The B30.2/SPRY domain is present in many proteins, including various members of the tripartite motif (TRIM) protein family such as TRIM5α, which mediates innate intracellular resistance to retroviruses in several primate species. This resistance is dependent on the integrity of the B30.2 domain that evolves rapidly in primates and exhibits species-specific anti-viral activity. TRIM22 is another positively selected TRIM gene. Particularly, the B30.2 domain shows rapid evolution in the primate lineage and recently published data indicate an anti-viral function of TRIM22. We show here that human and rhesus TRIM22 localise to different subcellular compartments and that this difference can be assigned to the positively selected B30.2 domain. Moreover, we could demonstrate that amino acid changes in two variable loops (VL1 and VL3) are responsible for the different subcellular localisations.
TRIM22; Retrovirus; B30.2 domain
The tripartite motif 5α protein (TRIM5α) is one of several factors expressed by mammalian cells that inhibit retrovirus replication. Human TRIM5α (huTRIM5α) inhibits infection by N-tropic murine leukemia virus (N-MLV) but is inactive against human immunodeficiency virus type 1 (HIV-1). However, we show that replacement of a small segment in the carboxy-terminal B30.2/SPRY domain of huTRIM5α with its rhesus macaque counterpart (rhTRIM5α) endows it with the ability to potently inhibit HIV-1 infection. The B30.2/SPRY domain and an additional domain in huTRIM5α, comprising the amino-terminal RING and B-box components of the TRIM motif, are required for N-MLV restriction activity, while the intervening coiled-coil domain is necessary and sufficient for huTRIM5α multimerization. Truncated huTRIM5α proteins that lack either or both the N-terminal RING/B-Box or the C-terminal B30.2/SPRY domain form heteromultimers with full-length huTRIM5α and are dominant inhibitors of its N-MLV restricting activity, suggesting that homomultimerization of intact huTRIM5α monomers is necessary for N-MLV restriction. However, localization in large cytoplasmic bodies is not required for inhibition of N-MLV by huTRIM5α or for inhibition of HIV-1 by chimeric or rhTRIM5α.
Tripartite motif (TRIM) proteins are composed of RING, B-box 2, and coiled coil domains. Some TRIM proteins, such as TRIM5α, also possess a carboxy-terminal B30.2(SPRY) domain and localize to cytoplasmic bodies. TRIM5α 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 TRIM5α was analyzed by amplifying, cloning, and sequencing nonhuman primate TRIM5 orthologs. Lineage-specific expansion and sequential duplication occurred in the TRIM5α 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 TRIM5α 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 TRIM5α in innate immunity against retroviruses.
Tripartite motif 5α (TRIM5α) restricts some retroviruses, including human immunodeficiency virus type 1 (HIV-1), from infecting the cells of particular species. TRIM5α is a member of the TRIM family of proteins, which contain RING, B-box, coiled-coil (CC), and, in some cases, B30.2(SPRY) domains. Here we investigated the abilities of domains from TRIM proteins (TRIM6, TRIM34, and TRIM21) that do not restrict HIV-1 infection to substitute for the domains of rhesus monkey TRIM5α (TRIM5αrh). The RING, B-box 2, and CC domains of the paralogous TRIM6 and TRIM34 proteins functionally replaced the corresponding TRIM5αrh domains, allowing HIV-1 restriction. By contrast, similar chimeras containing the components of TRIM21, a slightly more distant relative of TRIM5, did not restrict HIV-1 infection. The TRIM21 B-box 2 domain and its flanking linker regions contributed to the functional defectiveness of these chimeras. All of the chimeric proteins formed trimers. All of the chimeras that restricted HIV-1 infection bound the assembled HIV-1 capsid complexes. These results indicate that heterologous RING, B-box 2, and CC domains from related TRIM proteins can functionally substitute for TRIM5αrh domains.
The TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection.
