Proteasome function is required for rhTRIM5α-mediated loss of HIV-1 virions. HeLa cells or rhTRIM5α-HA–expressing HeLa cells were synchronously infected with S15-mCherry– and GFP-Vpr–labeled virions in the presence of 1 μg/ml MG132 or 20 nM BafA or in the absence of drug as indicated. (A and B) Cells were fixed for 1 (A) or 4 h (B) after the initiation of infection, and the number of fused (S15⁺; black bars) and unfused (S15⁻; gray bars) virions per cell was calculated. All values are expressed as a percentage of the total virions per cell (fused and unfused) present in the BafA sample at 1 h. The mean results from three independent experiments examining a total of 493 cells and 11,727 virions are graphed. Error bars represent the SEM from three independent experiments. The difference in values indicated by the asterisk are statistically significant (*, P = 0.0152). (C) HeLa cells stably expressing rhTRIM5α were continuously infected with GFP-Vpr–labeled HIV-1 virions (green) in the presence of 1 μg/ml MG132 for 6 h. Cells were then fixed and immunostained with anti-HA antibody (red) and antiubiquitin antibody (blue). The provided image is one z section from a deconvolved z-stack image.