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Anna Marie Skalka - One of the best experts on this subject based on the ideXlab platform.
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The cellular protein daxx interacts with Avian Sarcoma virus integrase and viral DNA to repress viral transcription.
Journal of Virology, 2005Co-Authors: James G. Greger, Richard A. Katz, Alexander M. Ishov, Gerd G. Maul, Anna Marie SkalkaAbstract:The cellular protein Daxx was identified as an interactor with Avian Sarcoma virus (ASV) integrase (IN) in a yeast two-hybrid screen. After infection, Daxx-IN interactions were detected by coimmunoprecipitation. An association between Daxx and viral DNA, likely mediated by IN, was also detected by chromatin immunoprecipitation. Daxx was not required for early events in ASV replication, including integration, as Daxx-null cells were transduced as efficiently as Daxx-expressing cells. However, viral reporter gene expression from ASV-based vectors was substantially higher in the Daxx-null cells than in Daxx-complemented cells. Consistent with this observation, histone deacetylases (HDACs) were found to associate with viral DNA in Daxx-complemented cells but not in Daxx-null cells. Furthermore, Daxx protein was induced in an interferon-like manner upon ASV infection. We conclude that Daxx interacts with an IN-viral DNA complex early after infection and may mediate the repression of viral gene expression via the recruitment of HDACs. Our findings provide a novel example of cellular immunity against viral replication in which viral transcription is repressed via the recruitment of antiviral proteins to the viral DNA.
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genome wide analyses of Avian Sarcoma virus integration sites
Journal of Virology, 2004Co-Authors: Anna Narezkina, Anna Marie Skalka, Konstantin Taganov, Samuel Litwin, Radka Stoyanova, Junpei Hayashi, Christoph Seeger, Richard A. KatzAbstract:The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 Avian Sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected. However, RNA polymerase II transcription units (protein-encoding genes) appear to be favored targets of ASV integration. The integration frequency within genes is similar to that previously described for murine leukemia virus but distinct from the higher frequency observed with human immunodeficiency virus type 1. We found no evidence for preferred ASV integration sites over the length of genes and immediate flanking regions. Microarray analysis of uninfected HeLa cells revealed that the expression levels of ASV target genes were similar to the median level for all genes represented in the array. Although expressed genes were targets for integration, we found no preference for integration into highly expressed genes. Our results provide a more detailed description of the chromosomal features that may influence ASV integration and support the idea that distinct, virus-specific mechanisms mediate integration site selection. Such differences may be relevant to viral pathogenesis and provide utility in retroviral vector design.
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transduction of terminally differentiated neurons by Avian Sarcoma virus
Journal of Virology, 2004Co-Authors: James G. Greger, Richard A. Katz, Anna Marie Skalka, Konstantin Taganov, Glenn F RallAbstract:Recent studies have demonstrated that Avian Sarcoma virus (ASV) can transduce cycle-arrested cells. Here, we have assessed quantitatively the transduction efficiency of an ASV vector in naturally arrested mouse hippocampal neurons. This efficiency was determined by comparing the number of transduced cells after infection of differentiated neurons versus dividing progenitor cells. The results indicate that ASV is able to transduce these differentiated neurons efficiently and that this activity is not the result of infection of residual dividing cells. The transduction efficiency of the ASV vector was found to be intermediate between the relatively high and low efficiencies obtained with human immunodeficiency virus type 1 and murine leukemia virus vectors, respectively.
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functional oligomeric state of Avian Sarcoma virus integrase
Journal of Biological Chemistry, 2003Co-Authors: Kogan K Bao, Anna Marie Skalka, Hong Wang, Jamie K Miller, Dorothy A Erie, Isaac WongAbstract:Retroviral integrase, one of only three enzymes encoded by the virus, catalyzes the essential step of inserting a DNA copy of the viral genome into the host during infection. Using the Avian Sarcoma virus integrase, we demonstrate that the enzyme functions as a tetramer. In presteady-state active site titrations, four integrase protomers were required for a single catalytic turnover. Volumetric determination of integrase-DNA complexes imaged by atomic force microscopy during the initial turnover additionally revealed substrate-induced assembly of a tetramer. These results suggest that tetramer formation may be a requisite step during catalysis with ramifications for antiviral design strategies targeting the structurally homologous human immunodeficiency virus, type 1 (HIV-1) integrase.
