HIV-1 virions sequestered within rhTRIM5α cytoplasmic bodies after proteasome inhibition contain p24^CA protein. (A and B) HeLa cells stably expressing HA-tagged rhTRIM5α were spinoculated with VSV-G–pseudotyped HIV_FCM containing FlAsH-labeled capsid and mCherry-Vpr for 2 h at 17°C. Unbound virus was removed and replaced with media, and cells were incubated for 6 h at 37°C in the presence of MG132. Cells were then fixed, stained with an HA-specific antibody, and analyzed by fluorescent microscopy to determine the total number of mCherry-Vpr⁺ virions/cell and the percentage of virions that associated with rhTRIM5α signal (B). (C) The fluorescent FlAsH intensity of mCherry-Vpr virions associated with rhTRIM5α signal in the MG132-treated sample was compared with the intensity of virions observed in the untreated and BafA infections. The mean FlAsH intensity of each population is indicated by black bars. (D) HIV_FCM particles positive for both FlAsH-labeled capsid (green) and mCherry-Vpr (red) within rhTRIM5α cytoplasmic bodies (blue). The provided image is one z section from a deconvolved z-stack image.