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1.  Discordant Evolution of the Adjacent Antiretroviral Genes TRIM22 and TRIM5 in Mammals 
PLoS Pathogens  2007;3(12):e197.
TRIM5α provides a cytoplasmic block to retroviral infection, and orthologs encoded by some primates are active against HIV. Here, we present an evolutionary comparison of the TRIM5 gene to its closest human paralogs: TRIM22, TRIM34, and TRIM6. We show that TRIM5 and TRIM22 have a dynamic history of gene expansion and loss during the evolution of mammals. The cow genome contains an expanded cluster of TRIM5 genes and no TRIM22 gene, while the dog genome encodes TRIM22 but has lost TRIM5. In contrast, TRIM6 and TRIM34 have been strictly preserved as single gene orthologs in human, dog, and cow. A more focused analysis of primates reveals that, while TRIM6 and TRIM34 have evolved under purifying selection, TRIM22 has evolved under positive selection as was previously observed for TRIM5. Based on TRIM22 sequences obtained from 27 primate genomes, we find that the positive selection of TRIM22 has occurred episodically for approximately 23 million years, perhaps reflecting the changing pathogenic landscape. However, we find that the evolutionary episodes of positive selection that have acted on TRIM5 and TRIM22 are mutually exclusive, with generally only one of these genes being positively selected in any given primate lineage. We interpret this to mean that the positive selection of one gene has constrained the adaptive flexibility of its neighbor, probably due to genetic linkage. Finally, we find a striking congruence in the positions of amino acid residues found to be under positive selection in both TRIM5α and TRIM22, which in both proteins fall predominantly in the β2-β3 surface loop of the B30.2 domain. Astonishingly, this same loop is under positive selection in the multiple cow TRIM5 genes as well, indicating that this small structural loop may be a viral recognition motif spanning a hundred million years of mammalian evolution.
Author Summary
The intrinsic immunity protein TRIM5α provides a post-entry defense against retroviral infection, which depends on its specific ability to recognize retroviral capsids. TRIM5α has been locked in genetic conflict with retroviruses throughout most of primate evolution, characterized by a higher than expected rate of amino acid change, referred to as positive selection. Here, we find that one of TRIM5's closest human paralogs, TRIM22, has also undergone positive selection in primates. However, we find that its close linkage to TRIM5 has resulted in an anti-correlated pattern of positive selection, with primate lineages generally showing positive selection in either TRIM5 or TRIM22, but not both. Amino acid positions in TRIM22 found to be under positive selection are in remarkable proximity to the “antiviral specificity patch” previously described for TRIM5α. TRIM5 and TRIM22 evolution appears to be equally discordant in other mammals; the cow genome contains an expanded cluster of TRIM5 genes and no TRIM22 gene, while the dog genome encodes TRIM22 but has lost TRIM5. Our analyses highlight TRIM22 as bearing all the evolutionary hallmarks of a candidate intrinsic immunity gene.
doi:10.1371/journal.ppat.0030197
PMCID: PMC2151084  PMID: 18159944
2.  Evolution of a TRIM5-CypA Splice Isoform in Old World Monkeys 
PLoS Pathogens  2008;4(2):e1000003.
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.
Author Summary
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.
doi:10.1371/journal.ppat.1000003
PMCID: PMC2279257  PMID: 18389077
3.  Crystal structure of the TRIM25 B30.2 (PRYSPRY) domain: a key component of antiviral signalling 
The Biochemical journal  2013;456(2):231-240.
TRIM (tripartite motif) proteins primarily function as ubiquitin E3 ligases that regulate the innate immune response to infection. TRIM25 [also known as Efp (oestrogen-responsive finger protein)] has been implicated in the regulation of oestrogen receptor α signalling and in the regulation of innate immune signalling via RIG-I (retinoic acid-inducible gene-I). RIG-I senses cytosolic viral RNA and is subsequently ubiquitinated by TRIM25 at its N-terminal CARDs (caspase recruitment domains), leading to type I interferon production. The interaction with RIG-I is dependent on the TRIM25 B30.2 domain, a protein-interaction domain composed of the PRY and SPRY tandem sequence motifs. In the present study we describe the 1.8 Å crystal structure of the TRIM25 B30.2 domain, which exhibits a typical B30.2/SPRY domain fold comprising two N-terminal α-helices, thirteen β-strands arranged into two β-sheets and loop regions of varying lengths. A comparison with other B30.2/SPRY structures and an analysis of the loop regions identified a putative binding pocket, which is likely to be involved in binding target proteins. This was supported by mutagenesis and functional analyses, which identified two key residues (Asp488 and Trp621) in the TRIM25 B30.2 domain as being critical for binding to the RIG-I CARDs.
