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TRIM5α and TRIMCyp form apparent hexamers and their multimeric state is not affected by exposure to restriction-sensitive viruses or by treatment with pharmacological inhibitors
© Nepveu-Traversy et al; licensee BioMed Central Ltd. 2009
- Received: 27 August 2009
- Accepted: 03 November 2009
- Published: 03 November 2009
Proteins of the TRIM5 family, such as TRIM5α and the related TRIMCyp, are cytoplasmic factors that can inhibit incoming retroviruses. This type of restriction requires a direct interaction between TRIM5 proteins and capsid proteins that are part of mature, intact retroviral cores. In such cores, capsids are arranged as hexameric units. Multiple lines of evidence imply that TRIM5 proteins themselves interact with retroviral cores as multimers. Accordingly, stabilization by crosslinking agents has revealed that TRIM5α and TRIMCyp are present as trimers in mammalian cells. We report here that TRIM5 proteins seem to form dimers, trimers, hexamers and multimers of higher complexity in mammalian cells. The hexameric form in particular seems to be the most abundant multimer. Multimerization did not involve disulfide bridges and was not affected by infection with restriction-sensitive viruses or by treatment with the known TRIM5 inhibitors arsenic trioxide, MG132 and cyclosporine A. We conclude that TRIM5 multimerization results from more than one protein-protein interface and that it is seemingly not triggered by contact with retroviral cores.
- Capsid Protein
- Arsenic Trioxide
- TRIM5 Protein
- Glutaraldehyde Concentration
- Crosslinking Assay
TRIM proteins form a family with dozens of members, most of them bearing a tripartite motif composed of a RING, B-box and Coiled-coil domains . Restriction of retroviruses by members of the TRIM5 subfamily of TRIM proteins, which comprises the primate proteins TRIM5α and TRIMCyp [2–4], is initiated by physical recognition of the incoming retrovirus by TRIM5 proteins. This interaction occurs within the first hours following virus entry  and involves determinants present in the N-terminal domain of the capsid proteins which constitute the retroviral outer core structure [6–8]. Retroviral capsid cores are assembled from hundreds of capsid proteins and the basic capsomer is a hexamer [9–11]. Restriction necessitates capsid proteins of the incoming retrovirus to be correctly maturated by the retroviral protease [12, 13]. This is a required step for the core to adopt its final structure. In addition, mutations that affect the stability of the retroviral core interfere with the efficiency of restriction [12, 13]. Virus-free capsid proteins, which do not multimerize to form cores, do not interact with TRIM5 proteins in cells . That TRIM5-mediated restriction requires assembled retroviral cores brings the question of whether TRIM5 proteins themselves must be present as multimers. TRIM proteins are known to homomultimerize through their coiled-coil domain , which is required for restriction . TRIM5 proteins from different species can interact with each other and in doing so can interfere with each other's restriction activity . TRIM5α has also been shown to trap incoming retroviral particles inside cytoplasmic bodies, which further suggests that TRIM5 proteins interact with their targets as multimers . TRIM5α and TRIMCyp have been stabilized as trimers by treatment with cross-linking agents [18–23]. Some undefined higher-order multimers have been occasionally observed [18, 19]. The relevance of trimerization was confirmed by the fact that modified TRIMCyp, in which the coiled-coil domain is substituted by that of a trimeric heterologous protein, restricted HIV-1, although at much lower levels than wild-type TRIMCyp did . A recombinant TRIM5 protein expressed in insect cells was observed as dimers  and minor amounts of dimeric TRIM5α have been observed in cells . However, dimerization/trimerization of TRIM5 proteins fails to explain the formation of cytoplasmic bodies or the sequestration of incoming restricted virus in such structures. Thus, we analyzed TRIM5α/TRIMCyp multimerization in the presence or absence of restriction-sensitive viruses and upon treatment with various drugs that inhibit the restriction process.
It was recently reported that TRIM5α and TRIMCyp are degraded in a proteasome-dependent pathway following infection with a restriction-sensitive retrovirus . Thus, it was conceivable that in our previous experiment HIV-1 modulated the multimerization of only a part of the cellular TRIMCyp proteins which were then degraded by the proteasome. To address that possibility, we repeated the experiment in the presence of the proteasomal inhibitor MG132, thereby preventing virus-induced TRIMCyp targeting to the proteasome (not shown). In addition we infected with a higher dose of the HIV-1 vector, leading to 20% infected cells. TRIMCyp restriction activity was significantly saturated at this MOI, implying that most TRIMCyp proteins that were restriction-competent at the time of infection were indeed engaging incoming HIV-1 . However, MG132 did not appreciably modify the multimerization profile of TRIMCyp in the absence or presence of HIV-1 (Fig. 2B). Like before, dimers, trimers and higher-order multimers were formed. The band corresponding to putative hexamers was less well-defined compared with previous experiments, but this is probably due to technical reasons unrelated to the effects of MG132 on TRIM5.
We find that in addition to the dimeric and trimeric forms previously described, TRIM5α and TRIMCyp can form apparent hexamers and more complex multimers. Why discrete hexamers were not previously seen by others is probably only related to the difficulty of resolving high molecular weight complexes in acrylamide gels, although we cannot totally exclude that the C-terminus FLAG tag used in our constructs may somehow interfere with protein multimerization. Because of low expression levels in mammalian cells, it is not possible at this point to perform the biochemical experiments that would be needed to ascertain that the various multimers seen here are composed of TRIM5 proteins only. For instance, a trimer of TRIM5 could associate with a heterologous cellular protein, yielding a band resembling a TRIM5 hexamer. Thus, other approaches will be needed. The hexamer model is obviously appealing because capsid proteins are themselves organized as hexamers in mature retroviral cores. Thus, a hexamer of TRIM5 proteins could be needed to recognize a retroviral capsomer. Formation of dimers, trimers and hexamers, however, does not seem to be triggered by contact with a restricted retrovirus. It remains possible that the nature and number of some specific higher order multimers not resolved in our gels could be modulated during the restriction process. Not surprisingly, the coiled-coil domain of TRIM5 proteins has been found to be required for the formation of trimers [18, 23]. However, this does not imply that a single protein:protein interface present in this domain is responsible for the various multimeric forms observed. Rather, it is more likely that one interface would lead to dimerization and another one to trimerization; together they would be responsible for hexamerization. Perhaps yet other determinants within TRIM5α and TRIMCyp lead to the formation of very high molecular weight multimers. Consistent with the existence of more than one molecular site of TRIM5:TRIM5 interactions, Li and Sodroski have recently reported that point mutants in the B-Box domain show normal multimerization patterns in crosslinking assays while being less efficient at engaging in protein:protein interactions through co-immunoprecipitation assays . Regardless of what the exact molecular mechanism of TRIM5 multimerization is, our data suggest that TRIM5 multimerization is complex but that formation of low molecular weight multimers is not influenced by contact with a restricted retrovirus.
We thank Mélodie B. Plourde for help with drafting the manuscript. This work was supported by the Canadian Institutes for Health Research and by the Canada Research Chairs program.
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