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Anna Marie Skalka - One of the best experts on this subject based on the ideXlab platform.
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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.
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subcellular localization of avian Sarcoma Virus and human immunodeficiency Virus type 1 integrases
Journal of Virology, 1997Co-Authors: George Kukolj, K S Jones, Anna Marie SkalkaAbstract:The composition and subcellular trafficking of subviral preintegration complexes are reported to vary among the different retroViruses. The process by which the avian Sarcoma Virus (ASV) preintegration complex gains access to target chromatin remains unknown. Here we report that ASV integrase (IN) expressed as a fusion to beta-galactosidase accumulates in the nuclei of transfected COS-1 cells. In contrast, human immunodeficiency type 1 (HIV-1) IN-beta-galactosidase fusions expressed similarly are predominantly cytoplasmic. To identify the region of ASV IN that specifies nuclear localization, various subdomains of the protein were expressed as beta-galactosidase fusions and their subcellular locations were assessed cytochemically and by indirect immunofluorescence. These analyses showed that the ASV IN protein possesses a functional nuclear localization signal that spans amino acids 206 to 235 and displays limited homology with known nuclear transport signals.
Michael F Summers - One of the best experts on this subject based on the ideXlab platform.
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solution structure and dynamics of the rous Sarcoma Virus capsid protein and comparison with capsid proteins of other retroViruses
Journal of Molecular Biology, 2000Co-Authors: Ramon Camposolivas, John L Newman, Michael F SummersAbstract:Abstract The solution structure and dynamics of the recombinant 240 amino acid residue capsid protein from the Rous Sarcoma Virus has been determined by NMR methods. The structure was determined using 2200 distance restraints and 330 torsion angle restraints, and the dynamics analysis was based on 15N relaxation parameters (R1, R2, and 1H-15N NOE) measured for 153 backbone amide groups. The monomeric protein consists of independently folded N- and C-terminal domains that comprise residues Leu14-Leu146 and Ala150-Gln226, respectively. The domains exhibit different rotational correlation times (16.6(±0.1) ns and 12.6(±0.1) ns, respectively), are connected by a flexible linker (Ala147-Pro149), and do not give rise to inter-domain NOE values, indicating that they are dynamically independent. Despite limited sequence similarity, the structure of the Rous Sarcoma Virus capsid protein is similar to the structures determined recently for the capsid proteins of retroViruses belonging to the lentiVirus and human T-cell leukemia Virus/bovine leukemia Virus genera. Structural differences that exist in the C-terminal domain of Rous Sarcoma Virus capsid relative to the other capsid proteins appear to be related to the occurrence of conserved cysteine residues. Whereas most genera of retroViruses contain a pair of conserved and essential cysteine residues in the C-terminal domain that appear to function by forming an intramolecular disulfide bond during assembly, the Rous Sarcoma Virus capsid protein does not. Instead, the Rous Sarcoma Virus capsid protein contains a single cysteine residue that appears to be conserved among the avian C-type retroViruses and is positioned in a manner that might allow the formation of an intermolecular disulfide bond during capsid assembly.
Richard A. Katz - One of the best experts on this subject based on the ideXlab platform.
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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.
Hideki Aihara - One of the best experts on this subject based on the ideXlab platform.
