TRIM5alpha-mediated ubiquitin chain conjugation is required for inhibition of HIV-1 reverse transcription and capsid destabilization.

Rhesus macaque TRIM5 alpha (rhTRIM5 alpha) is a retroviral restriction factor that inhibits HIV-1 infection. Previous studies have revealed that TRIM5 alpha restriction occurs via a two-step process. The first step is restriction factor binding, which is sufficient to inhibit infection. The second step, which is sensitive to proteasome inhibition, prevents the accumulation of reverse transcription products in the target cell. However, because of the pleotropic effects of proteasome inhibitors, the molecular mechanisms underlying the individual steps in the restriction process have remained poorly understood. In this study, we have fused the small catalytic domain of herpes simplex virus UL36 deubiquitinase (DUb) to the N-terminal RING domain of rhTRIM5 alpha, which results in a ubiquitination-resistant protein. Cell lines stably expressing this fusion protein inhibited HIV-1 infection to the same degree as a control fusion to a catalytically inactive DUb. However, reverse transcription products were substantially increased in the DUb-TRIM5 alpha fusion relative to the catalytically inactive control or the wild-type (WT) TRIM5 alpha. Similarly, expression of DUb-rhTRIM5 alpha resulted in the accumulation of viral cores in target cells following infection, while the catalytically inactive control and WT rhTRIM5 alpha induced the abortive disassembly of viral cores, indicating a role for ubiquitin conjugation in rhTRIM5 alpha-mediated destabilization of HIV-1 cores. Finally, DUb-rhTRIM5 alpha failed to activate NF-kappa B signaling pathways compared to controls, demonstrating that this ubiquitination-dependent activity is separable from the ability to restrict retroviral infection.