NBS1 is required to efficiently complete the integration of viral DNA and to avoid integrase-dependent apoptosis. (A) Completed integration in NBS1-deficient vs. normal control cells. Alu-PCR was performed to detect viral-host DNA junctions. In this nested PCR technique, genomic DNA was extracted from HIV-1-infected NBS1-deficient and control cells at 3 dpi. The first round of PCR was performed with one primer targeting the virus LTR region, and the other primer targeting cellular Alu sequences. The second round utilized two LTR primers. Top – the amplified viral sequences were detected by southern blotting. Bottom – Southern was quantified by densitometry. (B) PARP cleavage in infected cells. Normal and NBS1-deficient cells were infected as described in the Experimental Procedures. Two days post-infection, cells were harvested, lysed and cell lysates subjected to western blotting with an anti-PARP antibody. wt – cells infected with an integration-competent HIV-1-based vector, D64V – cells infected with the vector carrying the D64V mutation in the integrase protein.
Smith et al. Virology Journal 2008 5:11 doi:10.1186/1743-422X-5-11