The rhesus monkey intrinsic immunity factor TRIM5alpha(rh) recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5alpha proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we developed methods for expressing and purifying an active chimeric TRIM5alpha(rh) protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5alpha proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5alpha proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5alpha proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins.

FIG. TRIM5-21R binds CA-NC assemblies. (A) Schematic summary (above) and SDS-PAGE/Coomassie analyses (below) of purified full length dimeric TRIM5-21R (lane 3, denoted WT), a dimeric TRIM5-21R construct missing part of the V1 loop (lane 2, denoted deltaV1) and a monomeric TRIM5-21R construct missing the entire SPRY domain (lane 1, denoted deltaSPRY). (B) TRIM5-21R protein binding to CA-NC/DNA assemblies. Monomeric (M) or dimeric (D) TRIM5-21R (WT, lanes 3,4 and 7,8), TRIM5-21RdeltaV1 (deltaV1, lanes 2 and 6) or TRIM5-21RdeltaSPRY (deltaSPRY, lanes 1 and 5) proteins were incubated alone (lanes 1-4) or with CA-NC/DNA assemblies (lanes 5-8) and then centrifuged through a 70% sucrose cushion to separate CA-NC assemblies and bound proteins (pellet) from unbound and lower molecular weight proteins (supernatant). Western blots show the distribution of TRIM (a-FLAG, panels 1, 3, and 5) and CA-NC a-CA, panels 2, 4, and 6). Note that CA-NC assemblies bound full length TRIM5-21R constructs better than either TRIM5-21RdeltaV1 or TRIM5-21RdeltaSPRY. (C) CA-NC/DNA assemblies bind better to dimeric (D) TRIM5-21R proteins than to monomeric (M) proteins under stringent conditions. Monomeric (M, lanes 2, 4, and 6) or dimeric (D, lanes 1, 3, and 5) proteins were incubated alone (lanes 1 and 2) or with CA-NC/DNA assemblies (lanes 3-6) and then centrifuged through a 70% sucrose cushion to separate CA-NC assemblies and bound proteins (pellet) from unbound and lower molecular weight proteins (supernatant). Western blots show the distribution of TRIM5-21R (a-FLAG, panels 1, 3, and 5) and CA-NC (a-CA, panels 2, 4, and 6). The concentrations of TRIM5-21R proteins in lanes 5 and 6 were 3.33-fold higher than in lanes 3 and 4, and all binding assays were performed at lower protein concentrations than those shown in part (B) Note that under these conditions, CA-NC assemblies bound dimeric TRIM5-21R better than monomeric TRIM5-21R.