doi:10.1042/BJ20121425
PMCID: PMC4012390  PMID: 24015671
B30.2; E3 ligase; interferon; oestrogen-responsive finger protein (Efp); retinoic acid-inducible gene-I (RIG-I); tripartite motif (TRIM); tripartite motif-containing 25 (TRIM25)
4.  Species-Specific Variation in the B30.2(SPRY) Domain of TRIM5α Determines the Potency of Human Immunodeficiency Virus Restriction 
Journal of Virology  2005;79(5):3139-3145.
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.
doi:10.1128/JVI.79.5.3139-3145.2005
PMCID: PMC548447  PMID: 15709033
5.  Genomic analysis of the TRIM family reveals two groups of genes with distinct evolutionary properties 
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2148-8-225
PMCID: PMC2533329  PMID: 18673550
6.  Human Tripartite Motif 5α Domains Responsible for Retrovirus Restriction Activity and Specificity 
Journal of Virology  2005;79(14):8969-8978.
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.
doi:10.1128/JVI.79.14.8969-8978.2005
PMCID: PMC1168745  PMID: 15994791
7.  SUMO-Interacting Motifs of Human TRIM5α are Important for Antiviral Activity 
PLoS Pathogens  2011;7(4):e1002019.
Human TRIM5α potently restricts particular strains of murine leukemia viruses (the so-called N-tropic strains) but not others (the B- or NB-tropic strains) during early stages of infection. We show that overexpression of SUMO-1 in human 293T cells, but not in mouse MDTF cells, profoundly blocks N-MLV infection. This block is dependent on the tropism of the incoming virus, as neither B-, NB-, nor the mutant R110E of N-MLV CA (a B-tropic switch) are affected by SUMO-1 overexpression. The block occurred prior to reverse transcription and could be abrogated by large amounts of restricted virus. Knockdown of TRIM5α in 293T SUMO-1-overexpressing cells resulted in ablation of the SUMO-1 antiviral effects, and this loss of restriction could be restored by expression of a human TRIM5α shRNA-resistant plasmid. Amino acid sequence analysis of human TRIM5α revealed a consensus SUMO conjugation site at the N-terminus and three putative SUMO interacting motifs (SIMs) in the B30.2 domain. Mutations of the TRIM5α consensus SUMO conjugation site did not affect the antiviral activity of TRIM5α in any of the cell types tested. Mutation of the SIM consensus sequences, however, abolished TRIM5α antiviral activity against N-MLV. Mutation of lysines at a potential site of SUMOylation in the CA region of the Gag gene reduced the SUMO-1 block and the TRIM5α restriction of N-MLV. Our data suggest a novel aspect of TRIM5α-mediated restriction, in which the presence of intact SIMs in TRIM5α, and also the SUMO conjugation of CA, are required for restriction. We propose that at least a portion of the antiviral activity of TRIM5α is mediated through the binding of its SIMs to SUMO-conjugated CA.
Author Summary
TRIM5α is an intrinsic immunity protein that provides a post-entry block of retroviral infection, which depends on its specific ability to recognize retroviral capsid (CA). Human TRIM5α is able to recognize and block infection by N-tropic murine leukemia virus (N-MLV) as well as other viruses. The exact mechanism by which TRIM5α exerts its action is still controversial. In this study, we have identified a new aspect of TRIM5α-mediated restriction of N-MLV: the involvement of the SUMO conjugation machinery. SUMO conjugation of MLV CA is an important step during viral infection, and our data suggest that innate immunity takes advantage of this process to restrict viral infection. We show that human TRIM5α protein contains two SUMO interacting motifs (SIMs) that are required for its antiviral activity against N-MLV. We propose that at least a portion of the antiviral activity of TRIM5α is mediated through the binding of its SIMs to SUMO-conjugated CA.
doi:10.1371/journal.ppat.1002019
PMCID: PMC3072370  PMID: 21490953
8.  A Specific Region of 37 Amino Acid Residues in the SPRY (B30.2) Domain of African Green Monkey TRIM5α Determines Species-Specific Restriction of Simian Immunodeficiency Virus SIVmac Infection 
Journal of Virology  2005;79(14):8870-8877.
