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Bernard Moss - One of the best experts on this subject based on the ideXlab platform.
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Vaccinia Virus strain differences in cell attachment and entry
Virology, 2009Co-Authors: Zain Bengali, Alan C Townsley, Bernard MossAbstract:Vaccinia Virus (VACV) strain WR can enter cells by a low pH endosomal pathway or direct fusion with the plasma membrane at neutral pH. Here, we compared attachment and entry of five VACV strains in six cell lines and discovered two major patterns. Only WR exhibited pH 5-enhanced rate of entry following neutral pH adsorption to cells, which correlated with sensitivity to bafilomycin A1, an inhibitor of endosomal acidification. Entry of IHD-J, Copenhagen and Elstree strains were neither accelerated by pH 5 treatment nor prevented by bafilomycin A1. Entry of the Wyeth strain, although not augmented by pH 5, was inhibited by bafilomycin A1. WR and Wyeth were both relatively resistant to the negative effects of heparin on entry, whereas the other strains were extremely sensitive due to inhibition of cell binding. The relative sensitivities of individual Vaccinia Virus strains to heparin correlated inversely with their abilities to bind to and enter glycosaminoglycan-deficient sog9 cells but not other cell lines tested. These results suggested that that IHD-J, Copenhagen and Elstree have a more limited ability than WR and Wyeth to use the low pH endosomal pathway and are more dependent on binding to glycosaminoglycans for cell attachment.
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protein composition of the Vaccinia Virus mature virion
Virology, 2007Co-Authors: Wolfgang Resch, Kim K Hixson, Ronald J Moore, Mary S Lipton, Bernard MossAbstract:The protein content of Vaccinia Virus mature virions, purified by rate zonal and isopycnic centrifugations and solubilized by SDS or a solution of urea and thiourea, was determined by the accurate mass and time tag technology which uses both tandem mass spectrometry and Fourier transform-ion cyclotron resonance mass spectrometry to detect tryptic peptides separated by high-resolution liquid chromatography. Eighty Vaccinia Virus-encoded proteins representing 37% of the 218 genes annotated in the complete genome sequence were detected in at least three analyses. Ten proteins accounted for approximately 80% of the virion mass. Thirteen identified proteins were not previously reported as components of virions. On the other hand, 8 previously described virion proteins were not detected here, presumably due to technical reasons including small size and hydrophobicity. In addition to Vaccinia Virus-encoded proteins, 24 host proteins omitting isoforms were detected. The most abundant of these were cytoskeletal proteins, heat shock proteins and proteins involved in translation.
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Vaccinia Virus entry into cells via a low ph dependent endosomal pathway
Journal of Virology, 2006Co-Authors: Alan C Townsley, Andrea S Weisberg, Timothy R Wagenaar, Bernard MossAbstract:Previous studies established that Vaccinia Virus could enter cells by fusion with the plasma membrane at neutral pH. However, low pH triggers fusion of Vaccinia Virus-infected cells, a hallmark of Viruses that enter by the endosomal route. Here, we demonstrate that entry of mature Vaccinia virions is accelerated by brief low-pH treatment and severely reduced by inhibitors of endosomal acidification, providing evidence for a predominant low-pH-dependent endosomal pathway. Entry of Vaccinia Virus cores into the cytoplasm, measured by expression of firefly luciferase, was increased more than 10-fold by exposure to a pH of 4.0 to 5.5. Furthermore, the inhibitors of endosomal acidification bafilomycin A1, concanamycin A, and monensin each lowered Virus entry by more than 70%. This reduction was largely overcome by low-pH-induced entry through the plasma membrane, confirming the specificities of the drugs. Entry of Vaccinia Virus cores with or without brief low-pH treatment was visualized by electron microscopy of thin sections of immunogold-stained cells. Although some Virus particles fused with the plasma membrane at neutral pH, 30 times more fusions and a greater number of cytoplasmic cores were seen within minutes after low-pH treatment. Without low-pH exposure, the number of released cores lagged behind the number of virions in vesicles until 30 min posttreatment, when they became approximately equal, perhaps reflecting the time of endosome acidification and Virus fusion. The choice of two distinct pathways may contribute to the ability of Vaccinia Virus to enter a wide range of cells.