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transduction of interphase cells by Avian Sarcoma virus
Journal of Virology, 2002Co-Authors: Richard A. Katz, James G. Greger, Anna Marie Skalka, Glenn F Rall, Kristen Darby, Pamela BoimelAbstract:It has been generally believed that oncoretroviruses are dependent on mitosis for efficient nuclear entry of viral DNA. We previously identified a nuclear localization signal in the integrase protein of an oncoretrovirus, Avian Sarcoma virus (ASV), suggesting an active import mechanism for the integrase-DNA complex (G. Kukolj, R. A. Katz, and A. M. Skalka, Gene 223:157-163, 1998). Here, we have evaluated the requirement for mitosis in nuclear import and integration of ASV DNA. Using a modified ASV encoding a murine leukemia virus amphotropic env gene and a green fluorescent protein (GFP) reporter gene, DNA nuclear import was measured in cell cycle-arrested Avian (DF-1) as well as human (HeLa) and mouse cells. The results showed efficient accumulation of nuclear forms of ASV DNA in γ-irradiation-arrested cells. Efficient transduction of a GFP reporter gene was also observed after infection of cells that were arrested with γ-irradiation, mitomycin C, nocodazole, or aphidicolin, confirming that nuclear import and integration of ASV DNA can occur in the absence of mitosis. By monitoring GFP expression in individual cells, we also obtained evidence for nuclear import of viral DNA during interphase in cycling cells. Lastly, we observed that ASV can transduce postmitotic mouse neurons. These results support an active nuclear import mechanism for the oncoretrovirus ASV and suggest that this mechanism can operate in both nondividing and dividing cells.
Richard A. Katz - One of the best experts on this subject based on the ideXlab platform.
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The cellular protein daxx interacts with Avian Sarcoma virus integrase and viral DNA to repress viral transcription.
Journal of Virology, 2005Co-Authors: James G. Greger, Richard A. Katz, Alexander M. Ishov, Gerd G. Maul, Anna Marie SkalkaAbstract:The cellular protein Daxx was identified as an interactor with Avian Sarcoma virus (ASV) integrase (IN) in a yeast two-hybrid screen. After infection, Daxx-IN interactions were detected by coimmunoprecipitation. An association between Daxx and viral DNA, likely mediated by IN, was also detected by chromatin immunoprecipitation. Daxx was not required for early events in ASV replication, including integration, as Daxx-null cells were transduced as efficiently as Daxx-expressing cells. However, viral reporter gene expression from ASV-based vectors was substantially higher in the Daxx-null cells than in Daxx-complemented cells. Consistent with this observation, histone deacetylases (HDACs) were found to associate with viral DNA in Daxx-complemented cells but not in Daxx-null cells. Furthermore, Daxx protein was induced in an interferon-like manner upon ASV infection. We conclude that Daxx interacts with an IN-viral DNA complex early after infection and may mediate the repression of viral gene expression via the recruitment of HDACs. Our findings provide a novel example of cellular immunity against viral replication in which viral transcription is repressed via the recruitment of antiviral proteins to the viral DNA.
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genome wide analyses of Avian Sarcoma virus integration sites
Journal of Virology, 2004Co-Authors: Anna Narezkina, Anna Marie Skalka, Konstantin Taganov, Samuel Litwin, Radka Stoyanova, Junpei Hayashi, Christoph Seeger, Richard A. KatzAbstract:The chromosomal features that influence retroviral integration site selection are not well understood. Here, we report the mapping of 226 Avian Sarcoma virus (ASV) integration sites in the human genome. The results show that the sites are distributed over all chromosomes, and no global bias for integration site selection was detected. However, RNA polymerase II transcription units (protein-encoding genes) appear to be favored targets of ASV integration. The integration frequency within genes is similar to that previously described for murine leukemia virus but distinct from the higher frequency observed with human immunodeficiency virus type 1. We found no evidence for preferred ASV integration sites over the length of genes and immediate flanking regions. Microarray analysis of uninfected HeLa cells revealed that the expression levels of ASV target genes were similar to the median level for all genes represented in the array. Although expressed genes were targets for integration, we found no preference for integration into highly expressed genes. Our results provide a more detailed description of the chromosomal features that may influence ASV integration and support the idea that distinct, virus-specific mechanisms mediate integration site selection. Such differences may be relevant to viral pathogenesis and provide utility in retroviral vector design.
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transduction of terminally differentiated neurons by Avian Sarcoma virus
Journal of Virology, 2004Co-Authors: James G. Greger, Richard A. Katz, Anna Marie Skalka, Konstantin Taganov, Glenn F RallAbstract:Recent studies have demonstrated that Avian Sarcoma virus (ASV) can transduce cycle-arrested cells. Here, we have assessed quantitatively the transduction efficiency of an ASV vector in naturally arrested mouse hippocampal neurons. This efficiency was determined by comparing the number of transduced cells after infection of differentiated neurons versus dividing progenitor cells. The results indicate that ASV is able to transduce these differentiated neurons efficiently and that this activity is not the result of infection of residual dividing cells. The transduction efficiency of the ASV vector was found to be intermediate between the relatively high and low efficiencies obtained with human immunodeficiency virus type 1 and murine leukemia virus vectors, respectively.
