used to infect foreskin fragments. These fragments were implanted into athymic mice. HPV 59 was detected in the foreskin xenografts removed 4 months later by DNA in situ hybridization and PCR/reverse blot assay. Thus, the complete viral growth cycle, including production on infectious virus, was demonstrated in the HPV 59 immortalized cells grown in a simple culture system.

  19. Influenza A virus PB1-F2 protein prolongs viral shedding in chickens lengthening the transmission window

    PubMed Central

    James, Joe; Howard, Wendy; Iqbal, Munir; Nair, Venugopal K.; Barclay, Wendy S.

    2016-01-01

    Avian influenza is a significant economic burden on the poultry industry in geographical regions where it is enzootic. It also poses a public health concern when avian influenza subtypes infect humans, often with high mortality. Understanding viral genetic factors which positively contribute to influenza A virus (IAV) fitness – infectivity, spread and pathogenesis – is of great importance both for human and livestock health. PB1-F2 is a small accessory protein encoded by IAV and in mammalian hosts has been implicated in a wide range of functions that contribute to increased pathogenesis. In the avian host, the protein has been understudied despite high-level full-length conservation in avian IAV isolates, which is in contrast to the truncations of the PB1-F2 length frequently found in mammalian host isolates. Here we report that the presence of a full-length PB1-F2 protein, from a low pathogenicity H9N2 avian influenza virus, prolongs infectious virus shedding from directly inoculated chickens, thereby enhancing transmission of the virus by lengthening the transmission window to contact birds. As well as extending transmission, the presence of a full-length PB1-F2 suppresses pathogenicity evidenced by an increased minimum lethal dose in embryonated chicken eggs and increasing survival in directly infected birds when compared to a virus lacking an ORF for PB1-F2. We propose that there is a positive pressure to maintain a full-length functional PB1-F2 protein upon infection of avian hosts as it contributes to the effective transmission of IAV in the field. PMID:27558742

  20. Peptide backbone circularization enhances antifreeze protein thermostability.

    PubMed

    Stevens, Corey A; Semrau, Joanna; Chiriac, Dragos; Litschko, Morgan; Campbell, Robert L; Langelaan, David N; Smith, Steven P; Davies, Peter L; Allingham, John S

    2017-10-01

    Antifreeze proteins (AFPs) are a class of ice-binding proteins that promote survival of a variety of cold-adapted organisms by decreasing the freezing temperature of bodily fluids. A growing number of biomedical, agricultural, and commercial products, such as organs, foods, and industrial fluids, have benefited from the ability of AFPs to control ice crystal growth and prevent ice recrystallization at subzero temperatures. One limitation of AFP use in these latter contexts is their tendency to denature and irreversibly lose activity at the elevated temperatures of certain industrial processing or large-scale AFP production. Using the small, thermolabile type III AFP as a model system, we demonstrate that AFP thermostability is dramatically enhanced via split intein-mediated N- and C-terminal end ligation. To engineer this circular protein, computational modeling and molecular dynamics simulations were applied to identify an extein sequence that would fill the 20-Å gap separating the free ends of the AFP, yet impose little impact on the structure and entropic properties of its ice-binding surface. The top candidate was then expressed in bacteria, and the circularized protein was isolated from the intein domains by ice-affinity purification. This circularized AFP induced bipyramidal ice crystals during ice growth in the hysteresis gap and retained 40% of this activity even after incubation at 100°C for 30 min. NMR analysis implicated enhanced thermostability or refolding capacity of this protein compared to the noncyclized wild-type AFP. These studies support protein backbone circularization as a means to expand the thermostability and practical applications of AFPs. © 2017 The Protein Society.

