FIG. 7.

Target cell TRIM5α does not affect infection by SIVagm. (A) Jurkat cells were transduced with an HIV-1-based vector that confers puromycin resistance and delivers an shRNA expression construct specific either for human TRIM5α (TR5-shRNA) or for luciferase (Luc-shRNA). VSV-G pseudotyped N- or B-tropic MLVGFP virions were normalized for titer with nonrestrictive Mus dunni cells and then used to infect Jurkat Luc-shRNA cells or Jurkat TR5-shRNA cells. The percentage of GFP-positive (infected) cells was determined by flow cytometry. Shown are representative results of a single experiment. Identical results were obtained on three separate occasions using independently produced viral stocks. (B) WT and vif-deficient (Vif−) SIVagm stocks were produced with untreated Jurkat cells and used to infect untreated Jurkat cells (normal), Jurkat TR5-shRNA cells (TRIM5α-KD), or Jurkat TR5-shRNA cells pretreated for 24 h with 2.5 μM CsA (CsA-treated TRIM5α-KD). Total DNA was harvested 24 h postinfection. Accumulation of full-length viral cDNA was determined by DNA PCR amplification. A primer set for the amplification of actin DNA was included in each reaction as an internal control (Actin). Heat, heat-inactivated WT SIVagm. (C) LuSIV cells were transduced with TR5-shRNA or Luc-shRNA vectors as described in the legend to panel A. TRIM5α silencing was measured by determining the relative sensitivity of the cells to infection by VSV-G pseudotyped B-tropic or N-tropic MLVGFP virions. (D) LuSIV TR5-shRNA cells were infected with equal amounts of the WT or the vif-defective [Vif(−)] SIVagm derived from infected Jurkat cells. Mock-infected cells were analyzed in parallel (mock). Infected cells were harvested 24 h after infection, and virus-induced luciferase activity was measured as described in Materials and Methods. Error bars reflect standard deviations calculated from three independent experiments.