Retroviral Restriction Factor TRIM5 Is a Trimer
http://www.100md.com
病菌学杂志 2005年第22期
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115
ABSTRACT
The retrovirus restriction factor TRIM5 targets the viral capsid soon after entry. Here we show that the TRIM5 protein oligomerizes into trimers. The TRIM5 coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5 mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5 to interact with threefold pseudosymmetrical structures on retroviral capsids.
TEXT
TRIM5 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). TRIM5 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 TRIM5 restricts retroviral infection remains unknown.
TRIM5 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 x complete EDTA-free protease inhibitor [Roche Diagnostics] in PBS) for 45 min at 4°C. Lysates were centrifuged at 14,000 x 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 -mercaptoethanol (-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).
TRIM5 isoforms include TRIM5, which consists of the RING, B-box 2, and coiled-coil domains, and TRIM5, which contains an additional C-terminal B30.2(SPRY) domain (26). The wild-type rhesus monkey TRIM5rh 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 -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, TRIM5rh exhibited a dimeric form both without and with GA treatment. Higher-order forms of TRIM5rh were evident after GA cross-linking, although trimers were not a dominant species. As TRIM5rh and TRIM5rh share 300 amino-terminal residues, these results suggest that the TRIM5rh sequences carboxy terminal to residue 300 can significantly affect the oligomerization state of the protein.
The oligomeric states of several TRIM5rh mutants lacking one or more domains was examined. TRIM5rh-HA 297, which consists of the B30.2(SPRY) domain alone, migrated as a monomer even after GA cross-linking (Fig. 1B). TRIM5rh-HA 93, which lacks the RING domain, and TRIM5rh-HA 132, 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 -ME concentrations (Fig. 1B) and was less evident when larger amounts of -ME were included in the sample buffer (Fig. 1C). We suspect that these gel-stable dimers result from artifactual oxidation of exposed TRIM5 cysteines upon cell lysis. Forms consistent with trimers were apparent for both TRIM5rh-HA 93 and TRIM5rh-HA 132 proteins after GA cross-linking (Fig. 1B). Treatment of the samples with higher concentrations of -ME demonstrated that the major higher-order product cross-linked by GA for both proteins was a trimer (Fig. 1C). Thus, the TRIM5rh 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 TRIM5 oligomer were cross-linked into gel-stable trimers suggested that many potential GA-reactive sites exist in the TRIM5 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).
TRIM5 mutants lacking the RING and B-box 2 domains have been shown to associate with wild-type TRIM5 and exert dominant-negative activity on retrovirus restriction (13a, 24a). Coexpression of the wild-type TRIM5rh-HA and TRIM5rh-HA 132 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 TRIM5 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 TRIM5 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.
We have demonstrated that the TRIM5 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 TRIM5rh are sufficient for trimerization. As expected from studies of other TRIM proteins (23, 24, 26), the TRIM5rh coiled coil apparently contributes to homo-oligomerization. This conclusion is supported by our observation that TRIM5rh-HA 132 can form trimers, whereas TRIM5rh-HA 297 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. TRIM5rh, which lacks the B30.2(SPRY) domain of the trimeric TRIM5 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 TRIM5 protein (2, 9, 15, 21, 22, 34). Thus, the retroviral capsid likely binds TRIM5, 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 TRIM5 binding sites. In both proposed modes of TRIM5 binding (Fig. 4), each of the lobes of the trilobed pocket accommodates a TRIM5 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 TRIM5 binding site. Cyclophilin A, which has been reported to modulate TRIM5-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 TRIM5 restriction (9, 13, 15, 21) is consistent with a model in which a trimeric TRIM5 protein binds in the trilobed hole. Future studies will test the validity of these models of TRIM5-capsid interaction.
ACKNOWLEDGMENTS
We thank Yvette McLaughlin and Sheri Farnum for manuscript preparation.
We acknowledge the support of grants (AI063987 and HL54785) from the National Institutes of Health and a Center for AIDS Research Award (AI28691). We also acknowledge the support of the International AIDS Vaccine Initiative, the Bristol-Myers Squibb Foundation, the William A. Haseltine Foundation for the Arts and Sciences, and the late William F. McCarty-Cooper. H.J. was supported by a fellowship from the Canadian Institutes of Health Research.