  1. Viral interaction with molecular chaperones: role in regulating viral infection.

    PubMed

    Xiao, Allen; Wong, Jerry; Luo, Honglin

    2010-07-01

    As essential effectors in protein quality control, molecular chaperones serve as the primary checkpoint to assist proper protein folding and prevent misfolded proteins from denaturation and aggregation. In addition, chaperones can function to direct terminally misfolded proteins to the proteolytic system for degradation. Viruses rely on host cell machineries for productive infection. Like for many other processes, various viruses have been shown to evolve mechanisms to utilize or subvert the host protein quality control machinery to support the completion of their life cycle. Furthermore, recent studies suggest that some viruses encode for their own chaperone-like proteins to enhance their infectivity. This review summarizes the current understanding of the interplay between molecular chaperones and viral proteins, highlights the chaperone activities of a number of viral proteins, and discusses potential antiviral therapeutic strategies targeting the virus-chaperone interactions.

  2. Protein Trans-Splicing as a Means for Viral Vector-Mediated In Vivo Gene Therapy

    PubMed Central

    Li, Juan; Sun, Wenchang; Wang, Bing; Xiao, Xiao

    2008-01-01

    Abstract Inteins catalyze protein splicing in a fashion similar to how self-splicing introns catalyze RNA splicing. Split-inteins catalyze precise ligation of two separate polypeptides through trans-splicing in a highly specific manner. Here we report a method of using protein trans-splicing to circumvent the packaging size limit of gene therapy vectors. To demonstrate this method, we chose a large dystrophin gene and an adeno-associated viral (AAV) vector, which has a small packaging size. A highly functional 6.3-kb Becker-form dystrophin cDNA was broken into two pieces and modified by adding appropriate split-intein coding sequences, resulting in split-genes sufficiently small for packaging in AAV vectors. The two split-genes, after codelivery into target cells, produced two polypeptides that spontaneously trans-spliced to form the expected Becker-form dystrophin protein in cell culture in vitro. Delivering the split-genes by AAV1 vectors into the muscle of a mouse model of Duchenne muscular dystrophy rendered therapeutic gene expression and benefits. PMID:18788906

  3. RNA binding protein CPEB1 remodels host and viral RNA landscapes

    PubMed Central

    Batra, Ranjan; Stark, Thomas J.; Clark, Elizabeth; Belzile, Jean-Philippe; Wheeler, Emily C.; Yee, Brian A.; Huang, Hui; Gelboin-Burkhart, Chelsea; Huelga, Stephanie C.; Aigner, Stefan; Roberts, Brett T.; Bos, Tomas J.; Sathe, Shashank; Donohue, John Paul; Rigo, Frank; Ares, Manuel; Spector, Deborah H.; Yeo, Gene W.

    2016-01-01

    Host and virus interactions at the post-transcriptional level are critical for infection but remain poorly understood. Human cytomegalovirus (HCMV) is a prevalent herpesvirus family member that causes severe complications in immunocompromised patients and newborns. Here, we perform comprehensive transcriptome-wide analyses revealing that HCMV infection results in widespread alternative splicing (AS), shorter 3′-untranslated regions (3′UTRs) and polyA tail lengthening in host genes. The host RNA binding protein cytoplasmic polyadenylation element binding protein 1 (CPEB1) is highly induced upon infection and ectopic expression of CPEB1 in non-infected cells recapitulates infection-related post-transcriptional changes. CPEB1 is also required for polyA-tail lengthening of viral RNAs important for productive infection. Strikingly, depletion of CPEB1 reverses infection-related cytopathology and post-transcriptional changes, and decreases productive HCMV titers. Host RNA processing is also altered in herpes simplex virus-2 (HSV-2) infected cells, indicating a common theme among herpesvirus infections. Our work is a starting point for therapeutic targeting of host RNA binding proteins in herpesvirus infections. PMID:27775709

  4. A Low Protein Binding Cationic Poly(2-oxazoline) as Non-Viral Vector

    PubMed Central

    He, Zhijian; Miao, Lei; Jordan, Rainer; S-Manickam, Devika; Luxenhofer, Robert; Kabanov, Alexander V