Here we defined the entire human TRIM family and also identified the TRIM sets of other vertebrate (mouse, rat, dog, cow, chicken, tetraodon, and zebrafish) and invertebrate species (fruitfly, worm, and ciona). By means of comparative analyses we found that, after assembly of the tripartite motif in an early metazoan ancestor, few types of C-terminal domains have been associated with this module during evolution and that an important increase in TRIM number occurred in vertebrate species concomitantly with the addition of the SPRY domain. We showed that the human TRIM family is split into two groups that differ in domain structure, genomic organization and evolutionary properties. Group 1 members present a variety of C-terminal domains, are highly conserved among vertebrate species, and are represented in invertebrates. Conversely, group 2 is absent in invertebrates, is characterized by the presence of a C-terminal SPRY domain and presents unique sets of genes in each mammal examined. The generation of independent sets of group 2 genes is also evident in the other vertebrate species. Comparing the murine and human TRIM sets, we found that group 1 and 2 genes evolve at different speeds and are subject to different selective pressures.
We found that the TRIM family is composed of two groups of genes with distinct evolutionary properties. Group 2 is younger, highly dynamic, and might act as a reservoir to develop novel TRIM functions. Since some group 2 genes are implicated in innate immune response, their evolutionary features may account for species-specific battles against viral infection.
The tripartite motif (TRIM) proteins are a family of more than 70 members in human. However, only a few of them have been well studied. The TRIM proteins contain the conserved RING, B-box, coiled-coil, and SPRY domains, most of which are involved in protein ubiquitination. TRIM38 is a member of the TRIM protein family, which we studied in more detail here as its functions are largely unknown.
Our study shows that, similar to other TRIM family members, TRIM38 is localized in the cytoplasm. TRIM38 increases ubiquitination of other cellular proteins and catalyzes self-ubiquitination. TRIM38 also promotes K63- and K48-linked ubiquitination of cellular proteins. An intact RING domain is important for the functions of TRIM38. In addition, enterovirus 71 infection induces TRIM38 degradation.
Our observations demonstrate that TRIM38 has E3 ubiquitin ligase activity and can be degraded during virus infection. These findings may provide insight into innate immune signaling pathways.
Retroviruses encounter dominant postentry restrictions in cells of particular species. Human immunodeficiency virus type 1 (HIV-1) is blocked in the cells of Old World monkeys by TRIM5α, a tripartite motif (TRIM) protein composed of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. Rhesus monkey TRIM5α (TRIM5αrh) more potently blocks HIV-1 infection than human TRIM5α (TRIM5αhu). Here, by studying chimeric TRIM5α proteins, we demonstrate that the major determinant of anti-HIV-1 potency is the B30.2(SPRY) domain. Analysis of species-specific variation in TRIM5α has identified three variable regions (v1, v2, and v3) within the B30.2 domain. The TRIM5α proteins of Old World primates exhibit expansion, duplication, and residue variation specifically in the v1 region. Replacement of three amino acids in the N terminus of the TRIM5αhu B30.2 v1 region with the corresponding TRIM5αrh residues resulted in a TRIM5α molecule that restricted HIV-1 nearly as efficiently as wild-type TRIM5αrh. Surprisingly, a single-amino-acid change in this region of TRIM5αhu allowed potent restriction of simian immunodeficiency virus, a phenotype not observed for either wild-type TRIM5αhu or TRIM5αrh. Some of the chimeric TRIM5α proteins that are >98% identical to the human protein yet mediate a strong restriction of HIV-1 infection may have therapeutic utility. These observations implicate the v1 variable region of the B30.2(SPRY) domain in TRIM5αrh antiviral potency.
The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5α has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA). We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3′ splice site upstream of exon 7, which may prevent or reduce expression of the α-isoform. All pig-tailed macaques (M. nemestrina) screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101) of rhesus macaques (M. mulatta). The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares ∼80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a remarkable example of convergent evolution. Based on the presence of the CypA insertion in separate macaque lineages, and its absence from sooty mangabeys, we estimate that the Macaca TRIM5-CypA variant appeared 5–10 million years ago in a common ancestor of the Asian macaques. Whether the formation of novel genes through alternative splicing has played a wider role in the evolution of the TRIM family remains to be investigated.
