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Daniel N. Hebert - One of the best experts on this subject based on the ideXlab platform.
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SV40 Late Protein VP4 Forms Toroidal Pores To Disrupt Membranes for Viral Release
Biochemistry, 2013Co-Authors: Smita Raghava, Kristina Giorda, Fabian B. Romano, Alejandro P. Heuck, Daniel N. HebertAbstract: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.
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The SV40 Late Protein VP4 is a viroporin that forms pores to disrupt membranes for viral release.
PLoS pathogens, 2011Co-Authors: Smita Raghava, Kristina Giorda, Fabian B. Romano, Alejandro P. Heuck, Daniel N. HebertAbstract:Nonenveloped viruses are generally released by the timely lysis of the host cell by a poorly understood process. For the nonenveloped virus SV40, virions assemble in the nucleus and then must be released from the host cell without being encapsuLated by cellular membranes. This process appears to involve the well-controlled insertion of viral Proteins into host cellular membranes rendering them permeable to large molecules. VP4 is a newly identified SV40 gene product that is expressed at Late times during the viral life cycle that corresponds to the time of cell lysis. To investigate the role of this Late expressed Protein in viral release, water-soluble VP4 was expressed and purified as a GST fusion Protein from bacteria. Purified VP4 was found to efficiently bind biological membranes and support their disruption. VP4 perforated membranes by directly interacting with the membrane bilayer as demonstrated by flotation assays and the release of fluorescent markers encapsuLated into large unilamellar vesicles or liposomes. The central hydrophobic domain of VP4 was essential for membrane binding and disruption. VP4 displayed a preference for membranes comprised of lipids that replicated the composition of the plasma membranes over that of nuclear membranes. Phosphatidylethanolamine, a lipid found at high levels in bacterial membranes, was inhibitory against the membrane perforation activity of VP4. The disruption of membranes by VP4 involved the formation of pores of ∼3 nm inner diameter in mammalian cells including permissive SV40 host cells. Altogether, these results support a central role of VP4 acting as a viroporin in the perforation of cellular membranes to trigger SV40 viral release.
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Simian Virus 40 Late Proteins Possess Lytic Properties That Render Them Capable of Permeabilizing Cellular Membranes
Journal of virology, 2006Co-Authors: Robert Daniels, Nasser M. Rusan, Anne-kathrin Wilbuer, Leonard C. Norkin, Patricia Wadsworth, Daniel N. HebertAbstract:Many nonenveloped viruses have evolved an infectious cycle that culminates in the lysis or permeabilization of the host to enable viral release. How these viruses initiate the lytic event is largely unknown. Here, we demonstrated that the simian virus 40 progeny accumuLated at the nuclear envelope prior to the permeabilization of the nuclear, endoplasmic reticulum, and plasma membranes at a time which corresponded with the release of the progeny. The permeabilization of these cellular membranes temporally correLated with Late Protein expression and was not observed upon the inhibition of their synthesis. To address whether one or more of the Late Proteins possessed an inherent capacity to induce membrane permeabilization, we examined the permeability of Escherichia coli that separately expressed the Late Proteins. VP2 and VP3, but not VP1, caused the permeabilization of bacterial membranes. Additionally, VP3 expression resulted in bacterial cell lysis. These findings demonstrate that VP3 possesses an inherent lytic property that is independent of eukaryotic signaling or cell death pathways.
Gerd Sutter - One of the best experts on this subject based on the ideXlab platform.
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double stranded rna binding Protein e3 controls translation of viral intermediate rna marking an essential step in the life cycle of modified vaccinia virus ankara
Journal of General Virology, 2006Co-Authors: Holger Ludwig, Yasemin Suezer, Zoe Waibler, Ulrich Kalinke, Barbara S Schnierle, Gerd SutterAbstract:Infection of human cells with modified vaccinia virus Ankara (MVA) activates the typical cascade-like pattern of viral early-, intermediate- and Late-gene expression. In contrast, infection of human HeLa cells with MVA deleted of the E3L gene (MVA-ΔE3L) results in high-level synthesis of intermediate RNA, but lacks viral Late transcription. The viral E3 Protein is thought to bind double-stranded RNA (dsRNA) and to act as an inhibitor of dsRNA-activated 2′-5′-oligoadenyLate synthetase (2′-5′OA synthetase)/RNase L and Protein kinase (PKR). Here, it is demonstrated that viral intermediate RNA can form RNase A/T1-resistant dsRNA, suggestive of activating both the 2′-5′OA synthetase/RNase L pathway and PKR in various human cell lines. Western blot analysis revealed that failure of Late transcription in the absence of E3L function resulted from the deficiency to produce essential viral intermediate Proteins, as demonstrated for vaccinia Late transcription factor 2 (VLTF 2). Substantial host cell-specific differences were found in the level of activation of either RNase L or PKR. However, both rRNA degradation and phosphorylation of eukaryotic translation initiation factor-2α (eIF2α) inhibited the synthesis of VLTF 2 in human cells. Moreover, intermediate VLTF 2 and Late-Protein production were restored in MVA-ΔE3L-infected mouse embryonic fibroblasts from Pkr 0/0 mice. Thus, both host-response pathways may be involved, but activity of PKR is sufficient to block the MVA molecular life cycle. These data imply that an essential function of vaccinia virus E3L is to secure translation of intermediate RNA and, thereby, expression of other viral genes.