    2015-01-01

    Developing safe and efficient non-viral gene delivery systems remains a major challenge. We present a new cationic poly(2-oxazoline) (CPOx) block copolymer for gene therapy that was synthesized by sequential polymerization of non-ionic 2-methyl-2-oxazoline and a new 2-oxazoline monomer, 2-(N-methyl, N-Boc-amino)-methyl-2-oxazoline, followed by deprotection of the pendant secondary amine groups. Upon mixing with plasmid DNA (pDNA), CPOx forms small (diameter ≈ 80 nm) and narrowly dispersed polyplexes (PDI < 0.2), which are stable upon dilution in saline and against thermal challenge. These polyplexes exhibited low plasma protein binding and very low cytotoxicity in vitro compared to the polyplexes of pDNA and poly(ethylene glycol)-b-poly(l-lysine) (PEG-b-PLL). CPOx/pDNA polyplexes at N/P = 5 bound considerably less plasma protein compared to polyplexes of PEG-b-PLL at the same N/P ratio. This is a unique aspect of the developed polyplexes emphasizing their potential for systemic delivery in vivo. The transfection efficiency of the polyplexes in B16 murine melanoma cells was low after 4 h but increased significantly for 10 h exposure time, indicative of slow internalization of polyplexes. Addition of Pluronic P85 boosted the transfection using CPOx/pDNA polyplexes considerably. The low protein binding of CPOx/pDNA polyplexes is particularly interesting for the future development of targeted gene delivery. PMID:25846127

  5. Molecular evolution of viral multifunctional proteins: the case of potyvirus HC-Pro.

    PubMed

    Hasiów-Jaroszewska, Beata; Fares, Mario A; Elena, Santiago F

    2014-01-01

    Our knowledge on the mode of evolution of the multifunctional viral proteins remains incomplete. To tackle this problem, here, we have investigated the evolutionary dynamics of the potyvirus multifunctional protein HC-Pro, with particular focus on its functional domains. The protein was partitioned into the three previously described functional domains, and each domain was analyzed separately and assembled. We searched for signatures of adaptive evolution and evolutionary dependencies of amino acid sites within and between the three domains using the entire set of available potyvirus sequences in GenBank. Interestingly, we identified strongly significant patterns of co-occurrence of adaptive events along the phylogenetic tree in the three domains. These patterns suggest that Domain I, whose main function is to mediate aphid transmission, has likely been coevolving with the other two domains, which are involved in different functions but all requiring the capacity to bind RNA. By contrast, episodes of positive selection on Domains II and III did not correlate, reflecting a trade-off between their evolvability and their evolutionary dependency likely resulting from their functional overlap. Covariation analyses have identified several groups of amino acids with evidence of concerted variation within each domain, but interdomain significant covariations were only found for Domains II and III, further reflecting their functional overlapping.

  6. Interferon-inducible GTPase: a novel viral response protein involved in rabies virus infection.

    PubMed

    Li, Ling; Wang, Hualei; Jin, Hongli; Cao, Zengguo; Feng, Na; Zhao, Yongkun; Zheng, Xuexing; Wang, Jianzhong; Li, Qian; Zhao, Guoxing; Yan, Feihu; Wang, Lina; Wang, Tiecheng; Gao, Yuwei; Tu, Changchun; Yang, Songtao; Xia, Xianzhu

    2016-05-01

    Rabies virus infection is a major public health concern because of its wide host-interference spectrum and nearly 100 % lethality. However, the interactions between host and virus remain unclear. To decipher the authentic response in the central nervous system after rabies virus infection, a dynamic analysis of brain proteome alteration was performed. In this study, 104 significantly differentially expressed proteins were identified, and intermediate filament, interferon-inducible GTPases, and leucine-rich repeat-containing protein 16C were the three outstanding groups among these proteins. Interferon-inducible GTPases were prominent because of their strong upregulation. Moreover, quantitative real-time PCR showed distinct upregulation of interferon-inducible GTPases at the level of transcription. Several studies have shown that interferon-inducible GTPases are involved in many biological processes, such as viral infection, endoplasmic reticulum stress response, and autophagy. These findings indicate that interferon-inducible GTPases are likely to be a potential target involved in rabies pathogenesis or the antiviral process.

