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Anna Marie Skalka - One of the best experts on this subject based on the ideXlab platform.
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Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors-0
2011Co-Authors: Mark Andrake, Monica M Sauter, Kim Boland, Andrew D Goldstein, Maryem Hussein, Anna Marie SkalkaAbstract:Sport mixture containing the ASV-BSA conjugate, the SV40-BSA conjugate, or Texas red-labeled BSA (TR-BSA). Top panels: Visualization of Texas red conjugates by fluorescence microscopy. Bottom panels: Differential interference contrast (DIC) microscopy of the same field to show preservation of cell integrity. . Digitonin permeabilized HeLa cells were untreated (no addition), treated with 50 μg/ml wheat germ agglutinin (WGA), or 50 units/ml apyrase (Apyrase) prior to incubation with complete transport mixture containing either the ASV-BSA or the SV40-BSA import substrates. . Free NLS peptides were added to the import reactions in molar excess of the import substrates as indicated. "Self" signifies competition with the homologous peptides; "Cross" indicates competition for ASV-BSA import by excess SV40TAg NLS peptide or competition for SV40-BSA import by excess ASV NLS peptide. The left column panels show import in the absence of competitor peptides. . Depletion of ASV-BSA import factor(s) from cytosolic extracts. All assays included Texas-Red labeled ASV-BSA except that shown in the lower left hand corner (panel 4) which included Texas-Red labeled SV40-BSA. Cytosol was either not treated (1; no depletion) or pretreated with glutathione-beads that bound GST alone (2) or fusion proteins of GST plus IN(1–207) which lacks the IN NLS (3), full-length IN(1–286) (5), or a fragment of IN(201–236) that contains the IN NLS (panels 4 and 6).Copyright information:Taken from "Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors"http://www.retrovirology.com/content/5/1/73Retrovirology 2008;5():73-73.Published online 7 Aug 2008PMCID:PMC2527327.
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Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors-3
2011Co-Authors: Mark Andrake, Monica M Sauter, Kim Boland, Andrew D Goldstein, Maryem Hussein, Anna Marie SkalkaAbstract:Ed to GFP (IBB-GFP) was examined in the absence (top) and presence (bottom) of excess unlabeled histone H1. Incubations were for 30 min and all exposure times were equivalent.Copyright information:Taken from "Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors"http://www.retrovirology.com/content/5/1/73Retrovirology 2008;5():73-73.Published online 7 Aug 2008PMCID:PMC2527327.
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Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors-5
2011Co-Authors: Mark Andrake, Monica M Sauter, Kim Boland, Andrew D Goldstein, Maryem Hussein, Anna Marie SkalkaAbstract:Sport mixture containing the ASV-BSA conjugate, the SV40-BSA conjugate, or Texas red-labeled BSA (TR-BSA). Top panels: Visualization of Texas red conjugates by fluorescence microscopy. Bottom panels: Differential interference contrast (DIC) microscopy of the same field to show preservation of cell integrity. . Digitonin permeabilized HeLa cells were untreated (no addition), treated with 50 μg/ml wheat germ agglutinin (WGA), or 50 units/ml apyrase (Apyrase) prior to incubation with complete transport mixture containing either the ASV-BSA or the SV40-BSA import substrates. . Free NLS peptides were added to the import reactions in molar excess of the import substrates as indicated. "Self" signifies competition with the homologous peptides; "Cross" indicates competition for ASV-BSA import by excess SV40TAg NLS peptide or competition for SV40-BSA import by excess ASV NLS peptide. The left column panels show import in the absence of competitor peptides. . Depletion of ASV-BSA import factor(s) from cytosolic extracts. All assays included Texas-Red labeled ASV-BSA except that shown in the lower left hand corner (panel 4) which included Texas-Red labeled SV40-BSA. Cytosol was either not treated (1; no depletion) or pretreated with glutathione-beads that bound GST alone (2) or fusion proteins of GST plus IN(1–207) which lacks the IN NLS (3), full-length IN(1–286) (5), or a fragment of IN(201–236) that contains the IN NLS (panels 4 and 6).Copyright information:Taken from "Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors"http://www.retrovirology.com/content/5/1/73Retrovirology 2008;5():73-73.Published online 7 Aug 2008PMCID:PMC2527327.