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transduction of interphase cells by Avian Sarcoma virus
Journal of Virology, 2002Co-Authors: Richard A. Katz, James G. Greger, Anna Marie Skalka, Glenn F Rall, Kristen Darby, Pamela BoimelAbstract:It has been generally believed that oncoretroviruses are dependent on mitosis for efficient nuclear entry of viral DNA. We previously identified a nuclear localization signal in the integrase protein of an oncoretrovirus, Avian Sarcoma virus (ASV), suggesting an active import mechanism for the integrase-DNA complex (G. Kukolj, R. A. Katz, and A. M. Skalka, Gene 223:157-163, 1998). Here, we have evaluated the requirement for mitosis in nuclear import and integration of ASV DNA. Using a modified ASV encoding a murine leukemia virus amphotropic env gene and a green fluorescent protein (GFP) reporter gene, DNA nuclear import was measured in cell cycle-arrested Avian (DF-1) as well as human (HeLa) and mouse cells. The results showed efficient accumulation of nuclear forms of ASV DNA in γ-irradiation-arrested cells. Efficient transduction of a GFP reporter gene was also observed after infection of cells that were arrested with γ-irradiation, mitomycin C, nocodazole, or aphidicolin, confirming that nuclear import and integration of ASV DNA can occur in the absence of mitosis. By monitoring GFP expression in individual cells, we also obtained evidence for nuclear import of viral DNA during interphase in cycling cells. Lastly, we observed that ASV can transduce postmitotic mouse neurons. These results support an active nuclear import mechanism for the oncoretrovirus ASV and suggest that this mechanism can operate in both nondividing and dividing cells.
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role of dna end distortion in catalysis by Avian Sarcoma virus integrase
Journal of Biological Chemistry, 2001Co-Authors: Richard A. Katz, George Kukolj, Paul Dicandeloro, Anna Marie SkalkaAbstract:Retroviral integrase (IN) recognizes linear viral DNA ends and introduces nicks adjacent to a highly conserved CA dinucleotide usually located two base pairs from the 3'-ends of viral DNA (the "processing" reaction). In a second step, the same IN active site catalyzes the insertion of these ends into host DNA (the "joining" reaction). Both DNA sequence and DNA structure contribute to specific recognition of viral DNA ends by IN. Here we used potassium permanganate modification to show that the Avian Sarcoma virus IN catalytic domain is able to distort viral DNA ends in vitro. This distortion activity is consistent with both unpairing and unstacking of the three terminal base pairs, including the processing site adjacent to the conserved CA. Furthermore, the introduction of mismatch mutations that destabilize the viral DNA ends were found to stimulate the IN processing reaction as well as IN-mediated distortion. End-distortion activity was also observed with mutant or heterologous DNA substrates. However, further analyses showed that using Mn(2+) as a cofactor, processing site specificity of these substrates was also maintained. Our results support a model whereby unpairing and unstacking of the terminal base pairs is a required step in the processing reaction. Furthermore, these results are consistent with our previous observations indicating that unpairing of target DNA promotes the joining reaction.
Paul Bates - One of the best experts on this subject based on the ideXlab platform.
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heptad repeat 2 based peptides inhibit Avian Sarcoma and leukosis virus subgroup a infection and identify a fusion intermediate
Journal of Virology, 2004Co-Authors: Robert C Netter, Judith M White, Laurie J Earp, John W Balliet, Sean M Amberg, Mark J Biscone, Arwen Vermeulen, Paul BatesAbstract:Fusion proteins of enveloped viruses categorized as class I are typified by two distinct heptad repeat domains within the transmembrane subunit. These repeats are important structural elements that assemble into the six-helix bundles characteristic of the fusion-activated envelope trimer. Peptides derived from these domains can be potent and specific inhibitors of membrane fusion and virus infection. To facilitate our understanding of retroviral entry, peptides corresponding to the two heptad repeat domains of the Avian Sarcoma and leukosis virus subgroup A (ASLV-A) TM subunit of the envelope protein were characterized. Two peptides corresponding to the C-terminal heptad repeat (HR2), offset from one another by three residues, were effective inhibitors of infection, while two overlapping peptides derived from the N-terminal heptad repeat (HR1) were not. Analysis of envelope mutants containing substitutions within the HR1 domain revealed that a single amino acid change, L62A, significantly reduced sensitivity to peptide inhibition. Virus bound to cells at 4°C became sensitive to peptide within the first 5 min of elevating the temperature to 37°C and lost sensitivity to peptide after 15 to 30 min, consistent with a transient intermediate in which the peptide binding site is exposed. In cell-cell fusion experiments, peptide inhibitor sensitivity occurred prior to a fusion-enhancing low-pH pulse. Soluble receptor for ASLV-A induces a lipophilic character in the envelope which can be measured by stable liposome binding, and this activation was found to be unaffected by inhibitory HR2 peptide. Finally, receptor-triggered conformational changes in the TM subunit were also found to be unaffected by inhibitory peptide. These changes are marked by a dramatic shift in mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, from a subunit of 37 kDa to a complex of about 80 kDa. Biotinylated HR2 peptide bound specifically to the 80-kDa complex, demonstrating a surprisingly stable envelope conformation in which the HR2 binding site is exposed. These experiments support a model in which receptor interaction promotes formation of an envelope conformation in which the TM subunit is stably associated with its target membrane and is able to bind a C-terminal peptide.