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protective immunity to Vaccinia Virus induced by vaccination with multiple recombinant outer membrane proteins of intracellular and extracellular virions
Journal of Virology, 2004Co-Authors: Christiana Fogg, Roselyn J. Eisenberg, Gary H. Cohen, Shlomo Lustig, Charles J Whitbeck, Bernard MossAbstract:Infectious intracellular and extracellular forms of Vaccinia Virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature Virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped Virus. The recombinant proteins, in 10-μg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live Vaccinia Virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live Vaccinia Virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of Vaccinia Virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge Virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular Virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live Vaccinia Virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine.
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highly attenuated smallpox vaccine protects mice with and without immune deficiencies against pathogenic Vaccinia Virus challenge
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Linda S Wyatt, Patricia L Earl, Leigh Anne Eller, Bernard MossAbstract:Modified Vaccinia Virus Ankara (MVA), developed >30 years ago as a highly attenuated candidate smallpox vaccine, was recloned from a 1974 passage and evaluated for safety and immunogenicity. Replication of MVA is impaired in most mammalian cells, and we found that mice with severe combined immunodeficiency disease remained healthy when inoculated with MVA at 1,000 times the lethal dose of Vaccinia Virus derived from the licensed Dryvax vaccine seed. In BALB/c mice inoculated intramuscularly with MVA, Virus-specific CD8+ T cells and antibodies to purified virions and membrane protein components of the intracellular and extracellular infectious forms of Vaccinia Virus were induced in a dose-dependent manner. After one or two inoculations of MVA, the T cell numbers and antibody titers equaled or exceeded those induced by percutaneous injection of Dryvax. Antibodies induced by MVA and Dryvax were neutralizing and inhibited Virus spread in cultured cells. Furthermore, vaccinated mice were protected against lethal intranasal challenge with a pathogenic Vaccinia Virus. B cell-deficient mice unable to generate antibodies and β2-microglobulin-deficient mice unable to express MHC class I molecules for a CD8+ T cell response were also protectively vaccinated by MVA. In contrast, mice with decreased CD4 or MHC class II expression and double-knockout mice deficient in MHC class I- and II-restricted activities were poorly protected or unprotected. This study confirmed the safety of MVA and demonstrated that the overlapping immune responses protected normal and partially immune-deficient animals, an encouraging result for this candidate attenuated smallpox vaccine.
Gerd Sutter - One of the best experts on this subject based on the ideXlab platform.
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modified Vaccinia Virus ankara history value in basic research and current perspectives for vaccine development
Advances in Virus Research, 2017Co-Authors: Asisa Volz, Gerd SutterAbstract:Safety tested Modified Vaccinia Virus Ankara (MVA) is licensed as third-generation vaccine against smallpox and serves as a potent vector system for development of new candidate vaccines against infectious diseases and cancer. Historically, MVA was developed by serial tissue culture passage in primary chicken cells of Vaccinia Virus strain Ankara, and clinically used to avoid the undesirable side effects of conventional smallpox vaccination. Adapted to growth in avian cells MVA lost the ability to replicate in mammalian hosts and lacks many of the genes orthopoxViruses use to conquer their host (cell) environment. As a biologically well-characterized mutant Virus, MVA facilitates fundamental research to elucidate the functions of poxVirus host-interaction factors. As extremely safe viral vectors MVA vaccines have been found immunogenic and protective in various preclinical infection models. Multiple recombinant MVA currently undergo clinical testing for vaccination against human immunodeficiency Viruses, Mycobacterium tuberculosis or Plasmodium falciparum. The versatility of the MVA vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the Middle East Respiratory Syndrome. Recent advances include promising results from the clinical testing of recombinant MVA-producing antigens of highly pathogenic avian influenza Virus H5N1 or Ebola Virus. This review summarizes our current knowledge about MVA as a unique strain of Vaccinia Virus, and discusses the prospects of exploiting this Virus as research tool in poxVirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology.