The TRIM5 gene encodes TRIM5α, a protein that blocks infection of the cell by retroviruses. We previously found that the TRIM5α protein of old world monkeys was highly polymorphic. Here, we describe a substitution in a highly conserved, non-coding element normally required for correct splicing of TRIM5α messenger RNA. While it is difficult to prove positive selection for a non-coding change, the frequency of this mutation in two different species of Asian monkeys (Macaca sp) raised the possibility that the mutation was once evolutionarily advantageous. As it turns out, monkeys carrying this substitution also carry a nearby cyclophilin-A (CypA) pseudogene, and these individuals express chimeric mRNA encoding a fusion between the TRIM5 and CypA sequences. Thus, the mutation, which interferes with expression of the normal TRIM5α protein, instead contributes to expression of a novel protein. Remarkably, this is the second example of the appearance of a TRIM5/CypA chimera during primate evolution, the other having occurred in a new world monkey lineage (Aotus sp). Cellular CypA binds to the capsid proteins of several lentiviruses, and we believe that TRIM5-CypA proteins were at one time selected for the ability to block infection by retroviral pathogens, possibly related to modern lentiviruses.
TRIM5α is a member of the tripartite motif family of proteins that restricts retroviral infection in a species-specific manner. The restriction requires an interaction between the viral capsid lattice and the B30.2/SPRY domain of TRIM5α. Previously, we determined that two SUMO interacting motifs (SIMs) present in the B30.2/SPRY domain of human TRIM5α (huTRIM5α) were important for the restriction of N-tropic Murine Leukemia Virus. Here, we examined whether SUMO expression and the SIM1 and SIM2 motifs in rhesus monkey TRIM5α (rhTRIM5α) are similarly important for Human Immunodeficiency Type 1 (HIV-) restriction.
We found that mutation of SIM1 and SIM2 of rhTRIM5α abolished the restriction of HIV-1 virus. Further, knockdown of SUMO-1 in rhTRIM5α expressing cells abolished restriction of HIV-1. These results may be due, in part, to the ability of SUMO-1 to stabilize rhTRIM5α protein expression, as SUMO-1 knockdown increased rhTRIM5α turnover and the mutations in SIM1 and SIM2 led to more rapid degradation than the wild type protein. The NF-κB signaling ability of rhTRIM5α was also attenuated by SUMO-1 knockdown. Finally, upon inhibition of CRM1-dependent nuclear export with Leptomycin B (LMB), wild type rhTRIM5α localized to SUMO-1 bodies in the nucleus, while the SIM1 and SIM2 mutants did not localize to SUMO-1.
Our results suggest that the rhTRIM5α B30.2/SPRY domain is not only important for the recognition of the HIV-1 CA, but it is also important for its association with SUMO-1 or SUMO-1 modified proteins. These interactions help to maintain TRIM5α protein levels and its nuclear localization into specific nuclear bodies.
SUMO-1; SIM; rhTRIM5α; HIV-1
The homeodomain transcription factor Phox2b is one of the key determinants involved in the development of noradrenergic (NA) neurons in both the central nervous system (CNS) and the peripheral nervous system (PNS). Using yeast two-hybrid screening, we isolated a Phox2b interacting protein, Trim11, which belongs to TRIM (Tripartite motif) or RBCC proteins family, and contains a RING domain, B-boxes, a coiled-coil domain, and the B30.2/SPRY domain. Protein-protein interaction assays showed that Phox2b was able to physically interact with Trim11. The B30.2/SPRY domain of Trim11 was required for the interaction with Phox2b. Expression of Phox2b and Trim11 was detected in the sympathetic ganglia (SG) of mouse embryos. Forced expression of Trim11 with Phox2b further increased mRNA levels of dopamine β-hydroxylase (DBH) gene in primary avian neural crest stem cell (NCSC) culture. This study suggests a potential role for Trim11 in the specification of NA phenotype by interaction with Phox2b.
