Contribution of E3-Ubiquitin Ligase Activity to HIV-1 Restriction by TRIM5αrh: Structure of the RING Domain of TRIM5α

TRIM5 alpha(rh) is a cytosolic protein that potently restricts HIV-1 before reverse transcription. TRIM5 alpha(rh) is composed of four different domains: RING, B-box 2, coiled coil, and B30.2(SPRY). The contribution of each of these domains to restriction has been extensively studied, with the exception of the RING domain. The RING domain of TRIM5 alpha exhibits E3-ubiquitin ligase activity, but the contribution of this activity to the restriction of HIV-1 is not known. To test the hypothesis that the E3-ubiquitin ligase activity of the RING domain modulates TRIM5 alpha(rh) restriction of HIV-1, we correlated the E3-ubiquitin ligase activity of a panel of TRIM5 alpha(rh) RING domain variants with the ability of these mutant proteins to restrict HIV-1. For this purpose, we first solved the nuclear magnetic resonance structure of the RING domain of TRIM5 alpha and defined potential functional regions of the RING domain by homology to other RING domains. With this structural information, we performed a systematic mutagenesis of the RING domain regions and tested the TRIM5 alpha RING domain variants for the ability to undergo self-ubiquitylation. Several residues, particularly the ones on the E2-binding region of the RING domain, were defective in their self-ubiquitylation ability. To correlate HIV-1 restriction to self-ubiquitylation, we used RING domain mutant proteins that were defective in self-ubiquitylation but preserve important properties required for potent restriction by TRIM5 alpha(rh), such as capsid binding and higher-order self-association. From these investigations, we found a set of residues that when mutated results in TRIM5 alpha molecules that lost both the ability to potently restrict HIV-1 and their self-ubiquitylation activity. Remarkably, all of these changes were in residues located in the E2-binding region of the RING domain. Overall, these results demonstrate a role for TRIM5 alpha self-ubiquitylation in the ability of TRIM5 alpha to restrict HIV-1.