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a study of low ph induced refolding of env of Avian Sarcoma and leukosis virus into a six helix bundle
Biophysical Journal, 2004Co-Authors: Ruben M Markosyan, Fredric S Cohen, Paul Bates, Grigory B MelikyanAbstract:The fusion protein of Avian Sarcoma and leukosis virus is likely to fold into a six-helix bundle as part of its final configuration. A peptide, R99, inhibits fusion, probably by binding into the grooves of the triple-stranded coiled coil that becomes the central core of the six-helix bundle. The stages at which the envelope protein (Env) of Avian Sarcoma and leukosis virus subgroup A folds into a bundle during low pH-induced fusion were determined. Effector cells expressing Env were bound to target cells expressing the cognate receptor Tva, and intermediates of fusion were created. R99 was added and the extent of fusion inhibition was used to distinguish between a prebundle state with exposed grooves and a state in which the grooves were no longer exposed. The native conformation of Env was not sensitive to R99. But adding a soluble form of Tva to effector cells conferred sensitivity. Acidic pH applied at low temperature created an intermediate state of local hemifusion. Surprisingly, R99 caused these locally hemifused membranes to separate. This indicates that the grooves of Env were still exposed, that prebundle configurations of Env stabilized hemifused states, and that binding of R99 altered the conformation of Env. In the presence of an inhibitory lipid that blocks fusion before hemifusion, applying low pH at 37°C created an intermediate in which R99 was without effect. This suggests that the six-helix bundle can form before hemifusion and that subsequent conformational changes, such as formation of the trimeric hairpin, are responsible for pore formation and/or growth.
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the Avian retrovirus Avian Sarcoma leukosis virus subtype a reaches the lipid mixing stage of fusion at neutral ph
Journal of Virology, 2003Co-Authors: Laurie J Earp, Paul Bates, Sue E Delos, Robert C Netter, Judith M WhiteAbstract:We previously showed that the envelope glycoprotein (EnvA) of Avian Sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at ≥22°C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and ≥22°C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate Avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37°C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.
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the Avian retrovirus Avian Sarcoma leukosis virus subtype a reaches the lipid mixing stage of fusion at neutral ph
Journal of Virology, 2003Co-Authors: Laurie J Earp, Paul Bates, Sue E Delos, Robert C Netter, Judith M WhiteAbstract:We previously showed that the envelope glycoprotein (EnvA) of Avian Sarcoma/leukosis virus subtype A (ASLV-A) binds to liposomes at neutral pH following incubation with its receptor, Tva, at >or=22 degrees C. We also provided evidence that ASLV-C fuses with cells at neutral pH. These findings suggested that receptor binding at neutral pH and >or=22 degrees C is sufficient to activate Env for fusion. A recent study suggested that two steps are necessary to activate Avian retroviral Envs: receptor binding at neutral pH, followed by exposure to low pH (W. Mothes et al., Cell 103:679-689, 2000). Therefore, we evaluated the requirements for intact ASLV-A particles to bind to target bilayers and fuse with cells. We found that ASLV-A particles bind stably to liposomes in a receptor- and temperature-dependent manner at neutral pH. Using ASLV-A particles biosynthetically labeled with pyrene, we found that ASLV-A mixes its lipid envelope with cells within 5 to 10 min at 37 degrees C. Lipid mixing was neither inhibited nor enhanced by incubation at low pH. Lipid mixing of ASLV-A was inhibited by a peptide designed to prevent six-helix bundle formation in EnvA; the same peptide inhibits virus infection and EnvA-mediated cell-cell fusion (at both neutral and low pHs). Bafilomycin and dominant-negative dynamin inhibited lipid mixing of Sindbis virus (which requires low pH for fusion), but not of ASLV-A, with host cells. Finally, we found that, although EnvA-induced cell-cell fusion is enhanced at low pH, a mutant EnvA that is severely compromised in its ability to support infection still induced massive syncytia at low pH. Our results indicate that receptor binding at neutral pH is sufficient to activate EnvA, such that ASLV-A particles bind hydrophobically to and merge their membranes with target cells. Possible roles for low pH at subsequent stages of viral entry are discussed.