Phox2b; Trim11; yeast two-hybrid screening; protein-protein interaction; noradrenergic neuron; neural crest stem cell
TRIM5α is a tripartite motif (TRIM) protein that consists of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. The TRIM5αrh protein from rhesus monkeys recognizes the human immunodeficiency virus type 1 (HIV-1) capsid as it enters the host cell and blocks virus infection prior to reverse transcription. HIV-1-restricting ability can be eliminated by disruption of the B-box 2 domain. Changes in the TRIM5αrh B-box 2 domain have been associated with alterations in TRIM5αrh turnover, the formation of cytoplasmic bodies and higher-order oligomerization. We present here the nuclear magnetic resonance structure of the TRIM5 B-box 2 domain and identify an unusual hydrophobic patch (cluster 1) on the domain surface. Alteration of cluster 1 or the flanking arginine 121 resulted in various degrees of inactivation of HIV-1 restriction, in some cases depending on compensatory changes in other nearby charged residues. For this panel of TRIM5αrh B-box 2 mutants, inhibition of HIV-1 infection was strongly correlated with higher-order self-association and binding affinity for capsid complexes but not with TRIM5αrh half-life or the formation of cytoplasmic bodies. Thus, promoting cooperative TRIM5αrh interactions with the HIV-1 capsid represents a major mechanism whereby the B-box 2 domain potentiates HIV-1 restriction.
The antagonistic interaction with host restriction proteins is a major driver of evolutionary change for viruses. We previously reported that polymorphisms of the TRIM5α B30.2/SPRY domain impacted the level of SIVsmm viremia in rhesus macaques. Viremia in macaques homozygous for the non-restrictive TRIM5α allele TRIM5Q was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5TFP/TFP or TRIM5Cyp/TFP. Using this model, we observed that despite an early impact on viremia, SIVsmm overcame TRIM5α restriction at later stages of infection and that increasing viremia was associated with specific amino acid substitutions in capsid. Two amino acid substitutions (P37S and R98S) in the capsid region were associated with escape from TRIM5TFP restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5Cyp. Introduction of these mutations into the original SIVsmE543 clone not only resulted in escape from TRIM5α restriction in vitro but the P37S and R98S substitutions improved virus fitness in macaques with homozygous restrictive TRIMTFP alleles in vivo. Similar substitutions were observed in other SIVsmm strains following transmission and passage in macaques, collectively providing direct evidence that TRIM5α exerts selective pressure on the cross-species transmission of SIV in primates.
Human immunodeficiency virus (HIV) resulted from the transmission of simian immunodeficiency viruses (SIV) from nonhuman primates followed by adaptation and expansion as a pandemic in humans. This required the virus to overcome a variety of intrinsic host restriction factors in humans in order to replicate efficiently. Similarly, SIV encounters restriction factors upon cross-species transmission between nonhuman primates, specifically from a natural host species such as sooty mangabey monkeys to rhesus macaques. Previously we observed significant differences in the levels of virus replication of SIV among rhesus macaques due to subtle differences in one of these restriction factors, TRIM5 among individual macaques. Although a restrictive version of TRIM5 resulted in lower viremia, we also observed that the virus spontaneously mutated in the viral capsid gene and that these mutations were associated with escape from TRIM5 restriction. In the present study, we found that introduction of these escape mutations into the parental virus confers resistance to TRIM5 both in tissue culture and in macaques. These studies provide direct evidence that TRIM5 is a critical factor influencing the cross-species transmission of SIV in primates.
Like all tripartite motif (TRIM) proteins, the retroviral restriction factor TRIM5α consists of RING, B-box 2 and coiled-coil domains, with a C-terminal B30.2(SPRY) domain. Although structures have been determined for some individual TRIM domains, the structure of an intact TRIM protein is unknown.
Here, we express and characterize a protease-resistant 29-kD core fragment containing the B-box 2, coiled coil and adjacent linker (L2) region of TRIM5α. This BCCL2 protein formed dimers and higher-order oligomers in solution. Approximately 40% of the BCCL2 secondary structure consisted of alpha helices. Partial loss of alpha-helical content and dissociation of dimers occurred at 42°C, with the residual alpha helices remaining stable up to 80°C.