  7. The Vpr protein from HIV-1: distinct roles along the viral life cycle

    PubMed Central

    Le Rouzic, Erwann; Benichou, Serge

    2005-01-01

    The genomes of human and simian immunodeficiency viruses (HIV and SIV) encode the gag, pol and env genes and contain at least six supplementary open reading frames termed tat, rev, nef, vif, vpr, vpx and vpu. While the tat and rev genes encode regulatory proteins absolutely required for virus replication, nef, vif, vpr, vpx and vpu encode for small proteins referred to "auxiliary" (or "accessory"), since their expression is usually dispensable for virus growth in many in vitro systems. However, these auxiliary proteins are essential for viral replication and pathogenesis in vivo. The two vpr- and vpx-related genes are found only in members of the HIV-2/SIVsm/SIVmac group, whereas primate lentiviruses from other lineages (HIV-1, SIVcpz, SIVagm, SIVmnd and SIVsyk) contain a single vpr gene. In this review, we will mainly focus on vpr from HIV-1 and discuss the most recent developments in our understanding of Vpr functions and its role during the virus replication cycle. PMID:15725353

  8. Negative Potentials Across Biological Membranes Promote Fusion by Class II and Class III Viral Proteins

    PubMed Central

    Markosyan, Ruben M.

    2010-01-01

    Voltage was investigated as a factor in the fusion of virions. Virions, pseudotyped with a class II, SFV E1 or VEEV E, or a class III protein, VSV G, were prepared with GFP within the core and a fluorescent lipid. This allowed both hemifusion and fusion to be monitored. Voltage clamping the target cell showed that fusion is promoted by a negative potential and hindered by a positive potential. Hemifusion occurred independent of polarity. Lipid dye movement, in the absence of content mixing, ceased before complete transfer for positive potentials, indicating that reversion of hemifused membranes into two distinct membranes is responsible for voltage dependence and inhibition of fusion. Content mixing quickly followed lipid dye transfer for a negative potential, providing a direct demonstration that hemifusion induced by class II and class III viral proteins is a functional intermediate of fusion. In the hemifused state, virions that fused exhibited slower lipid transfer than did nonfusing virions. All viruses with class II or III fusion proteins may utilize voltage to achieve infection. PMID:20427575

  9. Viral interference with DNA repair by targeting of the single-stranded DNA binding protein RPA.

    PubMed

    Banerjee, Pubali; DeJesus, Rowena; Gjoerup, Ole; Schaffhausen, Brian S

    2013-10-01

    Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

  10. Stability of Retroviral Vectors Against Ultracentrifugation Is Determined by the Viral Internal Core and Envelope Proteins Used for Pseudotyping.

    PubMed

    Kim, Soo-Hyun; Lim, Kwang-Il

    2017-05-31

    Retroviral and lentiviral vectors are mostly pseudotyped and often purified and concentrated via ultracentrifugation. In this study, we quantified and compared the stabilities of retroviral [murine leukemia virus (MLV)-based] and lentiviral [human immunodeficiency virus (HIV)-1-based] vectors pseudotyped with relatively mechanically stable envelope proteins, vesicular stomatitis virus glycoproteins (VSVGs), and the influenza virus WSN strain envelope proteins against ultracentrifugation. Lentiviral genomic and functional particles were more stable than the corresponding retroviral particles against ultracentrifugation when pseudotyped with VSVGs. However, both retroviral and lentiviral particles were unstable when pseudotyped with the influenza virus WSN strain envelope proteins. Therefore, the stabilities of pseudotyped retroviral and lentiviral vectors against ultracentrifugation process are a function of not only the type of envelope proteins, but also the type of viral internal core (MLV or HIV-1 core). In addition, the fraction of functional viral particles among genomic viral particles greatly varied at times during packaging, depending on the type of envelope proteins used for pseudotyping and the viral internal core.

