The RING domain of TRIM5α exhibits E3-ubiquitin ligase activity, but the contribution of this activity to the restriction of HIV-1 is not understood. Here we present the structure of the RING domain of human TRIM5α and use this information to direct a mutational analysis of the functional surfaces of the RING domain of TRIM5αrh
. To explore the role of the E3-ubiquitin ligase in restriction, we correlated the E3-ubiquitin ligase activity of TRIM5α with the different properties of the restriction factor TRIM5α, including HIV-1 restriction, binding to the HIV-1 capsid, inhibition of reverse transcription, and the ability to form higher-order complexes. We found a distinct set of TRIM5α variants located on the E2-binding surface of the RING domain, where the loss of E3-ubiquitin ligase activity correlated with a defect in HIV-1 restriction ability. Our results demonstrate that E3-ubiquitin ligase activity has a role in HIV-1 restriction by TRIM5α, as has been previously suggested by others (1
The RING domain of TRIM5α adopts a ββα RING fold containing shorter β-strands and a longer α-helix than the typical fold observed in RING domains. Comparison of the RING domain of TRIM5α with other RING domains revealed that this structure has two regions that are potentially important for RING domain function (). Similar to the RING domains of Cbl, CHIP, and cIAP2 (9
), the RING domain of TRIM5α presents a distinct E2-binding region composed of similar amino acids (A). Opposite to the E2-binding region is the RING-RING interaction region, which is similar to the interaction region that allows BRCA1 and BARD1 RING domains to form a heterodimer (6
) (B). The construct used to solve the NMR structure of the RING domain of TRIM5α did not include the last 10 amino acids; these residues are part of the association helix used by BRCA1 and BARD1 RING domains to heterodimerize. Longer constructs of the TRIM5α RING domain resulted in a poor HSQC spectrum.
The E2-binding region of RING domains is essential for docking of the E2 protein and allows the transfer of ubiquitin from E2 to the target protein. In order to disrupt this docking site in the RING domain of TRIM5α, we generated a series of mutations on the different surfaces of the RING domain (); these variants were tested for the different properties of the restriction factor TRIM5α. These experiments revealed that TRIM5α self-ubiquitylation requires an intact E2-binding region, which suggested that an intact E2 docking site in the RING domain is required for the self-ubiquitylation property of TRIM5α. Mutations in all of the residues of the E2-binding site of the RING domain affected self-ubiquitylation to a certain extent; in some cases, complete loss of self-ubiquitylation was observed. Mutations in the E2-binding region that resulted in a partial effect on self-ubiquitylation could be explained by the existence of complementation by a different RING domain. Hetero-oligomerization with a related RING domain could rescue ubiquitylation activity in a defective RING domain, as has been shown for ubiquitylation-deficient mutant proteins of the Mdm2 RING domain that can be rescued by hetero-oligomerization with the RING domain of MdmX (60
To exclude RING mutations that had effects on other properties that are important for HIV-1 restriction by TRIM5α, we quantitatively measure the binding of these variants to the HIV-1 capsid, as previously shown (15
). Similarly, we also measured the ability of the RING variants to undergo higher-order self-association, which is also required for potent restriction of HIV-1. Remarkably, TRIM5α self-ubiquitylation activity correlates with restriction activity on mutant proteins where binding to the HIV-1 capsid and higher-order self-association were not affected. This correlation supports the hypothesis that the E3-ubiquitin ligase activity of the RING domain is required for potent restriction of HIV-1 by TRIM5α.
Several observations have linked the restriction of HIV-1 by TRIM5α with the proteasome. The observation that proteasomal inhibitors allow the occurrence of reverse transcription without affecting restriction suggests that TRIM5α blocks restriction before and after reverse transcription (1
). The use of proteasome inhibitors in the fate of the capsid assay showed an increase in particulate capsid during infection in the presence of TRIM5α, which also suggests a role for the proteasome in restriction and uncoating (11
). The Aiken laboratory has demonstrated that TRIM5α is degraded in a proteasome-dependent manner in the presence of a restricted capsid, which links the proteasome with the HIV-1 restriction by TRIM5α (54
). The present work attempted to connect ubiquitylation, a process preceding proteasomal degradation, with the ability of TRIM5α to block HIV-1. Similar to what we observed for a panel of B-box 2 mutant proteins (15
), the levels of HIV-1 late reverse transcripts for this panel of RING mutant proteins inversely correlated with the degree of restriction.
Mutations in the RING-RING interaction region that removed the ability to undergo self-ubiquitylation might cause a defect in RING oligomerization, which is different from affecting the docking site of the E2 enzyme. Several mutations in the RING-RING interaction region also affected the self-ubiquitylation activity of TRIM5α without affecting folding measured by HIV-1 capsid binding and higher-order self-association. This is in agreement with findings suggesting that RING domain oligomerization enhances ubiquitylation (30
). Loss of RING domain oligomerization could account for the partial defect in self-ubiquitylation observed in some of the RING-RING interaction region variants. In some cases, loss of RING dimerization could result in complete loss of E3-ubiquitin ligase activity, as has been shown for the RING domain of RNF4 (41
). Even though concentration dependence experiments to test RING domain dimerization failed to prove homodimerization (data not shown), these experiments did not exclude the possibility that the RING domain hetero-oligomerizes with a different RING domain, as shown for BRCA-1 and BARD (6
Our results demonstrated that potent restriction of HIV-1 by TRIM5αrh
requires intact self-ubiquitylation activity. One could conceive a model in which the self-ubiquitylation of TRIM5α is required to remove TRIM5α when it is forming hexagonal structures on the surface of the capsid (19
); removal of TRIM5α from the surface of the capsid will allow a decrease on the amount of particulate capsid during infection, assisting a rapid uncoating process (10
). Further analysis destined to understand the nature of the endogenous E2 enzyme and the ubiquitylation substrate of TRIM5α will give new mechanistic insights into restriction.