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non plaque forming virions of modified Vaccinia Virus ankara express viral genes
Virology, 2016Co-Authors: Annatheresa Lulf, Gerd Sutter, Astrid Freudenstein, Lisa Marr, Asisa VolzAbstract:In cell culture infections with Vaccinia Virus the number of counted Virus particles is substantially higher than the number of plaques obtained by titration. We found that standard vaccine preparations of recombinant Modified Vaccinia Virus Ankara produce only about 20-30% plaque-forming virions in fully permissive cell cultures. To evaluate the biological activity of the non-plaque-forming particles, we generated recombinant Viruses expressing fluorescent reporter proteins under transcriptional control of specific viral early and late promoters. Live cell imaging and automated counting by fluorescent microscopy indicated that virtually all Virus particles can enter cells and switch on viral gene expression. Although most of the non-plaque-forming infections are arrested at the level of viral early gene expression, we detected activation of late viral transcription in 10-20% of single infected cells. Thus, non-plaque-forming particles are biologically active, and likely contribute to the immunogenicity of Vaccinia Virus vaccines.
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modified Vaccinia Virus ankara mva development as recombinant vaccine and prospects for use in veterinary medicine
Berliner Und Munchener Tierarztliche Wochenschrift, 2015Co-Authors: Asisa Volz, Robert Fux, M C Langenmayer, Gerd SutterAbstract:PoxViruses as expression vectors are widely used in medical research for the development of recombinant vaccines and molecular therapies. Here we review recent accomplishments in vaccine research using recombinant modified Vaccinia Virus ankara (MVA). MVA is a highly attenuated Vaccinia Virus strain that originated from serial tissue culture passage in chicken embryo fibroblasts more than 40 years ago. Growth adaptation to avian host cells caused deletions and mutations in the viral genome affecting about 15% of the original genetic information. In consequence, MVA is replication-deficient in cells of mammalian origin and fails to produce many of the virulence factors encoded by conventional Vaccinia Virus. Because of its safety for the general environment MVA can be handled under conditions of biosafety level one. Non-replicating MVA can enter any target cell and activate its molecular life cycle to express all classes of viral and recombinant genes. Therefore, recombinant MVA have been established as an extremely safe and efficient vector system for vaccine development in medical research. By now, various recombinant MVA vaccines have been found safe and immunogenic when used for phase I/II clinical testing in humans, and suitable for industrial scale production following good practice of manufacturing. Thus, there is an obvious usefulness of recombinant MVA vaccines for novel prophylactic and therapeutic approaches also in veterinary medicine. Results from first studies in companion and farm animals are highly promising.
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Modified Vaccinia Virus Ankara but not Vaccinia Virus induces chemokine expression in cells of the monocyte/macrophage lineage
Virology journal, 2015Co-Authors: Michael H. Lehmann, Philip J. R. Price, Christine Brandmüller, Gerd SutterAbstract:The orthopoxVirus strain Modified Vaccinia Virus Ankara (MVA) rapidly induces innate immune responses. Previously, we demonstrated that CCL2 and CCR1 are important players in MVA induced recruitment of leukocytes to the lung. Alveolar macrophages are sentinel cells in the lung, which are likely amongst the first cells of the immune system to encounter and respond to Virus during respiratory infection. Therefore we examined the potential of the murine alveolar macrophage MH-S cell line as a model to study chemokine expression during infection with MVA and Vaccinia Virus (VACV) strain Western Reserve (WR). MVA but not VACV infected MH-S cells increased the expression of the CXCR2 acting chemokine CXCL2. MH-S cells constitutively produced CCL2 and CCR1 acting chemokines CCL3, CCL5 and CCL9. Consequently, supernatants of mock treated and Virus infected MH-S cells induced chemotaxis of murine promyelocyte MPRO cells and human monocytic THP-1 cells at the same level. However, supernatants of MVA infected MH-S cells significantly increased chemotaxis of the CCR2 deficient human monocytic cell line U-937. Chemotaxis of all three cell types was inhibited by J 113863, a CCR1 antagonist. Additionally, we show that MVA but not VACV WR infection of THP-1 cells induces expression of C-C motif and C-X-C motif chemokines and generates a chemotactic activity for monocytes, which was J 113863 sensitive. These results extend our previous findings, demonstrating that MVA but not VACV WR induces chemokine production in alveolar macrophages and monocytes, which can induce recruitment of monocytes in a CCR1 dependent manner.
