Figure 3

Centrosome-associated HIV-1 pre-integration intermediate is inducible upon cell activation. A. HIV-1 reverse-transcribed viral cDNA localizes at the centrosome of resting MRC5 cells transduced with a DNAse-treated VSVg-pseudotyped NL4.3 virus, which was made using the NL4.3Luc plasmid, in which the env gene was replaced by the luciferase transgene, and a VSVg-expressor vector. Fluorescence in situ hybridization (FISH) was performed 4 days after transduction using the full-length proviral genome as a probe [32]. After FISH, immunostaining with anti-γ-tubulin ab was performed as described above. B. Viral gene expression resumes after reactivation of quiescent cells. Transduced resting MRC5 cells were sorted to recover only GFP-negative cells which were then stimulated to divide by splitting and serum addition. The percentage of GFP-expressing cells was determined at 48, 72 and 96 h after sorting and reactivation by flow cytometry.