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role of viral factor e3l in modified vaccinia virus ankara infection of human hela cells regulation of the virus life cycle and identification of differentially expressed host genes
Journal of Virology, 2005Co-Authors: Holger Ludwig, Jorg Mages, Caroline Staib, Michael H Lehmann, Roland Lang, Gerd SutterAbstract:Modified vaccinia virus Ankara (MVA) is a highly attenuated virus strain being developed as a vaccine for delivery of viral and recombinant antigens. The MVA genome lacks functional copies of numerous genes interfering with host response to infection. The interferon resistance gene E3L encodes one important viral immune defense factor still made by MVA. Here we demonstrate an essential role of E3L to allow for completion of the MVA molecular life cycle upon infection of human HeLa cells. A deletion mutant virus, MVA-ΔE3L, was found defective in Late Protein synthesis, viral Late transcription, and viral DNA replication in infected HeLa cells. Moreover, we detected viral early and continuing intermediate transcription associated with degradation of rRNA, indicating rapid activation of 2′-5′-oligoadenyLate synthetase/RNase L in the absence of E3L. Further molecular monitoring of E3L function by microarray analysis of host cell transcription in MVA- or MVA-ΔE3L-infected HeLa cells revealed an overall significant down regulation of more than 50% of cellular transcripts expressed under mock conditions already at 5 h after infection, with a more prominent shutoff following MVA-ΔE3L infection. Interestingly, a cluster of genes up reguLated exclusively in MVA-ΔE3L-infected cells could be identified, including transcripts for interleukin 6, growth arrest and DNA damage-inducible Protein β, and dual-specificity Protein phosphatases. Our data indicate that lack of E3L inhibits MVA antigen production in human HeLa cells at the level of viral Late gene expression and suggest that E3L can prevent activation of additional host factors possibly affecting the MVA molecular life cycle.
Wenjie Tan - One of the best experts on this subject based on the ideXlab platform.
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Non-replicating Vaccinia Virus TianTan Strain (NTV) Translation Arrest of Viral Late Protein Synthesis Associated With Anti-viral Host Factor SAMD9.
Frontiers in cellular and infection microbiology, 2020Co-Authors: Ying Zhao, Li Zhao, Panpan Huang, Jiao Ren, Peng Zhang, Houwen Tian, Wenjie TanAbstract:NTV is a highly attenuated virus that was created by genetically deleting 26 genes reLated to host range and virulence from TianTan strain. Since NTV is highly attenuated, it has been used widely as an optimizing viral vector. In this study, we explored the biological characteristics in vitro and the host restriction mechanism of NTV. Most cell lines do not support sufficient dissemination and replication of NTV, and in non-permissive cell line HeLa, the replication block of NTV occurred at the translation stage of viral Late Protein expression. Lack of PKR activity was not sufficient to rescue expression of viral Late Proteins and replication, even though the phosphorylation level of eIF2α increased in NTV-infected HeLa cells. Moreover, the translation inhibition of NTV in HeLa cells was dependent upon a SAMD9 signaling pathway, as demonstrated by silencing SAMD9 expression with siRNA and observing the colocalization of SAMD9 and AVGs. Reinserting C7L or K1L into NTV rescued the Late viral Protein expression and replication of NTV in HeLa cells. Among the genes deleted in NTV, C7L or/and K1L gene was mainly responsible for its replication defect. Protein C7 interacted with SAMD9, which antagonized the antiviral response of SAMD9 to ensure viral Protein translation and replication of NTV in non-permissive cell lines. Our finding will serve as a baseline for modification of NTV in future application.