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candidate influenza vaccines based on recombinant modified Vaccinia Virus ankara
Expert Review of Vaccines, 2009Co-Authors: Guus F Rimmelzwaan, Gerd SutterAbstract:Recombinant modified Vaccinia Virus Ankara (MVA) is attractive and promising as a novel viral vector for the expression of foreign genes of interest because it possesses unique properties. In particular, its excellent safety profile and the availability of versatile vector technologies have frequently made MVA the Vaccinia Virus of choice for preclinical and clinical studies. Owing to its avirulence and deficiency to productively replicate after in vivo inoculation, MVA can be used under biosafety level 1 conditions. In addition to a better safety profile than replication competent Vaccinia Viruses, the use of MVA leads to similar levels of gene expression and has better immunostimulatory properties and improved efficacy as a recombinant vaccine. In animal models, recombinant MVA vaccines were immunogenic and induced protective immunity against various infectious agents, including Viruses, bacteria and parasites. Here we review the progress that has been made in the development of recombinant MVA as a viral vector and candidate pandemic influenza H5N1 vaccine. Specifically, we will focus on the preclinical evaluation of recombinant MVA vector as pandemic influenza A/H5N1 vaccine candidates and discuss the possible future approaches for the use of these novel MVA-based vaccines.
Michael Merchlinsky - One of the best experts on this subject based on the ideXlab platform.
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smallpox vaccines induce antibodies to the immunomodulatory secreted Vaccinia Virus complement control protein
Journal of General Virology, 2009Co-Authors: Joan E Adamo, Jerry P. Weir, Clement A. Meseda, Michael MerchlinskyAbstract:Vaccination with Dryvax elicits a broad humoral response against many viral proteins. Human Vaccinia immune globulin was used to screen the secreted proteins from cells infected with Dryvax or the candidate smallpox vaccine LC16m8 to determine whether the protective humoral response included antibodies against secreted viral proteins. Many proteins were detected, with the primary band corresponding to a band of 28 or 30 kDa in cells infected with Dryvax or LC16m8, respectively. This was identified as the Vaccinia Virus complement protein (VCP), which migrated more slowly in LC16m8-infected cells due to post-translational glycosylation. Vaccinia Virus deleted in VCP, vVCPko, protected mice from a lethal intranasal challenge of Vaccinia Western Reserve strain. Mice vaccinated with purified VCP demonstrated a strong humoral response, but were not protected against a moderate lethal challenge of Vaccinia Virus, suggesting that the humoral response against VCP is not critical for protection.
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Vaccinia Virus nicking joining enzyme is encoded by k4l vacwr035
Journal of Virology, 2005Co-Authors: Dawn Eckert, Ollie Williams, Clement A. Meseda, Michael MerchlinskyAbstract:Vaccinia Virus encodes an enzyme with DNA modifying activity that cleaves and inefficiently cross-links cruciformic DNA. This enzyme is contained within the virion, expressed at late times postinfection, and processes DNA in an energy-independent, Mg2+ ion-independent manner. Viral nuclease activity was measured in extracts from cells infected with well-defined viral mutants. Since some viral extracts lacked nuclease activity, the gene encoding the activity was postulated to be one of the open reading frames absent in the Viruses lacking activity. Inducible expression of each candidate open reading frame revealed that only the gene VACWR035, or K4L, was required for nuclease activity. A recombinant Virus missing only the open reading frame for K4L lacked nuclease activity. Extracts from a recombinant Virus expressing K4L linked to a FLAG polypeptide were able to cleave and cross-link cruciformic DNA. There were no significant differences between the Virus lacking K4L and wild-type Vaccinia Virus WR with respect to infectivity, growth characteristics, or processing of viral replicative intermediate DNA, including both telomeric and cross-linked forms. Purification of the K4L FLAG polypeptide expressed in bacteria yielded protein containing nicking-joining activity, implying that K4L is the only Vaccinia Virus protein required for the nicking-joining enzymatic activity.
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Vaccinia Virus wr gene a5l is required for morphogenesis of mature virions
Journal of Virology, 1999Co-Authors: Ollie Williams, Andrea S Weisberg, Elizabeth J. Wolffe, Michael MerchlinskyAbstract:The Vaccinia Virus WR A5L open reading frame (corresponding to open reading frame A4L in Vaccinia Virus Copenhagen) encodes an immunodominant late protein found in the core of the Vaccinia virion. To investigate the role of this protein in Vaccinia Virus replication, we have constructed a recombinant Virus, vA5Li, in which the endogenous gene has been deleted and an inducible copy of the A5 gene dependent on isopropyl-β-d-thiogalactopyranoside (IPTG) for expression has been inserted into the genome. In the absence of inducer, the yield of infectious Virus was dramatically reduced. However, DNA synthesis and processing, viral protein expression (except for A5), and early stages in virion formation were indistinguishable from the analogous steps in a normal infection. Electron microscopy revealed that the major Vaccinia Virus structural form present in cells infected with vA5Li in the absence of inducer was immature virions. Viral particles were purified from vA5Li-infected cells in the presence and absence of inducer. Both particles contained viral DNA and the full complement of viral proteins, except for A5, which was missing from particles prepared in the absence of inducer. The particles prepared in the presence of IPTG were more infectious than those prepared in its absence. The A5 protein appears to be required for the immature virion to form the brick-shaped intracellular mature virion.
