Figure 3

Integrated HIV-1 DNA detected in TERT-2 nuclei. TERT-2 cells were grown in monolayers and inoculated with HIV-1 (IIIb or BaL). Some cells were sub-cultured after infection. At 0.5 to 72 h post inoculation, TERT-2 cell nuclei were isolated as described in Materials and Methods. DNA was extracted from the nuclei and analyzed for integrated HIV DNA, linear HIV DNA, and 2-LTR circular HIV DNA. β-actin was included as a loading control. PCR reactions were performed as described in Materials and Methods (Table 2). Negative controls include cells without HIV-1 (NV), cells inoculated with heat-inactivated HIV-1 (HV), cells pretreated with 500 μM AZT (AZT), cells pretreated with colchicine (Col), and PCR reactions with no template (W). PBMC media (uninfected with HIV-1) and samples that were amplified in the second PCR only were also negative for HIV DNA (data not shown). ACH-2 cells and HIV-1-infected PBMCs served as positive controls for detection of integrated HIV DNA, linear HIV DNA, and circular HIV DNA. These agarose gel data for HIV-1 IIIb infection are representative of three independent experiments.