These results indicate that the B-box 2, coiled-coil and linker 2 regions of TRIM5α form a core dimerization motif that exhibits a high level of alpha-helical content.
In mammals, the members of the tripartite motif (TRIM) protein family are involved in various cellular processes including innate immunity against viral infection. Viruses exert strong selective pressures on the defense system. Accordingly, antiviral TRIMs have diversified highly through gene expansion, positive selection and alternative splicing. Characterizing immune TRIMs in other vertebrates may enlighten their complex evolution.
We describe here a large new subfamily of TRIMs in teleosts, called finTRIMs, identified in rainbow trout as virus-induced transcripts. FinTRIMs are formed of nearly identical RING/B-box regions and C-termini of variable length; the long variants include a B30.2 domain. The zebrafish genome harbors a striking diversity of finTRIMs, with 84 genes distributed in clusters on different chromosomes. A phylogenetic analysis revealed different subsets suggesting lineage-specific diversification events. Accordingly, the number of fintrim genes varies greatly among fish species. Conserved syntenies were observed only for the oldest fintrims. The closest mammalian relatives are trim16 and trim25, but they are not true orthologs. The B30.2 domain of zebrafish finTRIMs evolved under strong positive selection. The positions under positive selection are remarkably congruent in finTRIMs and in mammalian antiviral TRIM5α, concentrated within a viral recognition motif in mammals. The B30.2 domains most closely related to finTRIM are found among NOD-like receptors (NLR), indicating that the evolution of TRIMs and NLRs was intertwined by exon shuffling.
The diversity, evolution, and features of finTRIMs suggest an important role in fish innate immunity; this would make them the first TRIMs involved in immunity identified outside mammals.
The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline-1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity.
The TRIM5 family of proteins contains a RING domain, one or two B boxes, and a coiled-coil domain. The TRIM5α isoform also encodes a C-terminal B30.2(SPRY) domain, differences within which define the breadth and potency of TRIM5α-mediated retroviral restriction. Because Macaca nemestrina animals are susceptible to some human immunodeficiency virus (HIV) isolates, we sought to determine if differences exist in the TRIM5 gene and transcripts of these animals. We identified a two-nucleotide deletion (Δ2) in the transcript at the 5′ terminus of exon 7 in all M. nemestrina TRIM5 cDNA clones examined. This frameshift results in a truncated protein of 300 amino acids lacking the B30.2(SPRY) domain, which we have named TRIM5θ. This deletion is likely due to a single nucleotide polymorphism that alters the 3′ splice site between intron 6 and exon 7. In some clones, a deletion of the entire 27-nucleotide exon 7 (Δexon7) resulted in the restoration of the TRIM5 open reading frame and the generation of another novel isoform, TRIM5η. There are 18 amino acid differences between M. nemestrina TRIM5η and Macaca mulatta TRIM5α, some of which are at or near locations previously shown to affect the breadth and potency of TRIM5α-mediated restriction. Infectivity assays performed on permissive CrFK cells stably transduced with TRIM5η or TRIM5θ show that these isoforms are incapable of restricting either HIV type 1 (HIV-1) or simian immunodeficiency virus infection. The expression of TRIM5 alleles incapable of restricting HIV-1 infection may contribute to the previously reported increased susceptibility of M. nemestrina to HIV-1 infection in vivo.
Trim33 (Tif1γ, ectodermin, moonshine), a member of the TIF1 family of transcriptional coactivators and corepressors, is a large nuclear protein that contains an N-terminal tripartite (Trim) domain composed of a RING domain, two B-box domains and a coiled coil domain. It has been suggested that Trim33 (Ectodermin) mediates ectodermal induction in the Xenopus by functioning as a Smad4 ubiquitin ligase, while in the zebrafish Trim33 (moonshine) has been reported to act as a R-Smad binding protein in induction of erythroid differentiation. Since the developmental role of Trim33 in mammals is currently unknown, we generated mice carrying the conditional Trim33 (Trim33FX) allele by flanking exons 2–4 encoding most of the functionally critical N-terminal tripartite domain by loxP sites. We confirmed the null genotype by using the EIIa-Cre transgenic approach to create mice that lack exons 2–4. Embryos deficient in Trim33 die during early somitogenesis, demonstrating that Trim33 plays an important non-redundant role in mammalian embryonic development.
