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.
Highlights
Post-integration repair (PIR) is an essential step in the retroviral lifecycle, and yet it remains incompletely understood
To establish if Nijmegen breakage syndrome1 protein (NBS1), ataxia telangiectasia-mutated (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 NBS1 protein is required for association of ATM, but not ATR, with viral DNA Normal and NBS1-deficient primary human fibroblasts were infected with the pseudotyped HIV-1-based vector at an m.o.i. of 0.1 and harvested at the indicated time points (Figure 1A)
Summary
Post-integration repair (PIR) is an essential step in the retroviral lifecycle, and yet it remains incompletely understood. We sought to examine the presence, interactions, and function of several DSB repair proteins in retroviral PIR, namely, the initial DSB sensor NBS1 and the ATM and ATR kinases. 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. 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. 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. Comparisons between two groups were performed using the two tailed Student t-test
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