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production and characterization of a soluble active form of tva the subgroup a Avian Sarcoma and leukosis virus receptor
Journal of Virology, 1999Co-Authors: John W Balliet, Joanne F Berson, Celina M Dcruz, Julie Huang, Joanne Crane, Joanna M Gilbert, Paul BatesAbstract:The receptor for the subgroup A Avian Sarcoma and leukosis viruses [ASLV(A)] is the cellular glycoprotein Tva. A soluble form of Tva, sTva, was produced and purified with a baculovirus expression system. Using this system, 7 to 10 mg of purified sTva per liter of cultured Sf9 cells was obtained. Characterization of the carbohydrate modification of sTva revealed that the three N glycosylation sites in sTva were differentially utilized; however, the O glycosylation common to Tva produced in mammalian and Avian cells was not observed. Purified sTva demonstrates significant biological activity, specifically blocking infection of Avian cells by ASLV(A) with a 90% inhibitory concentration of ∼25 pM. A quantitative enzyme-linked immunosorbent assay, developed to assess the binding of sTva to ASLV envelope glycoprotein, demonstrates that sTva has a high affinity for EnvA, with an apparent dissociation constant of approximately 0.3 nM. Once they are bound, a very stable complex is formed between EnvA and sTva, with an estimated complex half-life of 6 h. The soluble receptor protein described here represents a valuable tool for analysis of the receptor-envelope glycoprotein interaction and for structural analysis of Tva.
Mark J Federspiel - One of the best experts on this subject based on the ideXlab platform.
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Avian Sarcoma and leukosis virus envelope glycoproteins evolve to broaden receptor usage under pressure from entry competitors
Viruses, 2019Co-Authors: Audelia Munguia, Mark J FederspielAbstract:The subgroup A through E Avian Sarcoma and leukosis viruses (ASLV(A) through ASLV(E)) are a group of highly related alpharetroviruses that have evolved their envelope glycoproteins to use different receptors to enable efficient virus entry due to host resistance and/or to expand host range. Previously, we demonstrated that ASLV(A) in the presence of a competitor to the subgroup A Tva receptor, SUA-rIgG immunoadhesin, evolved to use other receptor options. The selected mutant virus, RCASBP(A)Δ155–160, modestly expanded its use of the Tvb and Tvc receptors and possibly other cell surface proteins while maintaining the binding affinity to Tva. In this study, we further evolved the Δ155–160 virus with the genetic selection pressure of a soluble form of the Tva receptor that should force the loss of Tva binding affinity in the presence of the Δ155–160 mutation. Viable ASLVs were selected that acquired additional mutations in the Δ155–160 Env hypervariable regions that significantly broadened receptor usage to include Tvb and Tvc as well as retaining the use of Tva as a receptor determined by receptor interference assays. A similar deletion in the hr1 hypervariable region of the subgroup C ASLV glycoproteins evolved to broaden receptor usage when selected on Tvc-negative cells.
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mutations in both the surface and transmembrane envelope glycoproteins of the rav 2 subgroup b Avian Sarcoma and leukosis virus are required to escape the antiviral effect of a secreted form of the tvbs3 receptor
Viruses, 2019Co-Authors: Deborah C Melder, William S Payne, Jerry B Dodgson, Mark J FederspielAbstract:The subgroup A through E Avian Sarcoma and leukosis viruses ASLV(A) through ASLV(E) are a group of highly related alpharetroviruses that have evolved to use very different host protein families as receptors. We have exploited genetic selection strategies to force the replication-competent ASLVs to naturally evolve and acquire mutations to escape the pressure on virus entry and yield a functional replicating virus. In this study, evolutionary pressure was exerted on ASLV(B) virus entry and replication using a secreted for of its Tvb receptor. As expected, mutations in the ASLV(B) surface glycoprotein hypervariable regions were selected that knocked out the ability for the mutant glycoprotein to bind the sTvbS3-IgG inhibitor. However, the subgroup B Rous associated virus 2 (RAV-2) also required additional mutations in the C-terminal end of the SU glycoprotein and multiple regions of TM highlighting the importance of the entire viral envelope glycoprotein trimer structure to mediate the entry process efficiently. These mutations altered the normal two-step ASLV membrane fusion process to enable infection.
