Lentiviral delivery of shRNAs targeting viral genes inhibits HIV_IIIB replication. HeLa-CD4 cells stably expressing shRNAs targeting luc, gag, rev, or vif generated by sorting GFP-positive cells 3 to 5 days after lentiviral transduction were infected with HIV_IIIB and analyzed 5 days later. (A) The level of infection was determined by flow cytometry after staining for intracellular p24 expression. Numbers indicate percentage of cells in the respective quadrants. (B) The 9.3-kb HIV RNA was detected on Northern blots using a gag RNA probe.

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.

HIV sequence variations in regions targeted by the shRNAs. HIV sequences in the Los Alamos HIV Sequence Database were analyzed for sequence variation at each of the 19 nt residues in the rev-, gag-, and vif-targeted regions. The number of isolates within each clade (A, B, C, D, or E) that contain mutations relative to the total number of isolates were determined and expressed as a percentage. For each sequence, 41 to 45 clade A isolates, 31 clade B isolates, 61 to 66 clade C isolates, 33 clade D isolates, and 9 clade E isolates were available.

Antiviral activity of shRNAs against clade B primary viruses. Sorted GFP-positive CD4⁺ T cells expressing luc, gag, rev, or vif shRNAs were infected with the IIIB, 89.6, or BaL lab-adapted strains or with primary HIV isolates from donors 214, 216, and 307 and analyzed by flow cytometry for p24 expression 6 days later. (A) Representative flow cytometry analysis of lentivirus-transduced CD4 T cells infected with primary virus from donor 216. (B) The extent of viral inhibition in cells expressing each of the 3 shRNAs was calculated as the reduction in the percent of p24-positive cells in cultures selected for HIV shRNA expression compared with cultures expressing luc shRNA. Values represent averages of 2 independent experiments, with the range indicated. The target sequences of the various viruses used are depicted in Table 2.

Antiviral effect of gag or vif shRNAs against viruses from diverse clades. GFP-positive CD4⁺ T cells expressing luc, gag, or vif shRNAs were infected with primary viruses representative of multiple HIV clades and analyzed for intracellular p24 expression by flow cytometry 6 days later. (A) Representative histogram of CD4 cells transduced with indicated shRNA and infected with clade C, Indian virus IN17. (B) The reduction in p24 expression in HIV shRNA-transduced cells relative to luc shRNA-transduced cultures is graphed. Data represent means and SD of 3 independent experiments. The target sequences of the various viruses used are depicted in Table 3.

Antiviral activity of mutated siRNAs corresponding to common variants in gag and vif targets. siRNAs with single, double, or triple mutations were designed based on the common gag and vif sequence variants present in the Los Alamos HIV Sequence Database. Cells transfected with 200 nM of the indicated synthetic siRNA were challenged with HIV_IIIB virus on day 1 after transfection. Three days later, supernatants were collected for evaluation of p24 production by ELISA (the p24 level was 10 ng/mL for the control mock-treated cells and was inhibited to 0.5 ng/mL by wild-type [wt] gag or vif siRNA). Five days after infection, cells were stained for intracellular p24 and the antiviral activity of mutant siRNAs was assessed by comparing the percent of p24-positive cells in cultures treated with the mutated siRNA with that in wt siRNA-treated HeLa-CD4 cells (35% to 40% of mock-treated cells and 3% to 5% of wt siRNA-transfected cells stained for p24). Values represent means and SD of 2 independent experiments. The bars and circles represent the levels of inhibition obtained by intracellular staining for p24 and ELISA, respectively. The sequences of the mutant siRNAs are shown in Table 1.