Cell biology of host restriction factor TRIM5 and its two-phase mechanism for blocking HIV-1 infection

TRIM5alpha was recently identified as the host restriction factor against HIV-1 infection in Old World Monkeys, such as rhesus macaques. The cell biology study of TRIM5alpha and the exploration of its function mechanism against viral infection were carried out in this work.; Using GFP-TRIM5alpha fusion protein to study its cellular distribution, we found that TRIM5alpha localizes in the cytoplasm in a punctate pattern and forms cytoplasmic bodies. Moreover, these bodies are highly mobile and display two kinds of movements, short salutatory movements or- uni-directional long distance movements. Further, the movements of TRIM5alpha are microtubule dependent. In the end, the FRAP analysis demonstrated that the cytoplasmic bodies of TRIM5alpha are not static, dead-end structures resulting from overexpression, but rather dynamic structures that turn over rapidly due to continuous exchange with soluble cytoplasmic TRIM5alpha and TRIM5alpha in similar neighboring bodies.; We further characterized the mechanism used by rhesus monkey TRIM5alpha (rhTRIM5alpha) to restrict HIV-1 replication in target cells by analyzing the effect of the proteasome inhibitor MG132 on the restriction ability of rhTRIM5alpha. We found that MG132 prevented the rhTRIM5alpha block to HIV-1 RT, allowing HIV-1 late RT products to now accumulate even though viral infection and nuclear 1-LTR and 2-LTR cDNA forms remained impaired. This finding suggests that rhTRIM5alpha restriction involves two phases in which rhTRIM5alpha first interacts with the viral capsid and obscures its normal trafficking, followed by proteasomal degradation of the viral core that blocks accumulation of RT products.; Finally, we found that rhTRIM5alpha shuttles between cytoplasm and nucleus, since treatment with the nuclear export inhibitor, LMB, resulted in the appearance of soluble rhTRIM5alpha in the nucleus. Our study also showed that LMB did not completely block tire nuclear export of rhTRIM5, suggesting that TRIM5alpha might employ multiple nuclear export pathways.