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reverse engineering provides insights on the evolution of subgroups a to e Avian Sarcoma and leukosis virus receptor specificity
Viruses, 2019Co-Authors: Mark J FederspielAbstract:The initial step of retrovirus entry—the interaction between the virus envelope glycoprotein trimer and a cellular receptor—is complex, involving multiple, noncontiguous determinants in both proteins that specify receptor choice, binding affinity and the ability to trigger conformational changes in the viral glycoproteins. Despite the complexity of this interaction, retroviruses have the ability to evolve the structure of their envelope glycoproteins to use a different cellular protein as receptors. The highly homologous subgroup A to E Avian Sarcoma and Leukosis Virus (ASLV) glycoproteins belong to the group of class 1 viral fusion proteins with a two-step triggering mechanism that allows experimental access to intermediate structures during the fusion process. We and others have taken advantage of replication-competent ASLVs and exploited genetic selection strategies to force the ASLVs to naturally evolve and acquire envelope glycoprotein mutations to escape the pressure on virus entry and still yield a functional replicating virus. This approach allows for the simultaneous selection of multiple mutations in multiple functional domains of the envelope glycoprotein that may be required to yield a functional virus. Here, we review the ASLV family and experimental system and the reverse engineering approaches used to understand the evolution of ASLV receptor usage.
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Model of the TVA Receptor Determinants Required for Efficient Infection by Subgroup A Avian Sarcoma and Leukosis Viruses
2016Co-Authors: Deborah C Melder, Gennett M Pike, Matthew W. Vanbrocklin, Mark J FederspielAbstract:The study of the interactions of subgroup A Avian Sarcoma and leucosis viruses [ASLV(A)] with the TVA receptor required to infect cells offers a powerful experimental model of retroviral entry. Several regions and specific residues in the TVA receptor have previously been identified to be critical determinants of the binding affinity with ASLV(A) envelope glycoproteins and to mediate efficient infection. Two homologs of the TVA receptor have been cloned: the original quail TVA receptor, which has been the basis for most of the initial characterization of the ASLV(A) TVA, and the chicken TVA receptor, which is 65 % identical to the quail receptor overall but identical in the region thought to be critical for infection. Our previous work characterized three mutant ASLV(A) isolates that could efficiently bind and infect cells using the chicken TVA receptor homolog but not using the quail TVA receptor homolog, with the infectivity of one mutant virus being>500-fold less with the quail TVA receptor. The mu-tant viruses containedmutations in the hr1 region of the surface glycoprotein. Using chimeras of the quail and chicken TVA re-ceptors, we have identified new residues of TVA critical for the binding affinity and entry of ASLV(A) using the mutant glycopro-teins and viruses to probe the function of those residues. The quail TVA receptor required changes at residues 10, 14, and 31 of the corresponding chicken TVA residues to bind wild-type andmutant ASLV(A) glycoproteins with a high affinity and recover the ability to mediate efficient infection of cells. A model of the TVA determinants critical for interacting with ASLV(A) glyco-proteins is proposed
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simple automated high resolution mass spectrometry method to determine the disulfide bond and glycosylation patterns of a complex protein subgroup a Avian Sarcoma and leukosis virus envelope glycoprotein
Journal of Biological Chemistry, 2011Co-Authors: Gennett M Pike, Benjamin J Madden, Deborah C Melder, Cristine M Charlesworth, Mark J FederspielAbstract:Enveloped viruses must fuse the viral and cellular membranes to enter the cell. Understanding how viral fusion proteins mediate entry will provide valuable information for antiviral intervention to combat associated disease. The Avian Sarcoma and leukosis virus envelope glycoproteins, trimers composed of surface (SU) and transmembrane heterodimers, break the fusion process into several steps. First, interactions between SU and a cell surface receptor at neutral pH trigger an initial conformational change in the viral glycoprotein trimer followed by exposure to low pH enabling additional conformational changes to complete the fusion of the viral and cellular membranes. Here, we describe the structural characterization of the extracellular region of the subgroup A Avian Sarcoma and leukosis viruses envelope glycoproteins, SUATM129 produced in chicken DF-1 cells. We developed a simple, automated method for acquiring high resolution mass spectrometry data using electron capture dissociation conditions that preferentially cleave the disulfide bond more readily than the peptide backbone amide bonds that enabled the identification of disulfide-linked peptides. Seven of nine disulfide bonds were definitively assigned; the remaining two bonds were assigned to an adjacent pair of cysteine residues. The first cysteine of surface and the last cysteine of the transmembrane form a disulfide bond linking the heterodimer. The surface glycoprotein contains a free cysteine at residue 38 previously reported to be critical for virus entry. Eleven of 13 possible SUATM129 N-linked glycosylation sites were modified with carbohydrate. This study demonstrates the utility of this simple yet powerful method for assigning disulfide bonds in a complex glycoprotein.
