Rhesus macaque TRIM5␣ (rhTRIM5␣) is a retroviral restriction factor that inhibits HIV-1 infection. Previous studies have revealed that TRIM5␣ 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␣, 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␣ fusion relative to the catalytically inactive control or the wild-type (WT) TRIM5␣. Similarly, expression of DUb-rhTRIM5␣ resulted in the accumulation of viral cores in target cells following infection, while the catalytically inactive control and WT rhTRIM5␣ induced the abortive disassembly of viral cores, indicating a role for ubiquitin conjugation in rhTRIM5␣-mediated destabilization of HIV-1 cores. Finally, DUb-rhTRIM5␣ failed to activate NF-B signaling pathways compared to controls, demonstrating that this ubiquitination-dependent activity is separable from the ability to restrict retroviral infection. IMPORTANCEThese studies provide direct evidence that ubiquitin conjugation to rhTRIM5␣-containing complexes is required for the second step of HIV-1 restriction. They also provide a novel tool by which the biological activities of TRIM family proteins might be dissected to better understand their function and underlying mechanisms of action. TRIM5␣ is a retroviral restriction factor that mediates a postentry block to infection (1, 2). The best-studied example of this restriction is the ability of the TRIM5␣ protein from rhesus macaques (rhTRIM5␣) to potently inhibit HIV-1 infection (1, 2). As a member of the TRIM family of proteins, TRIM5␣ possesses the canonical RING, B-Box, and coiled-coil (CC) domains that comprise the tripartite motif (TRIM) that define this family of proteins (3). The N-terminal RING domain acts as an E3 ubiquitin ligase (4-6), and together with the B-Box domain, also functions to mediate the self-association of TRIM5␣ dimers (7-9). The CC domain, in cooperation with the Linker2 (L2) region, mediates the dimerization of TRIM5␣ monomers and the formation of higher-order assemblies (10-14). TRIM5␣ also possesses a C-terminal SPRY domain which is known to recognize determinants in the assembled viral core to mediate restriction (15-17). Following core binding, TRIM5␣ induces the abortive disassembly of the viral core (18,19). The mechanism by which core disruption o...