  11. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    SciTech Connect

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory . E-mail: vchinchar@microbio.umsmed.edu

    2007-02-20

    Frog virus 3 (FV3) is a large DNA virus that encodes {approx} 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-II{alpha}). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-II{alpha} triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins.

  12. A second CRM1-dependent nuclear export signal in the influenza A virus NS2 protein contributes to the nuclear export of viral ribonucleoproteins.

    PubMed

    Huang, Shengping; Chen, Jingjing; Chen, Quanjiao; Wang, Huadong; Yao, Yanfeng; Chen, Jianjun; Chen, Ze

    2013-01-01

    Influenza A virus NS2 protein, also called nuclear export protein (NEP), is crucial for the nuclear export of viral ribonucleoproteins. However, the molecular mechanisms of NEP mediation in this process remain incompletely understood. A leucine-rich nuclear export signal (NES2) in NEP, located at the predicted N2 helix of the N-terminal domain, was identified in the present study. NES2 was demonstrated to be a transferable NES, with its nuclear export activity depending on the nuclear export receptor chromosome region maintenance 1 (CRM1)-mediated pathway. The interaction between NEP and CRM1 is coordinately regulated by both the previously reported NES (NES1) and now the new NES2. Deletion of the NES1 enhances the interaction between NEP and CRM1, and deletion of the NES1 and NES2 motifs completely abolishes this interaction. Moreover, NES2 interacts with CRM1 in the mammalian two-hybrid system. Mutant viruses containing NES2 alterations generated by reversed genetics exhibit reduced viral growth and delay in the nuclear export of viral ribonucleoproteins (vRNPs). The NES2 motif is highly conserved in the influenza A and B viruses. The results demonstrate that leucine-rich NES2 is involved in the nuclear export of vRNPs and contributes to the understanding of nucleocytoplasmic transport of influenza virus vRNPs.

  13. PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

    PubMed Central

    Zhang, Yong; Mao, Dailing; Roswit, William T.; Jin, Xiaohua; Patel, Anand C.; Patel, Dhara A.; Agapov, Eugene; Wang, Zhepeng; Tidwell, Rose M.; Atkinson, Jeffrey J.; Huang, Guangming; McCarthy, Ronald; Yu, Jinsheng; Yun, Nadezhda E.; Paessler, Slobodan; Lawson, T. Glen; Omattage, Natalie S.; Brett, Tom J.; Holtzman, Michael J.

    2015-01-01

    Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve interferon hyperresponsiveness without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depends on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acts on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to initiate their degradation via the immunoproteasome. Together, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway. PMID:26479788

  14. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.

    PubMed

    Sun, Yingjie; Dong, Luna; Yu, Shengqing; Wang, Xiaoxu; Zheng, Hang; Zhang, Pin; Meng, Chunchun; Zhan, Yuan; Tan, Lei; Song, Cuiping; Qiu, Xusheng; Wang, Guijun; Liao, Ying; Ding, Chan

    2017-04-01

    Mammalian cells respond to various environmental stressors to form stress granules (SGs) by arresting cytoplasmic mRNA, protein translation element, and RNA binding proteins. Virus-induced SGs function in different ways, depending on the species of virus; however, the mechanism of SG regulation of virus replication is not well understood. In this study, Newcastle disease virus (NDV) triggered stable formation of bona fide SGs on HeLa cells through activating the protein kinase R (PKR)/eIF2α pathway. NDV-induced SGs contained classic SG markers T-cell internal antigen (TIA)-1, Ras GTPase-activating protein-binding protein (G3BP)-1, eukaryotic initiation factors, and small ribosomal subunit, which could be disassembled in the presence of cycloheximide. Treatment with nocodazole, a microtubule disruption drug, led to the formation of relatively small and circular granules, indicating that NDV infection induces canonical SGs. Furthermore, the role of SGs on NDV replication was investigated by knockdown of TIA-1 and TIA-1-related (TIAR) protein, the 2 critical components involved in SG formation from the HeLa cells, followed by NDV infection. Results showed that depletion of TIA-1 or TIAR inhibited viral protein synthesis, reduced extracellular virus yields, but increased global protein translation. FISH revealed that NDV-induced SGs contained predominantly cellular mRNA rather than viral mRNA. Deletion of TIA-1 or TIAR reduced NP mRNA levels in polysomes. These results demonstrate that NDV triggers stable formation of bona fide SGs, which benefit viral protein translation and virus replication by arresting cellular mRNA.-Sun, Y., Dong, L., Yu, S., Wang, X., Zheng, H., Zhang, P., Meng, C., Zhan, Y., Tan, L., Song, C., Qiu, X., Wang, G., Liao, Y., Ding, C. Newcastle disease virus induces stable formation of bona fide stress granules to facilitate viral replication through manipulating host protein translation.

