shRNAs targeting viral genes inhibit HIV_IIIB replication in primary CD4 T cells. (A) FACS-sorted GFP-positive CD4 T cells stably expressing luc, gag, rev, or vif shRNAs were infected with HIV_IIIB (MOI, 0.5) and evaluated 6 days later for intracellular p24 expression by flow cytometry. Horizontal bars identify the percentage of p24 antigen–positive cells. (B) Inhibitory effect of shRNAs in CD4⁺ T cells challenged with increasing amounts of HIV-1. Mock- and shRNA-transduced CD4⁺ T cells were infected with HIV_IIIB at MOIs of 0.01 to 10, and the culture supernatants harvested at various time points were tested for p24 levels by ELISA. The extent of viral inhibition in culture supernatants from cells expressing each of the shRNAs was calculated as the reduction in p24 production with shRNA compared with corresponding cultures with no shRNA. The p24 levels in mock-transduced culture supernatants were 468, 576, 729, and 1610 ng/mL, respectively, at 0.01, 0.1, 1.0, and 10 MOI of HIV_IIIB on day 15 after infection. (C) shRNAs inhibit HIV replication in previously infected CD4⁺ T cells. (Left panel) HIV_IIIB-infected CD4⁺ T cells are shown by intracellular p24 staining (bold line, uninfected cultures; thin solid line, HIV-infected cells; dotted line, infected cells stained with control isotype-matched antibody). Horizontal bars identify the percentage of p24 antigen–positive cells. These infected cells were transduced with shRNA-expressing lentiviruses, and 2 weeks later p24 expression in GFP-positive (GFP+) and GFP-negative (GFP–) cells was assessed by FACS (right panel). The extent of viral inhibition was calculated as the reduction in the proportion of p24-positive cells in GFP-positive or GFP-negative cultures relative to the mock-transduced culture. Error bars denote the standard deviation from 3 independent experiments.