It is of great interest to understand the molecular details of the pathways that constitute species barriers to viral infection. The tripartite motif protein TRIM5α has emerged as an important mediator of species-specific retroviral replication and innate immunity. This review considers the role of TRIM5α as an antiviral protein in mammals. The methods used to identify species-specific restriction to retroviral infection, and the identification of TRIM5α itself, are outlined. TRIM5α mediates an early postentry block to sensitive retroviral infection, usually before viral DNA synthesis. Results from mutational analysis of TRIM5α and their contribution to a mechanistic model for TRIM5α antiviral activity are discussed. The antiviral role of other TRIM proteins is considered, as is the role of TRIM5α cytoplasmic bodies.
The tripartite motif (TRIM) protein, TRIM5α, is an endogenous factor in primates that recognizes the capsids of certain retroviruses after virus entry into the host cell. TRIM5α promotes premature uncoating of the capsid, thus blocking virus infection. Low levels of expression and tendencies to aggregate have hindered the biochemical, biophysical, and structural characterization of TRIM proteins. Here, a chimeric TRIM5α protein (TRIM5Rh-21R) with a RING domain derived from TRIM21 was expressed in baculovirus-infected insect cells and purified. Although a fraction of the TRIM5Rh-21R protein formed large aggregates, soluble fractions of the protein formed oligomers (mainly dimers), exhibited a protease-resistant core, and contained a high percentage of helical secondary structure. Cross-linking followed by negative staining and electron microscopy suggested a globular structure. The purified TRIM5Rh-21R protein displayed E3-ligase activity in vitro and also self-ubiquitylated in the presence of ubiquitin-activating and -conjugating enzymes. The purified TRIM5Rh-21R protein specifically associated with human immunodeficiency virus type 1 capsid-like complexes; a deletion within the V1 variable region of the B30.2(SPRY) domain decreased capsid binding. Thus, the TRIM5Rh-21R restriction factor can directly recognize retroviral capsid-like complexes in the absence of other mammalian proteins.
The mammalian tripartite motif protein, TRIM5α, recognizes retroviral capsids entering the cytoplasm and blocks virus infection. Depending on the particular TRIM5α protein and retrovirus, complete disruption of the TRIM5α RING domain decreases virus-restricting activity to various degrees. TRIM5α exhibits RING domain-dependent E3 ubiquitin ligase activity, but the specific role of this activity in viral restriction is unknown. We created a panel of African green monkey TRIM5α (TRIM5αAGM) mutants, many of which are specifically altered in RING domain E3 ubiquitin ligase function, and characterized the phenotypes of these mutants with respect to restriction of simian and human immunodeficiency viruses (SIVmac and HIV-1, respectively). TRIM5αAGM ubiquitin ligase activity was essential for both the accelerated disassembly of SIVmac capsids and the disruption of reverse transcription. The levels of SIVmac particulate capsids in the cytosol of target cells expressing the TRIM5α variants strongly correlated with the levels of viral late reverse transcripts. RING-mediated ubiquitylation and B30.2(SPRY) domain-determined capsid binding independently contributed to the potency of SIVmac restriction by TRIM5αAGM. In contrast, TRIM5α proteins attenuated in RING ubiquitin ligase function still accelerated HIV-1 capsid disassembly, inhibited reverse transcription, and blocked infection. Replacement of the helix-4/5 loop in the SIVmac capsid with the corresponding region of the HIV-1 capsid diminished the dependence of restriction on TRIM5α RING function. Thus, ubiquitylation mediated by the RING domain of TRIM5αAGM is essential for blocking SIVmac infection at the stage of capsid uncoating.
