Retroviral Restriction Factor TRIM5α Is a Trimer

Abstract

The retrovirus restriction factor TRIM5α targets the viral capsid soon after entry. Here we show that the TRIM5α protein oligomerizes into trimers. The TRIM5α coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5α mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5α, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5α to interact with threefold pseudosymmetrical structures on retroviral capsids.

TRIM5α is a constitutively expressed cytoplasmic protein that allows the cells of primates to resist infection by particular retroviruses, including human immunodeficiency virus type 1 (HIV-1) (10, 14, 25, 31, 32, 37). TRIM5α is thought to target the incoming retroviral capsid soon after entry into the cells (9, 11, 13, 15, 19, 21, 22, 29, 34). The specific mechanism by which TRIM5α restricts retroviral infection remains unknown.

TRIM5α is a member of the tripartite motif (TRIM) family of proteins which contain RING, B-box, and coiled-coil domains (26). Many TRIM proteins self-associate to form homo-oligomers; less frequently, hetero-oligomerization is observed (26). Structural predictions suggest that the coiled coils of TRIM proteins exhibit a propensity to form both dimers and trimers (6, 7, 17). There is only limited information available about the oligomeric state of TRIM proteins. Oligomerization has been shown to be important for the function of the nuclear TRIM28 (KAP-1) protein (23, 24). In this case, the RING, B-box, and coiled-coil domains were shown to contribute to trimerization. The coiled coil of TRIM7 is essential for oligomerization (39). Here we examine the oligomeric state of TRIM5.

The hemagglutinin (HA)-tagged TRIM5 variants in Fig. 1A were expressed transiently in 293T cells or stably in HeLa cells. Cells were washed in phosphate-buffered saline (PBS) and lysed in NP-40 lysis buffer (0.5% Nonidet P40 [NP-40], 1 × complete EDTA-free protease inhibitor [Roche Diagnostics] in PBS) for 45 min at 4°C. Lysates were centrifuged at 14,000 × g for 15 min at 4°C. The cleared lysates were not stored or frozen but rather were directly cross-linked. Approximately 100 to 200 μl of cleared lysates was diluted with PBS plus 1 mM EDTA to a final volume of 400 μl. Lysates were cross-linked with various concentrations (up to 10 mM) of glutaraldehyde (GA) for 5 min at room temperature and centrifuged briefly in a table-top centrifuge. The reaction mix was quenched with 0.1 M Tris-HCl, pH 7.5, and briefly centrifuged. The cleared, cross-linked lysates were precipitated with the anti-HA antibody HA.11 (Covance) and protein A-Sepharose beads (Amersham) for 2 h at 4°C; final volumes for the immunoprecipitation were greater than 700 μl. The beads were washed four times with NP-40 wash buffer (10 mM Tris-HCl, pH 7.5, 0.5 M NaCl, 0.5% NP-40) and boiled in LDS sample buffer (106 mM Tris-HCl, 141 mM Tris base, pH 8.5, 0.51 mM EDTA, 10% glycerol, 2% LDS, 0.22 mM SERVA Blue G250, 0.175 mM phenol red [Invitrogen]) with different concentrations of β-mercaptoethanol (β-ME) for 10 min. Precipitated proteins were separated on 8% or 12% Tris-glycine gels, transferred to a polyvinylidene difluoride membrane, and detected with the horseradish peroxidase-conjugated 3F10 anti-HA antibody (Roche Diagnostics) and the ECL Plus Western blotting detection system (Amersham).

FIG. 1.

Oligomeric state of rhesus monkey TRIM5α variants. (A) A diagram of the TRIM5α_rh protein with the carboxy-terminal HA tag is shown, with the domains labeled and domain boundaries numbered according to the amino acid residue. The amino-terminal truncation mutants and TRIM5γ_rh are depicted beneath the wild-type TRIM5α_rh. (B) HeLa cells stably expressing the indicated TRIM5α_rh variants or 293T cells expressing the TRIM5α_rh-HA Δ297 mutant were lysed and cross-linked with different concentrations (mM) of GA. After precipitation with an anti-HA antibody, samples were boiled in LDS sample buffer with 0.01% β-mercaptoethanol prior to gel electrophoresis and Western blotting with an anti-HA antibody. m, monomer; d, dimer; t, trimer. Molecular mass markers (in kDa) are indicated. (C) The TRIM5α-HA Δ93 and TRIM5α_rh-HA Δ132 proteins were stably expressed in HeLa cells, which were lysed, treated with GA, and used for immunoprecipitation as described for panel B. The precipitated proteins were boiled in LDS sample buffer with 2.5% β-mercaptoethanol, analyzed on gels, and Western blotted as described for panel B. m, monomer; d, dimer; t, trimer.

