The dynamics and regulation of HIV-1 nuclear import and its intranuclear movements after import have not been studied. To elucidate these essential HIV-1 post-entry events, we labeled viral complexes with two fluorescently tagged virion-incorporated proteins (APOBEC3F or integrase), and analyzed the HIV-1 dynamics of nuclear envelope (NE) docking, nuclear import, and intranuclear movements in living cells. We observed that HIV-1 complexes exhibit unusually long NE residence times (1.5±1.6 hrs) compared to most cellular cargos, which are imported into the nuclei within milliseconds. Furthermore, nuclear import requires HIV-1 capsid (CA) and nuclear pore protein Nup358, and results in significant loss of CA, indicating that one of the viral core uncoating steps occurs during nuclear import. Our results showed that the CA-Cyclophilin A interaction regulates the dynamics of nuclear import by delaying the time of NE docking as well as transport through the nuclear pore, but blocking reverse transcription has no effect on the kinetics of nuclear import. We also visualized the translocation of viral complexes docked at the NE into the nucleus and analyzed their nuclear movements and determined that viral complexes exhibited a brief fast phase (<9 min), followed by a long slow phase lasting several hours. A comparison of the movement of viral complexes to those of proviral transcription sites supports the hypothesis that HIV-1 complexes quickly tether to chromatin at or near their sites of integration in both wild-type cells and cells in which LEDGF/p75 was deleted using CRISPR/cas9, indicating that the tethering interactions do not require LEDGF/p75. These studies provide novel insights into the dynamics of viral complex-NE association, regulation of nuclear import, viral core uncoating, and intranuclear movements that precede integration site selection.

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...

The intracellular restriction factor TRIM5α, inhibits infection by numerous retroviruses in a species-specific manner. The best characterized example of this restriction is the TRIM5α protein from rhesus macaques (rhTRIM5α), which potently inhibits HIV-1 infection. TRIM5α localizes to cytoplasmic assemblies of protein referred to as cytoplasmic bodies, though the role that these bodies play in retroviral restriction is unclear. We employed a series of truncation mutants to identify a discrete region, located within the Linker2 region connecting the coiled-coil and B30.2/PRYSPRY domains of TRIM5α, which is required for cytoplasmic body localization. Deletion of this region in the context of full-length rhTRIM5α abrogates cytoplasmic body localization. Alanine mutagenesis of the residues in this region identifies two stretches of amino acids that are required for both cytoplasmic body localization and retroviral restriction. This work suggests that the determinants that mediate TRIM5α localization to cytoplasmic bodies play a requisite role in retroviral restriction.

BackgroundThe TRIM5 proteins are cellular restriction factors that prevent retroviral infection in a species-specific manner. Multiple experiments indicate that restriction activity requires accessory host factors, including E2-enzymes. To better understand the mechanism of restriction, we conducted yeast-two hybrid screens to identify proteins that bind to two TRIM5 orthologues.ResultsThe only cDNAs that scored on repeat testing with both TRIM5 orthologues were the proteasome subunit PSMC2 and ubiquitin. Using co-immunoprecipitation assays, we demonstrated an interaction between TRIM5α and PSMC2, as well as numerous other proteasome subunits. Fluorescence microscopy revealed co-localization of proteasomes and TRIM5α cytoplasmic bodies. Forster resonance energy transfer (FRET) analysis indicated that the interaction between TRIM5 and PSMC2 was direct. Previous imaging experiments demonstrated that, when cells are challenged with fluorescently-labeled HIV-1 virions, restrictive TRIM5α orthologues assemble cytoplasmic bodies around incoming virion particles. Following virus challenge, we observed localization of proteasome subunits to rhTRIM5α cytoplasmic bodies that contained fluorescently labeled HIV-1 virions.ConclusionsTaken together, the results presented here suggest that localization of the proteasome to TRIM5α cytoplasmic bodies makes an important contribution to TRIM5α-mediated restriction.