Contribution of E3-Ubiquitin Ligase Activity to HIV-1 Restriction by TRIM5rh: Structure of the RING Domain of TRIM5

TRIM5rh is a cytosolic protein that potently restricts HIV-1 before reverse transcription. TRIM5rh 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 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 TRIM5rh restriction of HIV-1, we correlated the E3-ubiquitin ligase activity of a panel of TRIM5rh 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 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 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 t