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Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair.

Smith JA, Wang FX, Zhang H, Wu KJ, Williams KJ, Daniel R - Virol. J. (2008)

Bottom Line: In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages.Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction.Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infectious Diseases - Center for Human Virology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA. Johanna.Smith@jefferson.edu

ABSTRACT
Retroviral transduction involves integrase-dependent linkage of viral and host DNA that leaves an intermediate that requires post-integration repair (PIR). We and others proposed that PIR hijacks the host cell double-strand DNA break (DSB) repair pathways. Nevertheless, the geometry of retroviral DNA integration differs considerably from that of DSB repair and so the precise role of host-cell mechanisms in PIR remains unclear. In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages. Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction. Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. A model is proposed emphasizing similarities and differences between PIR and DSB repair. Differences between the pathways may eventually allow strategies to block PIR while still allowing DSB repair.

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NBS1 associates with viral DNA and is required for recruitment of ATM but not ATR. (A) Chromatin immunoprecipitation of infected NBS1-deficient and control cells. To establish if NBS1, ATM, and/or ATR associate with viral DNA, normal and NBS1-deficient cells were infected with the HIV-1-based vector at an m.o.i. of 0.1 and chromatin immunoprecipitation was performed with anti-NBS1, anti-ATM and anti-ATR antibodies as described in the Experimental Procedures. m – mock, uninfected cells. The immunoprecipitating antibody is indicated on the left side of the photograph of the gel. (B) Chromatin immunoprecipitation of infected NBS1-deficient and control cells, which were transfected with the normal NBS1 gene. Control and NBS1-deficient cells were transfected with the NBS1-coding plasmid or an empty vector. 48 hrs post-transfection, cells were infected with the HIV-1-based vector at an m.o.i. of 0.1 and chromatin immunoprecipitation was performed 24 hrs later with anti-NBS1 and anti-ATM antibodies as described in the Experimental Procedures. m – uninfected cells, v – cells infected with the HIV-1-based vector, N – cells transfected with the normal NBS1 gene and infected with the HIV-1-based vector, c – cells transfected with the empty plasmid vector and infected with the HIC-1-based vector.
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Figure 1: NBS1 associates with viral DNA and is required for recruitment of ATM but not ATR. (A) Chromatin immunoprecipitation of infected NBS1-deficient and control cells. To establish if NBS1, ATM, and/or ATR associate with viral DNA, normal and NBS1-deficient cells were infected with the HIV-1-based vector at an m.o.i. of 0.1 and chromatin immunoprecipitation was performed with anti-NBS1, anti-ATM and anti-ATR antibodies as described in the Experimental Procedures. m – mock, uninfected cells. The immunoprecipitating antibody is indicated on the left side of the photograph of the gel. (B) Chromatin immunoprecipitation of infected NBS1-deficient and control cells, which were transfected with the normal NBS1 gene. Control and NBS1-deficient cells were transfected with the NBS1-coding plasmid or an empty vector. 48 hrs post-transfection, cells were infected with the HIV-1-based vector at an m.o.i. of 0.1 and chromatin immunoprecipitation was performed 24 hrs later with anti-NBS1 and anti-ATM antibodies as described in the Experimental Procedures. m – uninfected cells, v – cells infected with the HIV-1-based vector, N – cells transfected with the normal NBS1 gene and infected with the HIV-1-based vector, c – cells transfected with the empty plasmid vector and infected with the HIC-1-based vector.

