Proteasome inhibition induces the accumulation of HIV-1 in rhTRIM5α cytoplasmic bodies. (A) rhTRIM5α-HA–expressing HeLa cells were continuously infected with GFP-Vpr–labeled HIV-1 virions (green) in the presence of 1 μg/ml MG132 proteasome inhibitor for 6 h. Cells were then fixed and immunostained with antibodies to HA (red) or the 20s proteasomal subunit (blue) and imaged. The boxed regions are enlarged in the insets to the right for better visualization of the described interaction. The provided image is one z section from a deconvolved z-stack image. (B) The percentage of HIV-1 virions associated with rhTRIM5α cytoplasmic bodies was calculated for virions lacking envelope protein (ΔEnv) or pseudotyped with the VSV-G envelope protein (VSV-G). The mean of two independent experiments is shown in which a total of 698 virions (VSV-G) and 837 virions (ΔEnv) were counted. (C) rhTRIM5α-HA–expressing HeLa cells were spinoculated with GFP-Vpr–labeled NL43 virions in the presence of 1 μg/ml MG132, and the percentage of HIV-1 virions associated with rhTRIM5α cytoplasmic bodies was calculated for virions infected in the presence of absence of AMD3100. The mean of two independent experiments is shown in which a total of 369 virions (MG132) and 418 virions (MG132 + AMD3100) were counted. (D) The ability of fused and unfused HIV-1 virions to associate with rhTRIM5α cytoplasmic bodies was analyzed using HIV-1 dually labeled with S15-mCherry and GFP-Vpr. Cells were infected in the presence of 1 μg/ml MG132 (gray bars) or 20 nM BafA (black bars). Error bars represent the SEM from three experiments in which a total of 2,578 or 1,445 virions were counted for cells treated with MG132 or BafA, respectively.