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Preliminary exploration of replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan
Chinese Journal of Clinical Hepatology, 2018Co-Authors: Panpan Huang, Ying Zhao, Li Zhao, Jiao Ren, Li Ruan, Wenjie TanAbstract:Objective To detect the expression level of early and Late Protein of vaccinia virus and to preliminarily explore replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan. Methods We constructed prokaryotic expression vector, expressed and purified homologous early Protein E3 and Late Protein A27 closely reLated to replication and prepared mouse polyclonal antiserum by immunizing mice with homologous Proteins. Early and Late Protein expression levels of NTV were detected. Results We have expressed and purified vaccinia virus Proteins respectively in E. coli expression system and prepared homologous mouse polyclonal antiserum. Early Protein E3 and Late Protein A27 could be highly efficient expression in NTV infected non-permissive Hela cells, while expression of Late Protein F17 was blocked detected by Western blot. Conclusions The expression limitation of Late Protein F17 may be an explanation for the replication-defective mechanism of NTV. Key words: Replication-defective vaccinia virus; Vaccinia virus vector; NTV; Replication-defective mechanism
Gillian Elliott - One of the best experts on this subject based on the ideXlab platform.
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nuclear cytoplasmic compartmentalization of the herpes simplex virus 1 infected cell transcriptome is co ordinated by the viral endoribonuclease vhs and cofactors to facilitate the translation of Late Proteins
PLOS Pathogens, 2018Co-Authors: Kathleen Pheasant, Carla S Mollerlevet, Juliet Jones, Daniel P Depledge, Judith Breuer, Gillian ElliottAbstract:HSV1 encodes an endoribonuclease termed virion host shutoff (vhs) that is produced Late in infection and packaged into virions. Paradoxically, vhs is active against not only host but also virus transcripts, and is involved in host shutoff and the temporal expression of the virus transcriptome. Two other virus Proteins—VP22 and VP16 –are proposed to reguLate vhs to prevent uncontrolled and lethal mRNA degradation but their mechanism of action is unknown. We have performed dual transcriptomic analysis and single-cell mRNA FISH of human fibroblasts, a cell type where in the absence of VP22, HSV1 infection results in extreme translational shutoff. In Wt infection, host mRNAs exhibited a wide range of susceptibility to vhs ranging from resistance to 1000-fold reduction, a variation that was independent of their relative abundance or transcription rate. However, vhs endoribonuclease activity was not found to be overactive against any of the cell transcriptome in Δ22-infected cells but rather was delayed, while its activity against the virus transcriptome and in particular Late mRNA was minimally enhanced. Intriguingly, immediate-early and early transcripts exhibited vhs-dependent nuclear retention Later in Wt infection but Late transcripts were cytoplasmic. However, in the absence of VP22, not only early but also Late transcripts were retained in the nucleus by a vhs-dependent mechanism, a characteristic that extended to cellular transcripts that were not efficiently degraded by vhs. Moreover, the ability of VP22 to bind VP16 enhanced but was not fundamental to the rescue of vhs-induced nuclear retention of Late transcripts. Hence, translational shutoff in HSV1 infection is primarily a result of vhs-induced nuclear retention and not degradation of infected cell mRNA. We have therefore revealed a new mechanism whereby vhs and its co-factors including VP22 elicit a temporal and spatial regulation of the infected cell transcriptome, thus co-ordinating efficient Late Protein production.
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nuclear cytoplasmic compartmentalization of the herpes simplex virus 1 infected cell transcriptome is co ordinated by the viral endoribonuclease vhs and cofactors to facilitate the translation of Late Proteins
bioRxiv, 2018Co-Authors: Kathleen Pheasant, Carla S Mollerlevet, Juliet Jones, Daniel P Depledge, Judith Breuer, Gillian ElliottAbstract:Abstract HSV1 encodes an endoribonuclease termed virion host shutoff (vhs) that is produced Late in infection and packaged into virions. Paradoxically, vhs is active against not only host but also virus transcripts, and is involved in host shutoff and the temporal expression of the virus transcriptome. Two other virus Proteins - VP22 and VP16 – are proposed to reguLate vhs to prevent uncontrolled and lethal mRNA degradation but their mechanism of action is unknown. We have performed dual transcriptomic analysis and single-cell mRNA FISH of human fibroblasts, a cell type where in the absence of VP22, HSV1 infection results in extreme translational shutoff. In Wt infection, host mRNAs exhibited a wide range of susceptibility to vhs ranging from resistance to 1000-fold reduction, a variation that was independent of their relative abundance or transcription rate. However, vhs endoribonuclease activity was not found to be overactive against any of the cell transcriptome in Δ22-infected cells but rather was delayed, while its activity against the virus transcriptome and in particular Late mRNA