Background:  TRIM5a has recently been identified as a host restriction factor against HIV-1 infection in Old World monkeys. When expressed with either HA tag or as a fusion with a fluorescent protein, TRIM5a form cytoplasmic bodies. However, the biological role of cytoplasmic bodies in HIV restriction remains controversial. Using a stable HeLa cell line expressing a YFP-rhTRIM5a fusion protein, we screened reagents for potential effects on TRIM5a localization. By observing differences in TRIM5a localization induced by these reagents, we hope to better understand the cell biology of TRIM5a.

Methods:  To screen drugs for potential effects on TRIM5a localization, YFP-rhTRIM5a-expressing cells were incubated with drugs. Then, the localization of TRIM5a was determined by deconvolution microscopy. The expression of TRIM5a was studied by Western blot. Pulse-chase experiment was performed to determine the effect of drugs on the rate of turnover of TRIM5a.

Results:  We identified 2 drugs that had a significant effect of TRIM5a localization after 5 hours of treatment. First, exposure to cyclohexamide, an inhibitor of protein synthesis, led to a large decrease in YFP-TRIM5a expression. Second, treatment with MG132, a proteasome inhibitor, caused an increase in the size and a decrease in the number of the cytoplasmic bodies formed by the YFP-TRIM5a. Similar effects were observed with 2 other proteasome inhibitors, b-lactone and expoxomicin. Moreover, treatment of cells stably expressing HA-tagged rhTRIM5a with MG132 also caused an increase in cytoplasmic body size relative to untreated cells. Next we analyzed the effect of cyclohexamide and MG132 on the expression of TRIM5a by Western blot:  5-hour treatment with cyclohexamide caused a large decrease in the levels of both rhTRIM5a and huTRIM5a, but treatment with MG132 did not significantly alter the levels of expression of TRIM5a. Pulse chase analysis of TRIM5a revealed that the half-life of TRIM5a was approximately 1 hour and that this half-life was not dramatically affected by MG132 treatment.

Conclusions:  TRIM5a turns over rapidly in the cell with a half-life of about 1 hour. MG132 treatment leads to bigger but fewer cytoplasmic bodies without obvious effect on expression level or turnover rate. Thus, proteasome activity does not regulate TRIM5a expression or turnover, but appears to be involved in regulating the cell biology of TRIM5a.