Uncoating kinetics of HIV-1 and HIV-2 cores in TRIM5α expressing HeLa cells.

<p><i>In situ</i> uncoating assay was performed in HeLa cells stably transfected with vector encoding HA-tagged cynomolgus monkey (CM) TRIM5α (TRIM5α; closed circles) or empty vector (Vc; opened circles). Cells were spinoculated with VSV-G-pseudotyped and GFP-Vpr/S15-dTomato-containing HIV-1 (NL-Nh/GFP-Vpr), or with VSV-G-pseudotyped GFP-Vpx/S15-dTomato-containing HIV-2 (GH123-Nh/GFP-Vpx or ASA-Nh/GFP-Vpx), and fixed at 0, 1, 2, or 4 hr after infection. CAs of the infected viruses were labeled by Cy5-conjugated viral-specific anti-CA antibodies, and the GFP punctae then were quantified and individually examined for the presence of dTomato (S15) and Cy-5 (CA) signals. The percentage of the number of productively entered (S15-negative) virions that stained for p24 or p26 CA among total number of productively entered virions (GFP-positive, S15-negative, and CA-positive particles/(GFP-positive, S15-negative, and CA-positive particles + GFP-positive, S15-negative, and CA-negative particles)) over time following fusion is shown in the Y-axis. The 0-hr time point and bafilomycin A-treated Vc-transfected cells (Vc+Baf; open squares) represent the total number of GFP-positive particles (both S15-positive and S15-negative) that stained positive for CA among total number of GFP-positive particles. The results shown are means and SDs of three independent cell lines at each time point. *, <i>p</i> value less than 0.02 when compared with values from Vc-transfected cells.