Biochemical characterization of a recombinant TRIM5alpha protein that restricts human immunodeficiency virus type 1 replication.
ABSTRACT 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 have 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.
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ABSTRACT: In addition to the conventional innate and acquired immune responses, complex organisms have evolved an array of dominant, constitutively expressed genes that suppress or prevent viral infections. Two major cellular defenses against infection by retroviruses are the Fv1 and TRIM5 class of inhibitors that target incoming retroviral capsids and the APOBEC3 class of cytidine deaminases that hypermutate and destabilize retroviral genomes. Additional, less well characterized activities also inhibit viral replication. Here, the present understanding of these 'intrinsic' immune mechanisms is reviewed and their role in protection from retroviral infection is discussed.Nature Immunology 12/2004; 5(11):1109-15. · 26.01 Impact Factor
Article: The B30.2(SPRY) domain of the retroviral restriction factor TRIM5alpha exhibits lineage-specific length and sequence variation in primates.[show abstract] [hide abstract]
ABSTRACT: Tripartite motif (TRIM) proteins are composed of RING, B-box 2, and coiled coil domains. Some TRIM proteins, such as TRIM5alpha, also possess a carboxy-terminal B30.2(SPRY) domain and localize to cytoplasmic bodies. TRIM5alpha has recently been shown to mediate innate intracellular resistance to retroviruses, an activity dependent on the integrity of the B30.2 domain, in particular primate species. An examination of the sequences of several TRIM proteins related to TRIM5 revealed the existence of four variable regions (v1, v2, v3, and v4) in the B30.2 domain. Species-specific variation in TRIM5alpha was analyzed by amplifying, cloning, and sequencing nonhuman primate TRIM5 orthologs. Lineage-specific expansion and sequential duplication occurred in the TRIM5alpha B30.2 v1 region in Old World primates and in v3 in New World monkeys. We observed substitution patterns indicative of selection bordering these particular B30.2 domain variable elements. These results suggest that occasional, complex changes were incorporated into the TRIM5alpha B30.2 domain at discrete time points during the evolution of primates. Some of these time points correspond to periods during which primates were exposed to retroviral infections, based on the appearance of particular endogenous retroviruses in primate genomes. The results are consistent with a role for TRIM5alpha in innate immunity against retroviruses.Journal of Virology 06/2005; 79(10):6111-21. · 5.40 Impact Factor
Article: Intracellular versus cell surface assembly of retroviral pseudotypes is determined by the cellular localization of the viral glycoprotein, its capacity to interact with Gag, and the expression of the Nef protein.[show abstract] [hide abstract]
ABSTRACT: Retroviral Gag and Env glycoproteins (GPs) are expressed from distinct cellular areas and need to encounter to interact and assemble infectious particles. Retroviral particles may also incorporate GPs derived from other enveloped viruses via active or passive mechanisms, a process known as "pseudotyping." To further understand the mechanisms of pseudotyping, we have investigated the capacity of murine leukemia virus (MLV) or lentivirus core particles to recruit GPs derived from different virus families: the G protein of vesicular stomatitis virus (VSV-G), the hemagglutinin from an influenza virus, the E1E2 glycoproteins of hepatitis C virus (HCV-E1E2), and the retroviral Env glycoproteins of MLV and RD114 cat endogenous virus. The parameters that influenced the incorporation of viral GPs onto retroviral core particles were (i) the intrinsic cell localization properties of both viral GP and retroviral core proteins, (ii) the ability of the viral GP to interact with the retroviral core, and (iii) the expression of the lentiviral Nef protein. Whereas the hemagglutinin and VSV-G glycoproteins were recruited by MLV and lentivirus core proteins at the cell surface, the HCV and MLV GPs were most likely recruited in late endosomes. In addition, whereas these glycoproteins could be passively incorporated on either retrovirus type, the MLV GP was also actively recruited by MLV core proteins, which, through interactions with the cytoplasmic tail of the latter GP, induced its localization to late endosomal vesicles. Finally, the expression of Nef proteins specifically enhanced the incorporation of the retroviral GPs by increasing their localization in late endosomes.Journal of Biological Chemistry 02/2006; 281(1):528-42. · 4.77 Impact Factor