  15. The conundrum of a unique protein encoded by citrus tristeza virus that is dispensable for infection of most hosts yet shows characteristics of a viral movement protein.

    PubMed

    Bak, Aurélie; Folimonova, Svetlana Y

    2015-11-01

    Citrus tristeza virus (CTV), one of the most economically important viruses, produces a unique protein, p33, which is encoded only in the genomes of isolates of CTV. Recently, we demonstrated that membrane association of the p33 protein confers virus ability to extend its host range. In this work we show that p33 shares characteristics of viral movement proteins. Upon expression in a host cell, the protein localizes to plasmodesmata and displays the ability to form extracellular tubules. Furthermore, p33 appears to traffic via the cellular secretory pathway and the actin network to plasmodesmata locations and is likely being recycled through the endocytic pathway. Finally, our study reveals that p33 colocalizes with a putative movement protein of CTV, the p6 protein. These results suggest a potential role of p33 as a noncanonical viral movement protein, which mediates virus translocation in the specific hosts. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Purification in an active form of the phage phi 29 protein p4 that controls the viral late transcription.

    PubMed Central

    Barthelemy, I; Lázaro, J M; Méndez, E; Mellado, R P; Salas, M

    1987-01-01

    The phage phi 29 protein p4, that controls viral late transcription, was highly purified from Escherichia coli cells harbouring a gene 4-containing plasmid. This protein, representing about 6% of the total cellular protein, was obtained in a highly purified form. The protein was characterized as p4 by amino acid analysis and NH2-terminal sequence determination. The purified protein was active in an in vitro transcription assay, allowing specific initiation of transcription at the phi 29 A3 late promoter in the presence of Bacillus subtilis sigma 43-RNA polymerase holoenzyme. Images PMID:3671066

  17. The Use of Nanotrap Particles Technology in Capturing HIV-1 Virions and Viral Proteins from Infected Cells

    PubMed Central

    Sampey, Gavin; Shafagati, Nazly; Van Duyne, Rachel; Iordanskiy, Sergey; Kehn-Hall, Kylene; Liotta, Lance; Petricoin, Emanuel; Young, Mary; Lepene, Benjamin; Kashanchi, Fatah

    2014-01-01

    HIV-1 infection results in a chronic but incurable illness since long-term HAART can keep the virus to an undetectable level. However, discontinuation of therapy rapidly increases viral burden. Moreover, patients under HAART frequently develop various metabolic disorders and HIV-associated neuronal disease. Today, the main challenge of HIV-1 research is the elimination of the residual virus in infected individuals. The current HIV-1 diagnostics are largely comprised of serological and nucleic acid based technologies. Our goal is to integrate the nanotrap technology into a standard research tool that will allow sensitive detection of HIV-1 infection. This study demonstrates that majority of HIV-1 virions in culture supernatants and Tat/Nef proteins spiked in culture medium can be captured by nanotrap particles. To determine the binding affinities of different baits, we incubated target molecules with nanotrap particles at room temperature. After short sequestration, materials were either eluted or remained attached to nanotrap particles prior to analysis. The unique affinity baits of nanotrap particles preferentially bound HIV-1 materials while excluded albumin. A high level capture of Tat or Tat peptide by NT082 and NT084 particles was measured by western blot (WB). Intracellular Nef protein was captured by NT080, while membrane-associated Nef was captured by NT086 and also detected by WB. Selective capture of HIV-1 particles by NT073 and NT086 was measured by reverse transcriptase assay, while capture of infectious HIV-1 by these nanoparticles was demonstrated by functional transactivation in TZM-bl cells. We also demonstrated specific capture of HIV-1 particles and exosomes-containing TAR-RNA in patients' serum by NT086 and NT082 particles, respectively, using specific qRT-PCR. Collectively, our data indicate that certain types of nanotrap particles selectively capture specific HIV-1 molecules, and we propose to use this technology as a platform to enhance HIV-1

