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.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 homooligomers; 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 , 1 ϫ complete EDTA-free protease inhibitor [Roche Diagnostics] in PBS) for 45 min at 4°C. Lysates were centrifuged at 14,000 ϫ 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 l of cleared lysates was diluted with PBS plus 1 mM EDTA to a final volume of 400 l. 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 l. 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 ...