Mentions: Chromatin Immunoprecipitation (ChIP) assays were performed as described previously [34]. 3 × 105 NBS1-deficient primary fibroblasts or control fibroblast cells were infected with our HIV-1-based vector (lacZ reporter) at m.o.i. 1. At the time points indicated, viral DNA and interacting proteins were cross-linked by the addition of formaldehyde (1% final concentration) to the cultures, which were then incubated for 30 min at room temperature. In the reconstitution experiment described in Figure 1B, cells were transfected with 50 μg of the NBS1 expression plasmid or the empty vector using the Lipofectamine™ 2000 reagent (Invitrogen, Cat no. 11668-027). 48 hrs after transfection, cells were infected with the HIV-1-based vector under conditions described above. Crosslinking was performed 24 hrs after addition of the virus. The cross-linking reaction was quenched with glycine (0.125 M final concentration). Plates were then washed with cold phosphate-buffered saline, and then scraped into phosphate-buffered saline containing protease inhibitors, and washed and lysed by addition of 0.5% Nonidet P-40, 5 mM PIPES, pH 8.0, 85 mM KCL and protease inhibitors. The intact nuclei were isolated by centrifugation at 5000 rpm at 4°C. Nuclei were then resuspended in a lysis buffer (1% SDS, 50 mM Tris-Cl, pH 8.1, 10 mM EDTA, protease inhibitors). Chromatin was sonicated to obtain DNA fragments of approximately 600 bp. Samples were subjected to centrifugation to remove debris and were precleared by shaking for 1 hr with salmon sperm DNA/protein A-agarose (Upstate, Temecula, CA, cat. no. 16–157), which were then removed and supernatants were diluted 10-fold with a dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8.1, 167 mM NaCl, protease inhibitors). Chromatin fragments were immunoprecipitated overnight with antibodies against ATM (Santa Cruz Biotechnology, sc-15392), ATR (Santa Cruz Biotechnology, sc-1887), NBS1 (Santa Cruz Biotechnology, sc-8580), or, as a control, the irrelevant protein PI-3K 110δ (Santa Cruz Biotechnology, sc-55589). Protein-DNA-antibody complexes were isolated by the addition of salmon sperm DNA/protein A-agarose. After 1 hr, complexes were collected by centrifugation and washed three times with buffer (100 mM Tris, pH 8, 500 mM LiCl, 1% Nonidet P-40, 1% deoxycholic acid). Pellets were eluted from salmon sperm DNA/protein A-agarose with 50 mM NaHCO3, 1% SDS for 15 min at room temperature. Clarified samples were incubated with RNase and 5 M NaCl at 67°C for 4–5 hr to reverse cross-links and then precipitated overnight with ethanol. Following centrifugation, pellets were resuspended in proteinase K buffer and treated with proteinase K to digest residual proteins. After phenol/chloroform extraction, the DNA was precipitated with ethanol. Viral sequences in these fractions were detected by PCR using primers targeting the HIV-1 long terminal repeats: M667, 5'-GGC TAA CTA GGG AAC CCA CTG-3'; AA55, 5'-CTG CTA GAG ATT TTC CAC ACT GAC-3'[35]. The PCR reaction was done as follows: 94C for 5 min, then 30 cycles of 94C – 1 min, 55C – 1 min, 72C – 1 min. Final extension was run for 5 min at 72C. PCR products were resolved on an ethidium bromide-stained 2% agarose gel.


Evidence that the Nijmegen breakage syndrome protein, an early sensor of double-strand DNA breaks (DSB), is involved in HIV-1 post-integration repair by recruiting the ataxia telangiectasia-mutated kinase in a process similar to, but distinct from, cellular DSB repair.

Smith JA, Wang FX, Zhang H, Wu KJ, Williams KJ, Daniel R - Virol. J. (2008)

NBS1 associates with viral DNA and is required for recruitment of ATM but not ATR. (A) Chromatin immunoprecipitation of infected NBS1-deficient and control cells. To establish if NBS1, ATM, and/or ATR associate with viral DNA, normal and NBS1-deficient cells were infected with the HIV-1-based vector at an m.o.i. of 0.1 and chromatin immunoprecipitation was performed with anti-NBS1, anti-ATM and anti-ATR antibodies as described in the Experimental Procedures. m – mock, uninfected cells. The immunoprecipitating antibody is indicated on the left side of the photograph of the gel. (B) Chromatin immunoprecipitation of infected NBS1-deficient and control cells, which were transfected with the normal NBS1 gene. Control and NBS1-deficient cells were transfected with the NBS1-coding plasmid or an empty vector. 48 hrs post-transfection, cells were infected with the HIV-1-based vector at an m.o.i. of 0.1 and chromatin immunoprecipitation was performed 24 hrs later with anti-NBS1 and anti-ATM antibodies as described in the Experimental Procedures. m – uninfected cells, v – cells infected with the HIV-1-based vector, N – cells transfected with the normal NBS1 gene and infected with the HIV-1-based vector, c – cells transfected with the empty plasmid vector and infected with the HIC-1-based vector.
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Related In: Results  -  Collection