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Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115
ABSTRACT
The retrovirus restriction factor TRIM5 targets the viral capsid soon after entry. Here we show that the TRIM5 protein oligomerizes into trimers. The TRIM5 coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5 mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5 to interact with threefold pseudosymmetrical structures on retroviral capsids.
TEXT
TRIM5 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). TRIM5 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 TRIM5 restricts retroviral infection remains unknown.
TRIM5 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 x complete EDTA-free protease inhibitor [Roche Diagnostics] in PBS) for 45 min at 4°C. Lysates were centrifuged at 14,000 x 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 -mercaptoethanol (-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).
TRIM5 isoforms include TRIM5, which consists of the RING, B-box 2, and coiled-coil domains, and TRIM5, which contains an additional C-terminal B30.2(SPRY) domain (26). The wild-type rhesus monkey TRIM5rh 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 -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, TRIM5rh exhibited a dimeric form both without and with GA treatment. Higher-order forms of TRIM5rh were evident after GA cross-linking, although trimers were not a dominant species. As TRIM5rh and TRIM5rh share 300 amino-terminal residues, these results suggest that the TRIM5rh sequences carboxy terminal to residue 300 can significantly affect the oligomerization state of the protein.
The oligomeric states of several TRIM5rh mutants lacking one or more domains was examined. TRIM5rh-HA 297, which consists of the B30.2(SPRY) domain alone, migrated as a monomer even after GA cross-linking (Fig. 1B). TRIM5rh-HA 93, which lacks the RING domain, and TRIM5rh-HA 132, 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 -ME concentrations (Fig. 1B) and was less evident when larger amounts of -ME were included in the sample buffer (Fig. 1C). We suspect that these gel-stable dimers result from artifactual oxidation of exposed TRIM5 cysteines upon cell lysis. Forms consistent with trimers were apparent for both TRIM5rh-HA 93 and TRIM5rh-HA 132 proteins after GA cross-linking (Fig. 1B). Treatment of the samples with higher concentrations of -ME demonstrated that the major higher-order product cross-linked by GA for both proteins was a trimer (Fig. 1C). Thus, the TRIM5rh 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 TRIM5 oligomer were cross-linked into gel-stable trimers suggested that many potential GA-reactive sites exist in the TRIM5 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).
TRIM5 mutants lacking the RING and B-box 2 domains have been shown to associate with wild-type TRIM5 and exert dominant-negative activity on retrovirus restriction (13a, 24a). Coexpression of the wild-type TRIM5rh-HA and TRIM5rh-HA 132 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 TRIM5 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 TRIM5 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.
We have demonstrated that the TRIM5 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 TRIM5rh are sufficient for trimerization. As expected from studies of other TRIM proteins (23, 24, 26), the TRIM5rh coiled coil apparently contributes to homo-oligomerization. This conclusion is supported by our observation that TRIM5rh-HA 132 can form trimers, whereas TRIM5rh-HA 297 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. TRIM5rh, which lacks the B30.2(SPRY) domain of the trimeric TRIM5 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 TRIM5 protein (2, 9, 15, 21, 22, 34). Thus, the retroviral capsid likely binds TRIM5, 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 TRIM5 binding sites. In both proposed modes of TRIM5 binding (Fig. 4), each of the lobes of the trilobed pocket accommodates a TRIM5 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 TRIM5 binding site. Cyclophilin A, which has been reported to modulate TRIM5-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 TRIM5 restriction (9, 13, 15, 21) is consistent with a model in which a trimeric TRIM5 protein binds in the trilobed hole. Future studies will test the validity of these models of TRIM5-capsid interaction.
ACKNOWLEDGMENTS
We thank Yvette McLaughlin and Sheri Farnum for manuscript preparation.
We acknowledge the support of grants (AI063987 and HL54785) from the National Institutes of Health and a Center for AIDS Research Award (AI28691). We also acknowledge the support of the International AIDS Vaccine Initiative, the Bristol-Myers Squibb Foundation, the William A. Haseltine Foundation for the Arts and Sciences, and the late William F. McCarty-Cooper. H.J. was supported by a fellowship from the Canadian Institutes of Health Research.
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