  18. A multi-scale mathematical modeling framework to investigate anti-viral therapeutic opportunities in targeting HIV-1 accessory proteins

    PubMed Central

    Suryawanshi, Gajendra W.; Hoffmann, Alexander

    2015-01-01

    Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif’s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that

  19. A multi-scale mathematical modeling framework to investigate anti-viral therapeutic opportunities in targeting HIV-1 accessory proteins.

    PubMed

    Suryawanshi, Gajendra W; Hoffmann, Alexander

    2015-12-07

    Human immunodeficiency virus-1 (HIV-1) employs accessory proteins to evade innate immune responses by neutralizing the anti-viral activity of host restriction factors. Apolipoprotein B mRNA-editing enzyme 3G (APOBEC3G, A3G) and bone marrow stromal cell antigen 2 (BST2) are host resistance factors that potentially inhibit HIV-1 infection. BST2 reduces viral production by tethering budding HIV-1 particles to virus producing cells, while A3G inhibits the reverse transcription (RT) process and induces viral genome hypermutation through cytidine deamination, generating fewer replication competent progeny virus. Two HIV-1 proteins counter these cellular restriction factors: Vpu, which reduces surface BST2, and Vif, which degrades cellular A3G. The contest between these host and viral proteins influences whether HIV-1 infection is established and progresses towards AIDS. In this work, we present an age-structured multi-scale viral dynamics model of in vivo HIV-1 infection. We integrated the intracellular dynamics of anti-viral activity of the host factors and their neutralization by HIV-1 accessory proteins into the virus/cell population dynamics model. We calculate the basic reproductive ratio (Ro) as a function of host-viral protein interaction coefficients, and numerically simulated the multi-scale model to understand HIV-1 dynamics following host factor-induced perturbations. We found that reducing the influence of Vpu triggers a drop in Ro, revealing the impact of BST2 on viral infection control. Reducing Vif׳s effect reveals the restrictive efficacy of A3G in blocking RT and in inducing lethal hypermutations, however, neither of these factors alone is sufficient to fully restrict HIV-1 infection. Interestingly, our model further predicts that BST2 and A3G function synergistically, and delineates their relative contribution in limiting HIV-1 infection and disease progression. We provide a robust modeling framework for devising novel combination therapies that target

  20. A promoter of Epstein-Barr virus that can function during latent infection can be transactivated by EBNA-1, a viral protein required for viral DNA replication during latent infection.

    PubMed Central

    Sugden, B; Warren, N

    1989-01-01

    A viral promoter that functions on recombinant plasmids in cells immortalized by Epstein-Barr virus was identified and characterized. It is identical to that mapped on the viral genome by Bodescot et al. (M. Bodescot, M. Perricaudet, and P.J. Farrell, J. Virol. 61:3424-3430, 1987) which functions during the latent phase of the viral life cycle in some but not all cells to encode several latent viral gene products. Experiments with these plasmids indicated that this promoter requires the enhancer within oriP of Epstein-Barr virus in cis to function efficiently. They also indicated that it requires the EBNA-1 gene in trans to function efficiently. The EBNA-1 gene therefore positively affects both viral DNA replication (J.L. Yates, N. Warren, and B. Sugden, Nature [London] 313:812-815, 1985) and viral transcription. Images PMID:2542577