Figure 3. Cocaine increases HIV-1 PIC-associated viral DNA integration activity.

(A) PCR amplification and purification of target DNA for HIV-1 PIC-associated in vitro integration assay. A 2.0 kb target DNA fragment was PCR amplified from the ΦX174 genome. The PCR product was detected by agarose gel electrophoresis by use of ethidium bromide staining. Then, the 2.0 kb fragment was purified by the gel extraction method. A portion of the purified PCR product was analyzed by agarose gel electrophoresis for purity. (B) HIV-1 PICs were isolated from HIV-1-infected SupT1 cells, and in vitro integration assays were carried out with use of the 2.0 kb target DNA. Integration of viral DNA into the target was measured by nested qPCR that specifically amplifies the junctions of integrated viral DNA in the target. PIC associated integration assay was performed in the presence of increased concentrations of cocaine (1–100 µM). Controls, such as PICs alone, PICs + cocaine, target DNA alone, cocaine + target DNA, lysates from uninfected cells, lysates from uninfected cells + cocaine, and lysates from uninfected cells + target DNA, showed no integration activity. Cocaine increased PICs associated viral DNA integration in a dose-dependent manner. Data are presented as mean values, with error bars indicating sd of triplicate measurements. *P < 0.05 is for the comparison treated cells versus untreated cells.