Barbara S Schnierle - One of the best experts on this subject based on the ideXlab platform.
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Vaccinia Virus double stranded rna binding protein e3 does not interfere with sirna mediated gene silencing in mammalian cells
Virus Research, 2007Co-Authors: Markus Lantermann, Gerd Sutter, Astrid Schwantes, Katja Sliva, Barbara S SchnierleAbstract:Vaccinia Virus (VACV) evolved several strategies to evade antiviral cellular defence. The Vaccinia Virus E3 protein for example binds and sequesters double stranded RNA (dsRNA) and counteracts interferon action. We were interested to find out whether and to what extend E3 interferes with RNA silencing mediated by short interfering RNA (siRNA) in mammalian cells. We could show that the expression of a VACV-encoded marker gene can be efficiently inhibited by siRNA independently of the presence of the E3 protein. In addition, expression of E3 had no impact on RNA polymerase III promoter-derived shRNA-induced silencing of a cellular gene in human cells. Both VACV early and late gene expression could be inhibited by siRNA. Furthermore, downregulation of the expression of the E3L gene itself by siRNA in VACV infected cells produced the previously described phenotype of a knock-out Virus, which illustrates the power of siRNA for Vaccinia Virus gene function analysis.
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Vaccinia Virus mediated inhibition of host protein synthesis involves neither degradation nor underphosphorylation of components of the cap binding eukaryotic translation initiation factor complex eif 4f
Virology, 1992Co-Authors: Barbara S Schnierle, Bernard MossAbstract:Recent reports indicated that Vaccinia Virus late mRNAs contain a unique 5' poly(A) leader sequence and that the in vitro translation of these mRNAs may be relatively cap-independent. These observations led us to examine the possibility that the mechanism of inhibition of host protein synthesis by Vaccinia Virus, like that of certain other Viruses, involves specific modifications of the cap-binding translation initiation factor complex eIF-4F. The eIF-4F complex was affinity-purified from human cells infected with Vaccinia Virus and analyzed by one- and two-dimensional electrophoresis and immunoblotting. No evidence of Vaccinia Virus-induced degradation of p220, as occurs during polioVirus infection, or alteration of phosphorylation of eIF-4E (p24), as occurs during adenoVirus infection, was detected at the time of severe inhibition of host protein synthesis.
Wen Chang - One of the best experts on this subject based on the ideXlab platform.
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Vaccinia Virus proteome identification of proteins in Vaccinia Virus intracellular mature virion particles
Journal of Virology, 2006Co-Authors: Che-sheng Chung, Cheng-yen Huang, Cheinhung Chen, Chunglin Liao, Wen ChangAbstract:Vaccinia Virus is a large enveloped poxVirus with more than 200 genes in its genome. Although many poxVirus genomes have been sequenced, knowledge of the host and viral protein components of the virions remains incomplete. In this study, we used gel-free liquid chromatography and tandem mass spectroscopy to identify the viral and host proteins in purified Vaccinia intracellular mature virions (IMV). Analysis of the proteins in the IMV showed that it contains 75 viral proteins, including structural proteins, enzymes, transcription factors, and predicted viral proteins not known to be expressed or present in the IMV. We also determined the relative abundances of the individual protein components in the IMV. Finally, 23 IMV-associated host proteins were also identified. This study provides the first comprehensive structural analysis of the infectious Vaccinia Virus IMV.
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Vaccinia Virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts.
Journal of virology, 2005Co-Authors: Che-sheng Chung, Cheng-yen Huang, Wen ChangAbstract:Vaccinia Virus infects a wide variety of mammalian cells from different hosts, but the mechanism of Virus entry is not clearly defined. The mature intracellular Vaccinia Virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular Vaccinia Virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-β-cyclodextrin, inhibited Vaccinia Virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a Virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after Virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4°C and was only observed when virion penetration occurred at 37°C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant Viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of Vaccinia virions through glycosaminoglycans is not required for lipid raft formation.
