FIG. 5.

p62 knockdown results in reduced rhTRIM5α-mediated restriction. (A) Cells expressing YFP-rhTRIM5α were transfected with control or p62-specific siRNA. Cells were harvested at numerous times following the second siRNA transfection, and p62 expression was analyzed by Western blotting. siRNA-transfected cells were plated in equivalent numbers and infected with VSV-g pseudotyped HIV-1 reporter virus overnight. (B) Eighteen hours following infection, HeLa and YFP-rhTRIM5α-expressing HeLa cells were harvested and viral DNA products were analyzed by quantitative PCR. Values were normalized to the amounts of β-actin DNA in identical samples. Error bars represent the standard deviations from triplicate samples. Values given above the columns represent the fold enhancement relative to the level for the untreated sample of that cell type. (C) Thirty-six to 48 h following infection with the HIV-1 GFP reporter virus, untreated (squares), control siRNA-transfected (circles), and p62 siRNA-transfected (triangles) cells were harvested and analyzed for GFP expression by flow cytometry. Ten thousand events/sample were analyzed. The percentage of GFP-positive cells is indicated for each sample. The dilution factor of virus used is given on the x axis. The data shown here are representative of three independent experiments.