Pathogenic viral infections have exerted selection pressure on their hosts to evolve cellular antiviral inhibitors referred to as restriction factors. Examples of such molecules are APOBEC3G, APOBEC3F and TRIM5α. APOBEC3G and APOBEC3F are cytidine deaminases that are able to strongly inhibit retroviral replication by at least two mechanisms. They are counteracted by the lentiviral Vif protein. TRIM5α binds to sensitive, incoming retroviruses via its C-terminal PRY/SPRY domain and rapidly recruits them to the proteasome before significant viral DNA synthesis can occur. Both of these proteins robustly block retroviral replication in a species-specific way. It remains an open but important question as to whether innate restriction factors such as these can be harnessed to inhibit HIV-1 replication in humans.
Human immunodeficiency virus type 1 (HIV-1) infects humans and chimpanzees but not old world monkeys (OWMs) such as the rhesus monkey (Rh) and cynomolgus monkey (CM). HIV-1 efficiently enters cells of OWMs but encounters a block before reverse transcription. This narrow host range is attributed to a barrier in the host cell. In 2004, the screening of a Rh cDNA library identified tripartite motif 5α (TRIM5α) as a cellular antiviral factor. TRIM5α is one of splicing variants produced by TRIM5 gene and TRIM5 proteins are members of the TRIM family containing RING, B-box 2, and coiled-coil domains. The RING domain is frequently found in E3 ubiquitin ligase and TRIM5α is degraded via the ubiquitin–proteasome-dependent pathway. Among TRIM5 splicing variants, TRIM5α alone has an additional C-terminal PRYSPRY (B30.2) domain. Previous studies have shown that sequence variation in variable regions of the PRYSPRY domain among different monkey species affects species-specific retrovirus infection, while amino acid sequence differences in the viral capsid protein determine viral sensitivity to restriction. TRIM5α recognizes the multimerized capsid proteins (viral core) of an incoming virus by its PRYSPRY domain and is thus believed to control retroviral infection. There are significant intraspecies variations in the Rh-TRIM5 gene. It has also been reported that some Rh and CM individuals have retrotransposed cyclophilin A open reading frame in the TRIM5 gene, which produces TRIM5–cyclophilin A fusion protein (TRIMCyp). TRIMCyp, which was originally identified as an anti-HIV-1 factor of New World owl monkeys, is an interesting example of the gain of a new function by retrotransposition. As different TRIM5 genotypes of Rh showed different levels of simian immunodeficiency virus replication in vivo, the TRIM5 genotyping is thought to be important in acquired immunodeficiency syndrome monkey models.
TRIM5α; TRIMCyp; HIV-1; HIV-2; SIV; rhesus monkey; cynomolgus monkey
The family of tripartite-motif (TRIM) proteins are involved in diverse cellular processes, but are often characterized by critical protein–protein interactions necessary for their function. TRIM16 is induced in different cancer types, when the cancer cell is forced to proceed down a differentiation pathway. We have identified TRIM16 as a DNA-binding protein with histone acetylase activity, which is required for the retinoic acid receptor β2 transcriptional response in retinoid-treated cancer cells. In this study, we show that overexpressed TRIM16 reduced neuroblastoma cell growth, enhanced retinoid-induced differentiation and reduced tumourigenicity in vivo. TRIM16 was only expressed in the differentiated ganglion cell component of primary human neuroblastoma tumour tissues. TRIM16 bound directly to cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells. TRIM16 reduced cell motility and this required downregulation of vimentin. Retinoid treatment and enforced overexpression caused TRIM16 to translocate to the nucleus, and bind to and downregulate nuclear E2F1, required for cell replication. This study, for the first time, demonstrates that TRIM16 acts as a tumour suppressor, affecting neuritic differentiation, cell migration and replication through interactions with cytoplasmic vimentin and nuclear E2F1 in neuroblastoma cells.