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Figure 1: NBS1 associates with viral DNA and is required for recruitment of ATM but not ATR. (A) Chromatin immunoprecipitation of infected NBS1-deficient and control cells. To establish if NBS1, ATM, and/or ATR associate with viral DNA, normal and NBS1-deficient cells were infected with the HIV-1-based vector at an m.o.i. of 0.1 and chromatin immunoprecipitation was performed with anti-NBS1, anti-ATM and anti-ATR antibodies as described in the Experimental Procedures. m – mock, uninfected cells. The immunoprecipitating antibody is indicated on the left side of the photograph of the gel. (B) Chromatin immunoprecipitation of infected NBS1-deficient and control cells, which were transfected with the normal NBS1 gene. Control and NBS1-deficient cells were transfected with the NBS1-coding plasmid or an empty vector. 48 hrs post-transfection, cells were infected with the HIV-1-based vector at an m.o.i. of 0.1 and chromatin immunoprecipitation was performed 24 hrs later with anti-NBS1 and anti-ATM antibodies as described in the Experimental Procedures. m – uninfected cells, v – cells infected with the HIV-1-based vector, N – cells transfected with the normal NBS1 gene and infected with the HIV-1-based vector, c – cells transfected with the empty plasmid vector and infected with the HIC-1-based vector.
Mentions: Chromatin Immunoprecipitation (ChIP) assays were performed as described previously [34]. 3 × 105 NBS1-deficient primary fibroblasts or control fibroblast cells were infected with our HIV-1-based vector (lacZ reporter) at m.o.i. 1. At the time points indicated, viral DNA and interacting proteins were cross-linked by the addition of formaldehyde (1% final concentration) to the cultures, which were then incubated for 30 min at room temperature. In the reconstitution experiment described in Figure 1B, cells were transfected with 50 μg of the NBS1 expression plasmid or the empty vector using the Lipofectamine™ 2000 reagent (Invitrogen, Cat no. 11668-027). 48 hrs after transfection, cells were infected with the HIV-1-based vector under conditions described above. Crosslinking was performed 24 hrs after addition of the virus. The cross-linking reaction was quenched with glycine (0.125 M final concentration). Plates were then washed with cold phosphate-buffered saline, and then scraped into phosphate-buffered saline containing protease inhibitors, and washed and lysed by addition of 0.5% Nonidet P-40, 5 mM PIPES, pH 8.0, 85 mM KCL and protease inhibitors. The intact nuclei were isolated by centrifugation at 5000 rpm at 4°C. Nuclei were then resuspended in a lysis buffer (1% SDS, 50 mM Tris-Cl, pH 8.1, 10 mM EDTA, protease inhibitors). Chromatin was sonicated to obtain DNA fragments of approximately 600 bp. Samples were subjected to centrifugation to remove debris and were precleared by shaking for 1 hr with salmon sperm DNA/protein A-agarose (Upstate, Temecula, CA, cat. no. 16–157), which were then removed and supernatants were diluted 10-fold with a dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-Cl, pH 8.1, 167 mM NaCl, protease inhibitors). Chromatin fragments were immunoprecipitated overnight with antibodies against ATM (Santa Cruz Biotechnology, sc-15392), ATR (Santa Cruz Biotechnology, sc-1887), NBS1 (Santa Cruz Biotechnology, sc-8580), or, as a control, the irrelevant protein PI-3K 110δ (Santa Cruz Biotechnology, sc-55589). Protein-DNA-antibody complexes were isolated by the addition of salmon sperm DNA/protein A-agarose. After 1 hr, complexes were collected by centrifugation and washed three times with buffer (100 mM Tris, pH 8, 500 mM LiCl, 1% Nonidet P-40, 1% deoxycholic acid). Pellets were eluted from salmon sperm DNA/protein A-agarose with 50 mM NaHCO3, 1% SDS for 15 min at room temperature. Clarified samples were incubated with RNase and 5 M NaCl at 67°C for 4–5 hr to reverse cross-links and then precipitated overnight with ethanol. Following centrifugation, pellets were resuspended in proteinase K buffer and treated with proteinase K to digest residual proteins. After phenol/chloroform extraction, the DNA was precipitated with ethanol. Viral sequences in these fractions were detected by PCR using primers targeting the HIV-1 long terminal repeats: M667, 5'-GGC TAA CTA GGG AAC CCA CTG-3'; AA55, 5'-CTG CTA GAG ATT TTC CAC ACT GAC-3'[35]. The PCR reaction was done as follows: 94C for 5 min, then 30 cycles of 94C – 1 min, 55C – 1 min, 72C – 1 min. Final extension was run for 5 min at 72C. PCR products were resolved on an ethidium bromide-stained 2% agarose gel.

Bottom Line: In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages.Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction.Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Infectious Diseases - Center for Human Virology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA. Johanna.Smith@jefferson.edu

ABSTRACT
Retroviral transduction involves integrase-dependent linkage of viral and host DNA that leaves an intermediate that requires post-integration repair (PIR). We and others proposed that PIR hijacks the host cell double-strand DNA break (DSB) repair pathways. Nevertheless, the geometry of retroviral DNA integration differs considerably from that of DSB repair and so the precise role of host-cell mechanisms in PIR remains unclear. In the current study, we found that the Nijmegen breakage syndrome 1 protein (NBS1), an early sensor of DSBs, associates with HIV-1 DNA, recruits the ataxia telangiectasia-mutated (ATM) kinase, promotes stable retroviral transduction, mediates efficient integration of viral DNA and blocks integrase-dependent apoptosis that can arise from unrepaired viral-host DNA linkages. Moreover, we demonstrate that the ATM kinase, recruited by NBS1, is itself required for efficient retroviral transduction. Surprisingly, recruitment of the ATR kinase, which in the context of DSB requires both NBS1 and ATM, proceeds independently of these two proteins. A model is proposed emphasizing similarities and differences between PIR and DSB repair. Differences between the pathways may eventually allow strategies to block PIR while still allowing DSB repair.

Show MeSH
Related in: MedlinePlus