Deletion of the TRIM5α RING domain or disruption of RING domain folding by alteration of the zinc-coordinating cysteine residues has been shown to result in varying effects on retroviral inhibition, depending on the TRIM5 protein and the restricted virus (12
). In this study, by using a panel of RING mutants with intact zinc-binding residues, we show that the E3 ubiquitin ligase function of TRIM5αAGM
is important for the restriction of SIVmac
infection. The relative anti-SIVmac
inhibitory activities of TRIM5α variants that differed in RING domain sequences strongly correlated with the E3 ubiquitin ligase activity measured in vitro
. The TRIM5αAGM
RING variants were shown to dimerize, bind HIV-1 and SIVmac
capsid complexes, and restrict HIV-1 infection, supporting their structural integrity. Thus, the RING-mediated E3 ubiquitin ligase activity represents an “effector function” for TRIM5α-mediated SIVmac
restriction, contributing additively with capsid binding to determine restriction potency. The Rh-RtVt and Rh-RpVp chimeric proteins provide an example of how equivalent levels of SIVmac
restriction can be achieved by combining robust RING and weak B30.2(SPRY) functions, or weak RING and robust B30.2(SPRY) functions, respectively.
The RING-associated E3 ubiquitin ligase activity of TRIM5α inversely correlated with the levels of the SIVmac
particulate capsids in the cytosol of the infected cells and with SIVmac
cDNA synthesis. Moreover, the levels of particulate cytosolic capsids and viral cDNAs correlated. As the capsid is the direct binding target of TRIM5α (31
), our data suggest a model in which the ubiquitin ligase activity of TRIM5α leads to accelerated disassembly of cytosolic SIVmac
capsids. As has been seen for unstable HIV-1 capsid mutants (17
), premature uncoating of the SIVmac
capsids by TRIM5α directly or indirectly leads to disruption of reverse transcription.
Our results indicate the importance of the E3 ubiquitin ligase activity of TRIM5αAGM
restriction. What might be the functionally important target of TRIM5α-mediated ubiquitylation, and what are the consequences of ubiquitylation of the potential target? RING-mediated E3 ubiquitin ligases simultaneously bind both the E2 enzyme partner and substrate to transfer the ubiquitin moiety from E2 to substrate; a few TRIM family members are known to transfer ubiquitin to their ligands, which are directly recognized by the B30.2(SPRY) domains (19
). Based on these examples, one might expect the ubiquitylation target of TRIM5α to be the ligand of the B30.2(SPRY) domain, i.e., the retroviral capsid. However, no ubiquitylation of the SIVmac
capsid protein in either particulate or soluble form was observed in the fate-of-capsid assay. Similarly, fate-of-capsid studies conducted with N-MLV restricted by human TRIM5α also allowed visualization of the solubilized capsid proteins that resulted from TRIM5α action; no modified form of the capsid protein was detected (10
). Thus, if the targeted capsid is ubiquitylated, only a very small fraction of the total protein population is modified. Another possibility is that TRIM5α autoubiquitylation contributes to retroviral restriction. It has been reported that infection of cells with a retrovirus susceptible to TRIM5α restriction leads to a decrease in the level of TRIM5α protein in the cell (66
). The aggregation of TRIM5α proteins on the capsid surface could favor ubiquitylation and proteasome-mediated turnover of a fraction of the TRIM5α protein in the cell, potentially leading to faster uncoating of the bound capsid. However, considering that the ubiquitin chains are also able to induce conformational changes and recruit several cellular factors, depending on the type of Ub-Ub chain linkages, further work is required to substantiate a role for TRIM5α autoubiquitylation in SIVmac
restriction. Finally, host cell factors could be ubiquitylated by TRIM5α. The identification of such host cell proteins and dissection of their roles in early postentry steps in SIVmac
infection are worthy goals of future studies.
Proteasome inhibition resulted in increases in the levels of SIVmac
particulate cytosolic capsids and reverse transcripts in infected cells expressing particular restricting TRIM5α proteins. These effects were observed for a TRIM5α protein, Rh-RtVp, with potent RING E3 ubiquitin ligase function and were not seen in cells expressing the matched Rh-RpVp TRIM5α protein with diminished RING activity. The phenotypic similarity of proteasome inhibition and mutagenic inactivation of the TRIM5α RING E3 ubiquitin ligase supports the involvement of ubiquitin in pre-reverse transcription blocks to SIVmac
infection. Previous studies suggested that, although proteasome activity is not required for TRIM5α antiviral activity per se
), treatment with proteasome inhibitors resulted in higher levels of HIV-1 and murine leukemia virus reverse transcripts and particulate capsids in TRIM5α-expressing target cells (2
). Interpretation of results with proteasome inhibitors, however, is complicated by the observation that the levels of retroviral cDNA and particulate capsids in the cytosol can be increased by proteasome inhibition even in cells not expressing a restricting TRIM5α protein (F and D) (10
). Moreover, proteasome inhibition causes the redistribution of TRIM5α into large cytoplasmic aggregates (12
), potentially introducing nonphysiologic artifacts into the restriction mechanism. Finally, proteasome inhibitors not only block the proteasome-mediated degradation pathway, but can also deplete the pool of free ubiquitin in cells by impeding the recycling of ubiquitin from protein conjugates (9
). Thus, although our results clearly implicate ubiquitylation in TRIM5α-mediated pre-reverse transcription blocks of SIVmac
infection, it is formally possible that ubiquitin ligation contributes to this restriction in a manner that is independent of proteasome-mediated degradation.
RING-mediated E3 ubiquitin ligase activity was apparently more important to SIVmac
inhibition than to HIV-1 inhibition by TRIM5αAGM
. In a recent report (48
), Maegawa and colleagues also observed that the phenotypes of TRIM5α mutants with changes in RING domain cysteines depended on the restricted virus. Although further work will be needed to understand the basis for the SIVmac
-HIV-1 difference, we found that the helix-4/5 loop on the capsid influenced the requirement for RING-mediated E3 ubiquitin ligase activity. As changes in this loop have been shown to modulate HIV-1 capsid stability and the rate of capsid disassembly in infected cells (15
), quantitative or qualitative differences between the subunit interactions in the HIV-1 and SIVmac
capsids may alter the requirements for the E3 ubiquitin ligase function of TRIM5α.
Two TRIM5α variants, Rh-RpVp and Rh-RpVt, with poor RING domain function restricted SIVmac
infection moderately, even though corresponding decreases in the levels of viral reverse transcripts or particulate capsids were not detected. Apparently, SIVmac restriction by these two TRIM5α proteins occurs after reverse transcription and without detectable increases in the rate of capsid disassembly. Rh-RpVp blocked SIVmac
infection more potently than Rh-RpVt, indicating that the strength of this apparent post-reverse transcription block is influenced by capsid-binding affinity. TRIM protein variants have been reported to block retroviral infection after viral cDNA synthesis (90
). The availability of well-characterized TRIM5α mutants should allow further exploration of the mechanism and relevance of these blocks.