TRIM16; EBBP; neuroblastoma; vimentin; E2F1; tumour suppressor
Tripartite motif proteins (TRIM) constitute a large family of proteins containing a RING-Bbox-Coiled Coil motif followed by different C-terminal domains. Involved in ubiquitination, TRIM proteins participate in many cellular processes including antiviral immunity. The TRIM family is ancient and has been greatly diversified in vertebrates and especially in fish. We analyzed the complete sets of trim genes of the large zebrafish genome and of the compact pufferfish genome. Both contain three large multigene subsets - adding the hsl5/trim35-like genes (hltr) to the ftr and the btr that we previously described - all containing a B30.2 domain that evolved under positive selection. These subsets are conserved among teleosts. By contrast, most human trim genes of the other classes have only one or two orthologues in fish. Loss or gain of C-terminal exons generated proteins with different domain organizations; either by the deletion of the ancestral domain or, remarkably, by the acquisition of a new C-terminal domain. Our survey of fish trim genes in fish identifies subsets with different evolutionary dynamics. trims encoding RBCC-B30.2 proteins show the same evolutionary trends in fish and tetrapods: they evolve fast, often under positive selection, and they duplicate to create multigenic families. We could identify new combinations of domains, which epitomize how new trim classes appear by domain insertion or exon shuffling. Notably, we found that a cyclophilin-A domain replaces the B30.2 domain of a zebrafish fintrim gene, as reported in the macaque and owl monkey antiretroviral TRIM5α. Finally, trim genes encoding RBCC-B30.2 proteins are preferentially located in the vicinity of MHC or MHC gene paralogues, which suggests that such trim genes may have been part of the ancestral MHC.
The antiviral factor tripartite interaction motif 5α (Trim5α) restricts a broad range of retroviruses in a species-specific manner. Although human Trim5α is unable to block HIV-1 infection in human cells, a modest inhibition of HIV-1 replication has been reported. Recently two polymorphisms in the Trim5 gene (H43Y and R136Q) were shown to affect the antiviral activity of Trim5α in vitro. In this study, participants of the Amsterdam Cohort studies were screened for polymorphisms at amino acid residue 43 and 136 of the Trim5 gene, and the potential effects of these polymorphisms on the clinical course of HIV-1 infection were analyzed. In agreement with the reported decreased antiviral activity of Trim5α that contains a Y at amino acid residue 43 in vitro, an accelerated disease progression was observed for individuals who were homozygous for the 43Y genotype as compared to individuals who were heterozygous or homozygous for the 43H genotype. A protective effect of the 136Q genotype was observed but only after the emergence of CXCR4-using (X4) HIV-1 variants and when a viral load of 104.5 copies per ml plasma was used as an endpoint in survival analysis. Interestingly, naive CD4 T cells, which are selectively targeted by X4 HIV-1, revealed a significantly higher expression of Trim5α than memory CD4 T cells. In addition, we observed that the 136Q allele in combination with the −2GG genotype in the 5′UTR was associated with an accelerated disease progression. Thus, polymorphisms in the Trim5 gene may influence the clinical course of HIV-1 infection also underscoring the antiviral effect of Trim5α on HIV-1 in vivo.
The clinical course of HIV-1 infection is highly variable between individuals, and host genetic variations may at least account for part of these differences. Recently two single nucleotide polymorphisms in the tripartite interaction motif 5 gene (Trim5) have been reported to affect the antiviral activity of the Trim5α protein. Here we analyzed the effect of these polymorphisms on the clinical course of HIV-1 infection in participants of the Amsterdam Cohort studies. We observed an accelerated disease progression for individuals who were homozygous for the 43Y genotype that has been associated with a decreased antiviral activity of Trim5α in vitro. The 136Q genotype has in vitro been associated with a slightly higher anti-HIV-1 activity. We observed a protective effect of the 136Q genotype only after the emergence of CXCR4-using HIV-1 variants using viral load above 104.5 copies per ml plasma as an endpoint in survival analysis. These results suggest that genetic variations in the Trim5 gene may influence the clinical course of HIV-1 infection and confirm a role of Trim5α on HIV-1 in vivo.