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Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors-4
2011Co-Authors: Mark Andrake, Monica M Sauter, Kim Boland, Andrew D Goldstein, Maryem Hussein, Anna Marie SkalkaAbstract:Ious times (labeled above each column) at 37°C prior to fixation with paraformaldehyde and staining with fluorescent antibody against GST. The fusion protein used in each row is labeled at the right and the properties described in the text. Fusion proteins that are imported with slower kinetics are grouped at the top (rows 1–4), and those with faster kinetics in the middle (rows 5 and 6). Control fusion proteins that are not imported into the nucleus are in rows 7 and 8.Copyright information:Taken from "Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors"http://www.retrovirology.com/content/5/1/73Retrovirology 2008;5():73-73.Published online 7 Aug 2008PMCID:PMC2527327.
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Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors-1
2011Co-Authors: Mark Andrake, Monica M Sauter, Kim Boland, Andrew D Goldstein, Maryem Hussein, Anna Marie SkalkaAbstract:Min at 37°C prior to fixation with paraformaldehyde and staining with fluorescent antibody against GST. Left column panels are import without added cytosol and right column panels with added HeLa cytosol extracts.Copyright information:Taken from "Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors"http://www.retrovirology.com/content/5/1/73Retrovirology 2008;5():73-73.Published online 7 Aug 2008PMCID:PMC2527327.
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.
Jonathan Leis - One of the best experts on this subject based on the ideXlab platform.
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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.
Alexander Wlodawer - One of the best experts on this subject based on the ideXlab platform.
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atomic resolution structures of the core domain of Avian Sarcoma Virus integrase and its d64n mutant
Biochemistry, 1999Co-Authors: Jacek Lubkowski, Anna Marie Skalka, Jerry Alexandratos, George Merkel, Zbigniew Dauter, Fan Yang, Alexander WlodawerAbstract:Six crystal structures of the core domain of integrase (IN) from Avian Sarcoma Virus (ASV) and its active-site derivative containing an Asp64 f Asn substitution have been solved at atomic resolution ranging 1.02-1.42 A. The high-quality data provide new structural information about the active site of the enzyme and clarify previous inconsistencies in the description of this fragment. The very high resolution of the data and excellent quality of the refined models explain the dynamic properties of IN and the multiple conformations of its disordered residues. They also allow an accurate description of the solvent structure and help to locate other molecules bound to the enzyme. A detailed analysis of the flexible active-site region, in particular the loop formed by residues 144 -154, suggests conformational changes which may be associated with substrate binding and enzymatic activity. The pH-dependent conformational changes of the active-site loop correlates with the pH vs activity profile observed for ASV IN.
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Atomic resolution structures of the core domain of Avian Sarcoma Virus integrase and its D64N mutant
1999Co-Authors: Jacek Lubkowski, Anna Marie Skalka, George Merkel, Zbigniew Dauter, Fan Yang, Jerry Alex, Alexander WlodawerAbstract:ABSTRACT: Six crystal structures of the core domain of integrase (IN) from Avian Sarcoma Virus (ASV) and its active-site derivative containing an Asp64 f Asn substitution have been solved at atomic resolution ranging 1.02-1.42 Å. The high-quality data provide new structural information about the active site of the enzyme and clarify previous inconsistencies in the description of this fragment. The very high resolution of the data and excellent quality of the refined models explain the dynamic properties of IN and the multiple conformations of its disordered residues. They also allow an accurate description of the solvent structure and help to locate other molecules bound to the enzyme. A detailed analysis of the flexible active-site region, in particular the loop formed by residues 144-154, suggests conformational changes which may be associated with substrate binding and enzymatic activity. The pH-dependent conformational changes of the active-site loop correlates with the pH vs activity profile observed for ASV IN. RetroViruses, such as human immunodeficiency Virus type 1 (HIV-1)1 or Avian Sarcoma Virus (ASV), encode in their genes three essential enzymes: reverse transcriptase (RT), protease (PR), and integrase (IN) (1). In rare cases a retroVirus such as feline immunodeficiency Virus and equin
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structural basis for inactivating mutations and ph dependent activity of Avian Sarcoma Virus integrase
Journal of Biological Chemistry, 1998Co-Authors: Jacek Lubkowski, Richard A. Katz, Anna Marie Skalka, Jerry Alexandratos, George Merkel, Fan Yang, Kelly Gravuer, Alexander WlodawerAbstract:Crystallographic studies of the catalytic core domain of Avian Sarcoma Virus integrase (ASV IN) have provided the most detailed picture so far of the active site of this enzyme, which belongs to an important class of targets for designing drugs against AIDS. Recently, crystals of an inactive D64N mutant were obtained under conditions identical to those used for the native enzyme. Data were collected at different pH values and in the presence of divalent cations. Data were also collected at low pH for the crystals of the native ASV IN core domain. In the structures of native ASV IN at pH 6.0 and below, as well as in all structures of the D64N mutants, the side chain of the active site residue Asx-64 (Asx denotes Asn or Asp) is rotated by approximately 150 degrees around the Calpha---Cbeta bond, compared with the structures at higher pH. In the new structures, this residue makes hydrogen bonds with the amide group of Asn-160, and thus, the usual metal-binding site, consisting of Asp-64, Asp-121, and Glu-157, is disrupted. Surprisingly, however, a single Zn2+ can still bind to Asp-121 in the mutant, without restoration of the activity of the enzyme. These structures have elucidated an unexpected mechanism of inactivation of the enzyme by lowering the pH or by mutation, in which a protonated side chain of Asx-64 changes its orientation and interaction partner.