Jonathan Leis - One of the best experts on this subject based on the ideXlab platform.
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2001. Proper processing of Avian Sarcoma/leukosis virus capsid proteins is required for infectivity
2016Co-Authors: Jonathan Leis, Yan Xiang, See Profile, Rebecca C Craven, Rebekah Thorick, Marcy L. Vana, Rebecca CravenAbstract:The formation of the mature carboxyl terminus of CA in Avian Sarcoma/leukemia virus is the result of a sequence of cleavage events at three PR sites that lie between CA and NC in the Gag polyprotein. The initial cleavage forms the amino terminus of the NC protein and releases an immature CA, named CA1, with a spacer peptide at its carboxyl terminus. Cleavage of either 9 or 12 amino acids from the carboxyl terminus creates two mature CA species, named CA2 and CA3, that can be detected in Avian Sarcoma/leukemia virus (R. B. Pepinsky, I. A. Papayannopoulos, E. P. Chow, N. K. Krishna, R. C. Craven, and V. M. Vogt, J. Virol. 69:6430–6438, 1995). To study the importance of each of the three CA proteins, we introduced amino acid substitutions into each CA cleavage junction and studied their effects on CA processing as well as virus assembly and infectivity. Preventing cleavage at any of the three sites produced noninfectious virus. In contrast, a mutant in which cleavage at site 1 was enhanced so that particles contained CA2 and CA3 but little detectabl
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Avian Sarcoma virus and human immunodeficiency virus, type 1 use different subsets of ESCRT proteins to facilitate the budding process.
The Journal of biological chemistry, 2008Co-Authors: Andrew Pincetic, Carol A Carter, Gisselle N. Medina, Jonathan LeisAbstract:Members of the Nedd4 family of E3 ubiquitin ligases bind the L domain in Avian Sarcoma virus (ASV) Gag and facilitate viral particle release. Translational fusion of ASV Gag with an L domain deletion (Δp2b) to proteins that comprise ESCRT-I, -II, and -III (the endocytic sorting complexes required for transport) rescued both Gag ubiquitination and particle release from cells. The ESCRT-I factors Vps37C or Tsg101 were more effective in rescue of Gag/Δp2b budding than the ESCRT-II factor Eap20 or the ESCRT-III component CHMP6. Thus ESCRT components can substitute for Nedd4 family members in ASV Gag release. Unlike wild type, ASV Gag/Δp2b -ESCRT chimeras failed to co-immunoprecipitate with co-expressed hemagglutinin-tagged Nedd4, indicating that Nedd4 was not stably associated with these Gag fusions. Release of the Gag-ESCRT-I or -II fusions was inhibited by a dominant negative mutant of Vps4 ATPase similar to wild type ASV Gag. In contrast to ASV Gag, HIV-1 Gag containing an L domain inactivating mutation (P7L) was efficiently rescued by fusion to a component of ESCRT-III (Chmp6) but not ESCRT-II (Eap20). Depletion of the endogenous pool of Eap20 (ESCRT-II) had little effect on HIV-1 Gag release but blocked ASV Gag release. In contrast, depletion of the endogenous pool of Vps37C (ESCRT-I) had little effect on ASV but blocked HIV-1 Gag release. Furthermore, an N-terminal fragment of Chmp6 inhibited both HIV-1 and ASV Gag release in a dominant negative manner. Taken together, these results indicate that ASV and HIV-1 Gag utilize different combinations of ESCRT proteins to facilitate the budding process, although they share some common elements.