was minimally enhanced. Intriguingly, immediate-early and early transcripts exhibited vhs-dependent nuclear retention Later in Wt infection but Late transcripts were cytoplasmic. However, in the absence of VP22, not only early but also Late transcripts were retained in the nucleus, a characteristic that extended to cellular transcripts that were not efficiently degraded by vhs. Moreover, the ability of VP22 to bind VP16 enhanced but was not fundamental to the rescue of vhs-induced nuclear retention of Late transcripts. Hence, translational shutoff in HSV1 infection is primarily a result of vhs-induced nuclear retention and not degradation of infected cell mRNA. We have therefore revealed a new mechanism whereby vhs and its co-factors including VP22 elicit a temporal and spatial regulation of the infected cell transcriptome, thus co-ordinating efficient Late Protein production. Author Summary Herpesviruses are large DNA viruses that replicate in the nucleus and express their genes by exploiting host cell mRNA biogenesis mechanisms including transcription, nuclear export, translation and turnover. As such, these viruses express multiple factors that enable the appropriation of cellular pathways for optimal virus production, and work in concert to shut off host gene expression and to overexpress virus genes in a well-described cascade that occurs in a temporal pattern of immediate-early, early and Late Proteins. We have analysed global and single cell changes in the host and virus transcriptome to uncover a novel mechanism by which the viral endoribonuclease, termed vhs, turns off early virus gene expression. This is achieved through the vhs-induced nuclear retention of the entire infected cell transcriptome at the onset of Late gene expression. To enable the switch from early to Late Protein production the virus then requires a second factor called VP22 to specifically inhibit the nuclear retention of Late transcripts allowing their translation in the cytoplasm. In this way, HSV1 elicits a temporal and spatial regulation of the infected cell transcriptome to co-ordinate efficient Late Protein production, a process that may be relevant to herpesviruses in general.
Bonita J. Biegalke - One of the best experts on this subject based on the ideXlab platform.
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Human Cytomegalovirus UL34 Early and Late Proteins Are Essential for Viral Replication
Viruses, 2014Co-Authors: Rico Rana, Bonita J. BiegalkeAbstract:UL34 is one of the ~50 genes of human cytomegalovirus (HCMV) required for replication in cell culture in human fibroblasts. UL34 encodes highly reLated early (UL34a) and Late (UL34b) Proteins that are virtually identical, with the early Protein containing an additional 21 amino terminal amino acids. The UL34 Proteins are sequence-specific DNA‑binding Proteins that localize to the nucleus. The HCMV genome contains 14 to 15 UL34 binding sites; two of the UL34 binding sites contribute to transcriptional regulation of two other viral genes, US3 and US9. The roles of the remaining binding sites and the requirement for both UL34 Proteins during viral infection remain unknown. We examined the contributions of the early and Late UL34 Proteins to viral replication by generating HCMV-containing bacterial artificial chromosomes with the initiation codon for the early or the Late Protein mutated. Neither virus was able to replicate, demonstrating that UL34 expression is required throughout the viral replication cycle. A marked decrease in viral gene expression for each of the mutants suggests that UL34 Proteins may contribute generally to transcriptional regulation. Intracellular localization studies demonstrated that UL34 colocalizes with the major immediate early Protein, IE2, and the viral DNA polymerase processivity factor, UL44, to viral DNA replication centers. In conclusion, sustained UL34 Protein expression is required for viral replication. The sequence-specific DNA binding ability of UL34 Proteins, their localization to viral DNA replication centers and their general effects on viral gene expressions suggests that UL34 Proteins contribute to the establishment of a nuclear environment necessary for viral gene expression and DNA replication.
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Identification of the functional domains of the essential human cytomegalovirus UL34 Proteins.
Virology, 2006Co-Authors: Erin Lester, Rico Rana, Ziqi Liu, Bonita J. BiegalkeAbstract:The human cytomegalovirus UL34 gene represses expression of the US3 immune evasion gene and is essential for viral replication in cell culture. Two highly reLated Proteins, an early and a Late Protein, are synthesized at different times during infection from the UL34 gene. The Late Protein differs from the early Protein only by the omission of 21 amino terminal amino acids. Both UL34 Proteins repress expression of the US3 promoter and bind specifically to a DNA element in the US3 gene. We have localized the DNA-binding domain of the UL34 open reading frame to amino acids 22 to 243. Surprisingly, this same region of the UL34 open reading frame was also sufficient for transcriptional repression of US3 expression. The UL34 gene is unusual in encoding Proteins that have extensively overlapping DNA-binding and transcriptional regulatory domains.