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structure of the catalytic domain of Avian Sarcoma Virus integrase with a bound hiv 1 integrase targeted inhibitor
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Jacek Lubkowski, Jerry Alexandratos, Alexander Wlodawer, George Merkel, Fan Yang, He Zhao, Terrence R Burke, Nouri Neamati, Yves Pommier, Anna Marie SkalkaAbstract:The x-ray structures of an inhibitor complex of the catalytic core domain of Avian Sarcoma Virus integrase (ASV IN) were solved at 1.9- to 2.0-A resolution at two pH values, with and without Mn2+ cations. This inhibitor (Y-3), originally identified in a screen for inhibitors of the catalytic activity of HIV type 1 integrase (HIV-1 IN), was found in the present study to be active against ASV IN as well as HIV-1 IN. The Y-3 molecule is located in close proximity to the enzyme active site, interacts with the flexible loop, alters loop conformation, and affects the conformations of active site residues. As crystallized, a Y-3 molecule stacks against its symmetry-related mate. Preincubation of IN with metal cations does not prevent inhibition, and Y-3 binding does not prevent binding of divalent cations to IN. Three compounds chemically related to Y-3 also were investigated, but no binding was observed in the crystals. Our results identify the structural elements of the inhibitor that likely determine its binding properties.
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binding of different divalent cations to the active site of Avian Sarcoma Virus integrase and their effects on enzymatic activity
Journal of Biological Chemistry, 1997Co-Authors: Grzegorz Bujacz, Mark Andrake, Richard A. Katz, Jerry Alexandratos, Alexander Wlodawer, George Merkel, Anna Marie SkalkaAbstract:Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of Avian Sarcoma Virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of Avian Sarcoma Virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.
Mark Andrake - One of the best experts on this subject based on the ideXlab platform.
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Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors-3
2011Co-Authors: Mark Andrake, Monica M Sauter, Kim Boland, Andrew D Goldstein, Maryem Hussein, Anna Marie SkalkaAbstract:Ed to GFP (IBB-GFP) was examined in the absence (top) and presence (bottom) of excess unlabeled histone H1. Incubations were for 30 min and all exposure times were equivalent.Copyright information:Taken from "Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors"http://www.retrovirology.com/content/5/1/73Retrovirology 2008;5():73-73.Published online 7 Aug 2008PMCID:PMC2527327.
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Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors-1
2011Co-Authors: Mark Andrake, Monica M Sauter, Kim Boland, Andrew D Goldstein, Maryem Hussein, Anna Marie SkalkaAbstract:Min at 37°C prior to fixation with paraformaldehyde and staining with fluorescent antibody against GST. Left column panels are import without added cytosol and right column panels with added HeLa cytosol extracts.Copyright information:Taken from "Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors"http://www.retrovirology.com/content/5/1/73Retrovirology 2008;5():73-73.Published online 7 Aug 2008PMCID:PMC2527327.
