FIG 4.
Pteropus alecto TRIM proteins do not restrict HIV-1 activity. (A to C) CrFK cells were transduced with lentiviral vector encoding HA epitope-tagged Pteropus alecto TRIM5 and selected with puromycin. (A) Stable TRIM5 expression was verified by immunoblotting for HA and α-tubulin. (B) Indirect immunofluorescence against the HA epitope was performed (blue, DAPI; red, anti-HA). Wild-type and TRIM5-expressing CrFK cells were infected with limiting dilutions of the indicated eGFP-expressing single-cycle retroviral vectors. (C) At 48 h postinfection, eGFP expression was quantified by flow cytometry and the percentages of GFP-positive cells were used to determine the infectious titer (transducing units per milliliter) for each vector on wild-type or TRIM5-expressing cells. (D and E) Immunoblotting (D) and indirect immunofluorescence (E) were performed as described for panels A and B with CrFK cells expressing additional HA epitope-tagged Pteropus alecto TRIM proteins. (F) Wild-type and stable TRIM-expressing CrFK cells were infected with HIV-1LTR Luc, and 72 h postinfection, cells were counted, lysed, and analyzed for luminescence. Relative light unit (RLU) data were normalized to the number of healthy cells and are shown as the average RLU reading ± standard deviation. (G and H) P. alecto kidney or fetus cells were transfected with TRIM5-specific siRNA 48 h prior to infection with a luciferase-encoding HIV-1 strain. TRIM5 mRNA expression (G) and virally encoded luciferase activity (H) were measured 48 h following viral infection. Luciferase activity was normalized to untreated cells. *, P < 0.01.