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Vaccinia Virus j1r protein a viral membrane protein that is essential for virion morphogenesis
Journal of Virology, 2002Co-Authors: Wenling Chiu, Wen ChangAbstract:Vaccinia Virus, a member of the poxVirus family, contains a conserved J1R open reading frame that encodes a late protein of 17.8 kDa. The 18-kDa J1R protein is associated mainly with the membrane fraction of intracellular mature Virus particles. This study examines the biological function of J1R protein in the Vaccinia Virus life cycle. A recombinant Vaccinia Virus was constructed to conditionally express J1R protein in an isopropyl-β-d-galactopyranoside (IPTG)-inducible manner. When J1R is not expressed during Vaccinia Virus infection, the Virus titer is reduced approximately 100-fold. In contrast, J1R protein is not required for viral gene expression, as indicated by protein pulse-labeling. J1R protein is also not required for DNA processing, as the resolution of the concatemer junctions of replicated viral DNA was detected without IPTG. A deficiency of J1R protein caused a severe delay in the processing of p4a and p4b into mature core proteins 4a and 4b, indicating that J1R protein participates in virion morphogenesis. Infected cells grown in the absence of IPTG contained very few intracellular mature virions in the cytoplasm, and enlarged viroplasm structures accumulated with viral crescents attached at the periphery. Abundant intermediate membrane structures of abnormal shapes were observed, and many immature virions were either empty or partially filled, indicating that J1R protein is important for DNA packaging into immature virions. J1R protein also coimmunoprecipited with A45R protein in infected cells. In summary, these results indicate that Vaccinia Virus J1R is a membrane protein that is required for Virus growth and plaque formation. J1R protein interacts with A45R protein and performs an important role during immature virion formation in cultured cells.
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isolation and characterization of a chinese hamster ovary mutant cell line with altered sensitivity to Vaccinia Virus killing
Journal of Virology, 1996Co-Authors: Chihorng Bair, Che-sheng Chung, I A Vasilevskaya, Wen ChangAbstract:The Chinese hamster ovary (CHO) cell line is nonpermissive for Vaccinia Virus, and translation of viral intermediate genes was reported to be blocked (A. Ramsey-Ewing and B. Moss, Virology 206:984-993, 1995). However, cells are readily killed by Vaccinia Virus. A Vaccinia Virus-resistant CHO mutant, VV5-4, was isolated by retroviral insertional mutagenesis. Parental CHO cells, upon infection with Vaccinia Virus, die within 2 to 3 days, whereas VV5-4 cells preferentially survive this cytotoxic effect. The survival phenotype of VV5-4 is partial and in inverse correlation with the multiplicity of infection used. In addition, viral infection fails to shut off host protein synthesis in VV5-4. VV5-4 was used to study the relationship of progression of the Virus life cycle and cell fate. We found that in parental CHO cells, Vaccinia Virus proceeds through expression of viral early genes, uncoating, viral DNA replication, and expression of intermediate and late promoters. In contrast, we detect only expression of early genes and uncoating in VV5-4 cells, whereas viral DNA replication appears to be blocked. Consistent with the cascade regulation model of viral gene expression, we detect little intermediate- and late-gene expression in VV5-4 cells. Since Vaccinia Virus is known to be cytolytic, isolation of this mutant therefore demonstrates a new mode of the cellular microenvironment that affects progression of the Virus life cycle, resulting in a different cell fate. This process appears to be mediated by a general mechanism, since VV5-4 is also resistant to Shope fibroma Virus and myxoma Virus killing. On the other hand, VV5-4 remains sensitive to cowpox Virus killing. To examine the mechanism of VV5-4 survival, we investigated whether apoptosis is involved. DNA laddering and staining of apoptotic nuclei with Hoechst 33258 were observed in both CHO and VV5-4 cells infected with Vaccinia Virus. We concluded that the cellular pathway, which blocks viral DNA replication and allows VV5-4 to survive, is independent of apoptosis. This mutant also provides evidence that an inductive signal for apoptosis upon Vaccinia Virus infection occurs prior to viral DNA replication.