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Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors-4
2011Co-Authors: Mark Andrake, Monica M Sauter, Kim Boland, Andrew D Goldstein, Maryem Hussein, Anna Marie SkalkaAbstract:Ious times (labeled above each column) at 37°C prior to fixation with paraformaldehyde and staining with fluorescent antibody against GST. The fusion protein used in each row is labeled at the right and the properties described in the text. Fusion proteins that are imported with slower kinetics are grouped at the top (rows 1–4), and those with faster kinetics in the middle (rows 5 and 6). Control fusion proteins that are not imported into the nucleus are in rows 7 and 8.Copyright information:Taken from "Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors"http://www.retrovirology.com/content/5/1/73Retrovirology 2008;5():73-73.Published online 7 Aug 2008PMCID:PMC2527327.
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Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors-5
2011Co-Authors: Mark Andrake, Monica M Sauter, Kim Boland, Andrew D Goldstein, Maryem Hussein, Anna Marie SkalkaAbstract:Sport mixture containing the ASV-BSA conjugate, the SV40-BSA conjugate, or Texas red-labeled BSA (TR-BSA). Top panels: Visualization of Texas red conjugates by fluorescence microscopy. Bottom panels: Differential interference contrast (DIC) microscopy of the same field to show preservation of cell integrity. . Digitonin permeabilized HeLa cells were untreated (no addition), treated with 50 μg/ml wheat germ agglutinin (WGA), or 50 units/ml apyrase (Apyrase) prior to incubation with complete transport mixture containing either the ASV-BSA or the SV40-BSA import substrates. . Free NLS peptides were added to the import reactions in molar excess of the import substrates as indicated. "Self" signifies competition with the homologous peptides; "Cross" indicates competition for ASV-BSA import by excess SV40TAg NLS peptide or competition for SV40-BSA import by excess ASV NLS peptide. The left column panels show import in the absence of competitor peptides. . Depletion of ASV-BSA import factor(s) from cytosolic extracts. All assays included Texas-Red labeled ASV-BSA except that shown in the lower left hand corner (panel 4) which included Texas-Red labeled SV40-BSA. Cytosol was either not treated (1; no depletion) or pretreated with glutathione-beads that bound GST alone (2) or fusion proteins of GST plus IN(1–207) which lacks the IN NLS (3), full-length IN(1–286) (5), or a fragment of IN(201–236) that contains the IN NLS (panels 4 and 6).Copyright information:Taken from "Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors"http://www.retrovirology.com/content/5/1/73Retrovirology 2008;5():73-73.Published online 7 Aug 2008PMCID:PMC2527327.
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Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors-0
2011Co-Authors: Mark Andrake, Monica M Sauter, Kim Boland, Andrew D Goldstein, Maryem Hussein, Anna Marie SkalkaAbstract:Sport mixture containing the ASV-BSA conjugate, the SV40-BSA conjugate, or Texas red-labeled BSA (TR-BSA). Top panels: Visualization of Texas red conjugates by fluorescence microscopy. Bottom panels: Differential interference contrast (DIC) microscopy of the same field to show preservation of cell integrity. . Digitonin permeabilized HeLa cells were untreated (no addition), treated with 50 μg/ml wheat germ agglutinin (WGA), or 50 units/ml apyrase (Apyrase) prior to incubation with complete transport mixture containing either the ASV-BSA or the SV40-BSA import substrates. . Free NLS peptides were added to the import reactions in molar excess of the import substrates as indicated. "Self" signifies competition with the homologous peptides; "Cross" indicates competition for ASV-BSA import by excess SV40TAg NLS peptide or competition for SV40-BSA import by excess ASV NLS peptide. The left column panels show import in the absence of competitor peptides. . Depletion of ASV-BSA import factor(s) from cytosolic extracts. All assays included Texas-Red labeled ASV-BSA except that shown in the lower left hand corner (panel 4) which included Texas-Red labeled SV40-BSA. Cytosol was either not treated (1; no depletion) or pretreated with glutathione-beads that bound GST alone (2) or fusion proteins of GST plus IN(1–207) which lacks the IN NLS (3), full-length IN(1–286) (5), or a fragment of IN(201–236) that contains the IN NLS (panels 4 and 6).Copyright information:Taken from "Nuclear import of Avian Sarcoma Virus integrase is facilitated by host cell factors"http://www.retrovirology.com/content/5/1/73Retrovirology 2008;5():73-73.Published online 7 Aug 2008PMCID:PMC2527327.