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identification of amino acids in hiv 1 and Avian Sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends
Journal of Biological Chemistry, 2006Co-Authors: Aiping Chen, Irene T Weber, Robert W Harrison, Jonathan LeisAbstract:A tetramer model for HIV-1 integrase (IN) with DNA representing 20 bp of the U3 and U5 long terminal repeats (LTR) termini was assembled using structural and biochemical data and molecular dynamics simulations. It predicted amino acid residues on the enzyme surface that can interact with the LTR termini. A separate structural alignment of HIV-1, simian Sarcoma virus (SIV), and Avian Sarcoma virus (ASV) INs predicted which of these residues were unique. To determine whether these residues were responsible for specific recognition of the LTR termini, the amino acids from ASV IN were substituted into the structurally equivalent positions of HIV-1 IN, and the ability of the chimeras to 3 ' process U5 HIV-1 or ASV duplex oligos was determined. This analysis demonstrated that there are multiple amino acid contacts with the LTRs and that substitution of ASV IN amino acids at many of the analogous positions in HIV-1 IN conferred partial ability to cleave ASV substrates with a concomitant loss in the ability to cleave the homologous HIV-1 substrate. HIV-1 IN residues that changed specificity include Val(72), Ser(153), Lys(160)-Ile(161), Gly(163)-Val(165), and His(171)-Leu(172). Because a chimera that combines several of these substitutions showed a specificity of cleavage of the U5 ASV substrate closer to wild type ASV IN compared with chimeras with individual amino acid substitutions, it appears that the sum of the IN interactions with the LTRs determines the specificity. Finally, residues Ser(153) and Val(72) in HIV-1 IN are among those that change in enzymes that develop resistance to naphthyridine carboxamide- and diketo acid-related inhibitors in cells. Thus, amino acid residues involved in recognition of the LTRs are among these positions that change in development of drug resistance.
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Replication of Avian Sarcoma Virus In Vivo Requires an Interaction between the Viral RNA and the TψC Loop of the tRNA Trp Primer
American Society for Microbiology, 2002Co-Authors: Shannon Morris, Michael Johnson, Ed Stavnezer, Jonathan LeisAbstract:ABSTRACT Reverse transcription in Avian Sarcoma virus (ASV) initiates from the 3′ end of a tRNA Trp primer, which anneals near the 5′ end of the RNA genome. The region around the primer-binding site (PBS) forms an elaborate stem structure composed of the U5-inverted repeat (U5-IR) stem, the U5-leader stem, and the association of the tRNA primer with the PBS. There is evidence for an additional interaction between the viral U5 RNA and the TψC loop of the tRNA Trp (U5-TψC). We now demonstrate that this U5-TψC interaction is necessary for efficient replication of ASV in culture. By randomizing specific biologically relevant regions of the viral RNA, thereby producing a library of mutant viruses, we are able to select, through multiple rounds of infection, those sequences imparting survival fitness to the virus. Randomizing the U5-TψC interaction region of the viral RNA results in selection of largely wild-type sequences after five rounds of infection. Also recovered are mutant viruses that maintain their ability to base pair with the TψC loop of the tRNA Trp . To prove this interaction is specific to the tRNA primer, we constructed a second library, in which we altered the PBS to anneal to tRNA Pro , while simultaneously randomizing the viral RNA U5-TψC region. After five rounds of infection, the consensus sequence 5′-GPuPuCPy-3′ emerged, which is complementary to the 5′-GGTTC-3′ sequence found in the TψC loop of tRNA Pro . These observations confirm the importance of the U5-TψC interaction in vivo.
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hmg protein family members stimulate human immunodeficiency virus type 1 and Avian Sarcoma virus concerted dna integration in vitro
Journal of Virology, 1999Co-Authors: Patrick Hindmarsh, Todd W Ridky, Ray Reeves, Mark Andrake, Anna Marie Skalka, Jonathan LeisAbstract:We have reconstituted concerted human immunodeficiency virus type 1 (HIV-1) integration in vitro with specially designed mini-donor HIV-1 DNA, a supercoiled plasmid acceptor, purified bacterium-derived HIV-1 integrase (IN), and host HMG protein family members. This system is comparable to one previously described for Avian Sarcoma virus (ASV) (A. Aiyar et al., J. Virol. 70:3571–3580, 1996) that was stimulated by the presence of HMG-1. Sequence analyses of individual HIV-1 integrants showed loss of 2 bp from the ends of the donor DNA and almost exclusive 5-bp duplications of the acceptor DNA at the site of integration. All of the integrants sequenced were inserted into different sites in the acceptor. These are the features associated with integration of viral DNA in vivo. We have used the ASV and HIV-1 reconstituted systems to compare the mechanism of concerted DNA integration and examine the role of different HMG proteins in the reaction. Of the three HMG proteins examined, HMG-1, HMG-2, and HMG-I(Y), the products formed in the presence of HMG-I(Y) for both systems most closely match those observed in vivo. Further analysis of HMG-I(Y) mutants demonstrates that the stimulation of integration requires an HMG-I(Y) domain involved in DNA binding. While complexes containing HMG-I(Y), ASV IN, and donor DNA can be detected in gel shift experiments, coprecipitation experiments failed to demonstrate stable interactions between HMG-I(Y) and ASV IN or between HMG-I(Y) and HIV-1 IN.