TRIM5 isoforms include TRIM5γ, which consists of the RING, B-box 2, and coiled-coil domains, and TRIM5α, which contains an additional C-terminal B30.2(SPRY) domain (26). The wild-type rhesus monkey TRIM5α_rh protein exhibited a molecular mass of 54 to 56 kDa, consistent with that of a monomer (Fig. 1B). Only a small amount of a higher-order form, probably a dimer, was evident in the absence of cross-linker. This putative dimer was sensitive to β-ME (data not shown). Cross-linking with increasing GA concentrations resulted in the progressive appearance of a 156- to 164-kDa species, consistent with a trimer. By contrast, TRIM5γ_rh exhibited a dimeric form both without and with GA treatment. Higher-order forms of TRIM5γ_rh were evident after GA cross-linking, although trimers were not a dominant species. As TRIM5α_rh and TRIM5γ_rh share 300 amino-terminal residues, these results suggest that the TRIM5α_rh sequences carboxy terminal to residue 300 can significantly affect the oligomerization state of the protein.

The oligomeric states of several TRIM5α_rh mutants lacking one or more domains was examined. TRIM5α_rh-HA Δ297, which consists of the B30.2(SPRY) domain alone, migrated as a monomer even after GA cross-linking (Fig. 1B). TRIM5α_rh-HA Δ93, which lacks the RING domain, and TRIM5α_rh-HA Δ132, which lacks the RING and B-box 2 domains, exhibited similar patterns upon GA cross-linking. A species consistent with a dimer was evident in the absence of cross-linker and after GA treatment of both proteins. This form was most apparent when the sample buffer contained low β-ME concentrations (Fig. 1B) and was less evident when larger amounts of β-ME were included in the sample buffer (Fig. 1C). We suspect that these gel-stable dimers result from artifactual oxidation of exposed TRIM5α cysteines upon cell lysis. Forms consistent with trimers were apparent for both TRIM5α_rh-HA Δ93 and TRIM5α_rh-HA Δ132 proteins after GA cross-linking (Fig. 1B). Treatment of the samples with higher concentrations of β-ME demonstrated that the major higher-order product cross-linked by GA for both proteins was a trimer (Fig. 1C). Thus, the TRIM5α_rh segment that includes the coiled coil and the B30.2(SPRY) domain is sufficient for trimerization.

The efficiency with which all three subunits of the wild-type TRIM5α oligomer were cross-linked into gel-stable trimers suggested that many potential GA-reactive sites exist in the TRIM5α subunits. Consistent with this, the use of another cross-linker, EGS [ethylene glycolbis(succinimidylsuccinate)], allowed visualization of gel-stable dimers as well as trimers (Fig. 2A).

FIG. 2.

TRIM5α_rh trimerization and formation of heterotrimers. (A) 293T cells transiently expressing TRIM5α_rh-HA were lysed and the cell lysates treated with the indicated concentrations of GA or EGS. After precipitation with an anti-HA antibody, samples were boiled in LDS sample buffer with 2.5% β-mercaptoethanol prior to gel electrophoresis and Western blotting with an anti-HA antibody. m, monomer, d, dimer, t, trimer. Molecular mass markers (in kDa) are indicated. (B) 293T cells transiently expressing the wild-type (w.t.) TRIM5α_rh-HA protein, TRIM5α_rh-HA Δ132, or a mixture of both proteins were lysed. Cell lysates were incubated in the absence of GA (−GA) or with 0.25 mM GA and then used for immunoprecipitation and Western blotting with an anti-HA antibody as described above. The gel-stable homodimers are labeled (w.t.)₂ or (Δ132)₂ and the gel-stable homotrimers (w.t.)₃ or (Δ132)₃. The positions of the heterodimer (hd) and heterotrimers (ht) are indicated. Molecular mass markers (in kDa) are indicated.

TRIM5α mutants lacking the RING and B-box 2 domains have been shown to associate with wild-type TRIM5α and exert dominant-negative activity on retrovirus restriction (13a, 24a). Coexpression of the wild-type TRIM5α_rh-HA and TRIM5α_rh-HA Δ132 proteins resulted in the formation of heterotrimers; small amounts of gel-stable heterodimers and heterotrimers were evident in the absence of cross-linker (Fig. 2B, left panel). Cross-linking with GA increased the amounts of detectable heterotrimers (Fig. 2B, right panel).