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cryo em structure of the rous Sarcoma Virus octameric cleaved synaptic complex intasome
Communications Biology, 2021Co-Authors: Krishan K Pandey, Sibes Bera, Hideki Aihara, Ke Shi, Michael J Rau, Amarachi V Oleru, James A J Fitzpatrick, Alan Engelman, Duane P. GrandgenettAbstract:Despite conserved catalytic integration mechanisms, retroviral intasomes composed of integrase (IN) and viral DNA possess diverse structures with variable numbers of IN subunits. To investigate intasome assembly mechanisms, we employed the Rous Sarcoma Virus (RSV) IN dimer that assembles a precursor tetrameric structure in transit to the mature octameric intasome. We determined the structure of RSV octameric intasome stabilized by a HIV-1 IN strand transfer inhibitor using single particle cryo-electron microscopy. The structure revealed significant flexibility of the two non-catalytic distal IN dimers along with previously unrecognized movement of the conserved intasome core, suggesting ordered conformational transitions between intermediates that may be important to capture the target DNA. Single amino acid substitutions within the IN C-terminal domain affected intasome assembly and function in vitro and infectivity of pseudotyped RSV virions. Unexpectedly, 17 C-terminal amino acids of IN were dispensable for Virus infection despite regulating the transition of the tetrameric intasome to the octameric form in vitro. We speculate that this region may regulate the binding of highly flexible distal IN dimers to the intasome core to form the octameric complex. Our studies reveal key steps in the assembly of RSV intasomes. Pandey, Bera, Shi et al. report the cryo-electron microscopy structure of the Rous Sarcoma Virus octameric intasome complex stabilized by a HIV-1 integrase strand transfer inhibitor. This new structure highlights the intrinsic flexibility of the distal integrase subunits and suggests that ordered conformational transitions occur within the conserved intasome core during the assembly process.
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crystal structure of the rous Sarcoma Virus intasome
Nature, 2016Co-Authors: Surajit Banerjee, Krishan K Pandey, Sibes Bera, Duane P. Grandgenett, Hideki AiharaAbstract:A crystal structure of the octameric integrase from Rous Sarcoma Virus in complex with viral and target DNAs. Integration of reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroViruses, catalysed by the protein integrase. Two studies, from the laboratories of Hideki Aihara and Alan Engelman, have used crystallography and cryo-electron microscopy to determine the structure of Rous Sarcoma Virus and mouse mammary tumour Virus intasomes, the complex containing integrase, viral DNA, and target DNA. They find that integrase is an octameric assembly — not a tetramer as previously reported. A pair of core integrase dimers engages the viral DNA ends for catalysis, while another pair of non-catalytic integrase dimers bridge the two viral DNA molecules and help capture target DNA. The flanking, unanticipated pair of integrase dimers are required for target capture and strand transfer. Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroViruses. RetroVirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome1. Integrase from HIV-1 and closely related retroViruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase–DNA complexes have been reported for integrase from prototype foamy Virus featuring an additional DNA-binding domain and longer interdomain linkers2,3,4,5, the architecture of a canonical three-domain integrase bound to DNA remained elusive. Here we report a crystal structure of the three-domain integrase from Rous Sarcoma Virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein–DNA and protein–protein contacts that show both conserved and distinct features compared with those observed for prototype foamy Virus integrase. Our work highlights the diversity of retroVirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroViruses.
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crystal structure of the rous Sarcoma Virus intasome
Nature, 2016Co-Authors: Zhiqi Yin, Surajit Banerjee, Krishan K Pandey, Sibes Bera, Duane P. Grandgenett, Ke Shi, Hideki AiharaAbstract:Integration of the reverse-transcribed viral DNA into the host genome is an essential step in the life cycle of retroViruses. RetroVirus integrase catalyses insertions of both ends of the linear viral DNA into a host chromosome. Integrase from HIV-1 and closely related retroViruses share the three-domain organization, consisting of a catalytic core domain flanked by amino- and carboxy-terminal domains essential for the concerted integration reaction. Although structures of the tetrameric integrase-DNA complexes have been reported for integrase from prototype foamy Virus featuring an additional DNA-binding domain and longer interdomain linkers, the architecture of a canonical three-domain integrase bound to DNA remained elusive. Here we report a crystal structure of the three-domain integrase from Rous Sarcoma Virus in complex with viral and target DNAs. The structure shows an octameric assembly of integrase, in which a pair of integrase dimers engage viral DNA ends for catalysis while another pair of non-catalytic integrase dimers bridge between the two viral DNA molecules and help capture target DNA. The individual domains of the eight integrase molecules play varying roles to hold the complex together, making an extensive network of protein-DNA and protein-protein contacts that show both conserved and distinct features compared with those observed for prototype foamy Virus integrase. Our work highlights the diversity of retroVirus intasome assembly and provides insights into the mechanisms of integration by HIV-1 and related retroViruses.
James G. Greger - One of the best experts on this subject based on the ideXlab platform.
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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.