TRIM5 protein variants from other monkey species were examined. The TRIM5α proteins from two subspecies of African green monkey also predominantly formed trimers detectable by cross-linking (Fig. 3A). Owl monkeys, a New World species, do not express a TRIM5α protein but instead express TRIMCyp, which consists of the RING, B-box 2, and coiled-coil domains of TRIM5 fused with cyclophilin A (20, 28). TRIMCyp restricts HIV-1 infection in owl monkey cells. Upon GA cross-linking, TRIMCyp exhibited mostly very-high-molecular-weight species as well as a lower level of trimers (Fig. 3B). These results support an effect of the carboxy-terminal TRIM5 sequences on the oligomeric state of the protein.

FIG. 3.

Oligomeric state of TRIM5 variants from other monkey species. HeLa cells stably expressing TRIM5α proteins from African green monkeys (AGM), either the tantalus (tan) or pygerythrus (pyg) subspecies (A) or the owl monkey TRIMCyp protein (B) were lysed. Cell lysates were cross-linked with the indicated concentrations of GA and precipitated with an anti-HA antibody. Precipitated proteins were boiled in LDS sample buffer with 2.5% β-mercaptoethanol and analyzed by Western blotting with an anti-HA antibody. Molecular mass markers are indicated. m, monomer; d, dimer; t, trimer.

We have demonstrated that the TRIM5α proteins from three Old World monkey species, all of which have been shown to restrict HIV-1 infection (1, 8, 11, 12), exist as trimers. The coiled-coil and B30.2(SPRY) domains of TRIM5α_rh are sufficient for trimerization. As expected from studies of other TRIM proteins (23, 24, 26), the TRIM5α_rh coiled coil apparently contributes to homo-oligomerization. This conclusion is supported by our observation that TRIM5α_rh-HA Δ132 can form trimers, whereas TRIM5α_rh-HA Δ297 is a monomer.

Surprisingly, the carboxyl terminus of TRIM5 influences the oligomerization state of variants with identical or closely related RING, B-box 2, and coiled-coil domains. TRIM5γ_rh, which lacks the B30.2(SPRY) domain of the trimeric TRIM5α isoform, formed dimers and some higher-order species but few or no trimers. TRIMCyp, in which the B30.2 domain is replaced by a cyclophilin A moiety (20, 28), formed some trimers but mostly a higher-order entity, possibly a dimer of trimers.

The capsid protein of retroviruses determines susceptibility to restriction by a particular TRIM5α protein (2, 9, 15, 21, 22, 34). Thus, the retroviral capsid likely binds TRIM5α, a model supported by the observation that virus-like particles with mature capsids can compete for restriction factors in the target cell (1-3, 19). Retroviral capsids are composed of hexamers that, through dimeric contacts, assemble into large arrays (5, 16, 18). Retroviral capsids, although organized into these large assemblies, are intrinsically asymmetric. Retroviral capsids thus contain imperfect twofold- and threefold-symmetry axes. Interestingly, cryoelectron microscope studies of the HIV-1 capsid have revealed two types of holes in the capsid surface: a roughly cylindrical hole formed at the center of the hexameric ring and a trilobed hole flanked by the spokes of the hexamers (Fig. 4) (16). Both holes are centered at threefold pseudosymmetry axes and could serve as potential TRIM5α binding sites. In both proposed modes of TRIM5α binding (Fig. 4), each of the lobes of the trilobed pocket accommodates a TRIM5α B30.2 domain, which has been implicated in the determination of TRIM5 antiviral potency (27, 33, 38). Some evidence appears to favor the trilobed hole as a TRIM5α binding site. Cyclophilin A, which has been reported to modulate TRIM5α-mediated restriction (9, 13, 21, 30, 35), binds the HIV-1 capsid near the threefold axis associated with the trilobed holes (4, 36). Moreover, the location of amino acid changes in the HIV-1 capsid that influence susceptibility to TRIM5α restriction (9, 13, 15, 21) is consistent with a model in which a trimeric TRIM5α protein binds in the trilobed hole. Future studies will test the validity of these models of TRIM5α-capsid interaction.

FIG. 4.

Possible modes for binding a retroviral capsid with a trimeric TRIM5α protein. The cryoelectron tomographic reconstruction of the HIV-1 capsid reported by Li et al. (16) is shown. Two holes on the capsid surface, each centered on threefold axes of pseudosymmetry, exist. A nearly cylindrical hole is formed by the ring of hexameric capsid proteins (blue). A possible binding mode in this region is depicted by the yellow ovoids, each representing a TRIM5α B30.2 domain. In this binding mode, the trimeric axis of TRIM5α would intersect the capsid surface at the position indicated by the yellow dot. A second possible binding mode involves the trilobed hole formed by three adjacent capsid hexamers (shaded red). A possible binding mode of three TRIM5α B30.2 domains (green) in this trilobed hole is shown. In this binding mode, the TRIM5α trimeric axis would intersect the capsid surface at the position indicated by the green dot.