Human immunodeficiency virus type 1 (HIV-1) efficiently enters cells of Old World monkeys but encounters a block before reverse transcription. This restriction is mediated by a dominant repressive factor. Recently, a member of the tripartite motif (TRIM) family proteins, TRIM5α, was identified as a blocking factor in a rhesus macaque cDNA library. Among Old World monkey cell lines, the African green monkey kidney cell line CV1 is highly resistant to not only HIV-1 but also simian immunodeficiency virus SIVmac infection. We analyzed TRIM5α of CV1 cells and HSC-F cells, a T-cell line from a cynomolgus monkey, and found that both CV1- and HSC-F-TRIM5αs could inhibit CD4-dependent HIV-1 infection, as well as vesicular stomatitis virus glycoprotein-mediated infection. CV1-TRIM5α could also inhibit SIVmac infection, whereas HSC-F-TRIM5α could not. In the SPRY (B30.2) domain of CV1-TRIM5α, there was a 20-amino-acid duplication that was not present in HSC-F-TRIM5α. A chimeric TRIM5α containing 37 amino acid residues from CV1-TRIM5α, which spanned the 20-amino-acid duplication, in the background of HSC-F-TRIM5α fully gained the ability to inhibit SIVmac infection. Conversely, the mutant CV1-TRIM5α lacking the 20-amino-acid duplication completely lost the ability to restrict SIVmac infection. These findings clearly indicated that a specific region of 37 amino acid residues in the SPRY domain of CV1-TRIM5α contained a determinant of species-specific restriction of SIVmac.
doi:10.1128/JVI.79.14.8870-8877.2005
PMCID: PMC1168783  PMID: 15994780
9.  The Interferon Response Inhibits HIV Particle Production by Induction of TRIM22 
PLoS Pathogens  2008;4(2):e1000007.
Treatment of human cells with Type 1 interferons restricts HIV replication. Here we report that the tripartite motif protein TRIM22 is a key mediator. We used transcriptional profiling to identify cellular genes that were induced by interferon treatment and identified TRIM22 as one of the most strongly up-regulated genes. We confirmed, as in previous studies, that TRIM22 over-expression inhibited HIV replication. To assess the role of TRIM22 expressed under natural inducing conditions, we compared the effects of interferon in cells depleted for TRIM22 using RNAi and found that HIV particle release was significantly increased in the knockdown, implying that TRIM22 acts as a natural antiviral effector. Further studies showed that TRIM22 inhibited budding of virus-like particles containing Gag only, indicating that Gag was the target of TRIM22. TRIM22 did not block the release of MLV or EIAV Gag particles. Inhibition was associated with diffuse cytoplasmic staining of HIV Gag rather than accumulation at the plasma membrane, suggesting TRIM22 disrupts proper trafficking. Mutational analyses of TRIM22 showed that the catalytic amino acids Cys15 and Cys18 of the RING domain are required for TRIM22 antiviral activity. These data disclose a pathway by which Type 1 interferons obstruct HIV replication.
Author Summary
Interferons are produced by cells in response to challenge by foreign pathogens such as viruses. The molecular mechanisms by which Type I interferons (e.g., IFNβ) inhibit the replication of HIV-1 are not fully clarified. We identified a gene called TRIM22 that belongs to the tripartite motif (TRIM) family that was strongly induced by IFNβ. Using RNA interference to reduce the expression of TRIM22, we showed that TRIM22 is a key mediator of the IFNβ response when expressed at natural levels. We demonstrate that TRIM22 blocks the intracellular trafficking of the viral structural protein Gag to the surface of the cell, and that the antiviral activity of TRIM22 is dependent on two cysteine residues (Cys15 and Cys18) that are critical for the E3 ligase activity of RING-containing proteins. This report describes a mechanism by which Type I interferons block HIV-1 replication.
doi:10.1371/journal.ppat.1000007
PMCID: PMC2279259  PMID: 18389079
10.  Restriction of HIV-1 by Rhesus TRIM5α Is Governed by Alpha Helices in the Linker2 Region 
Journal of Virology  2014;88(16):8911-8923.
ABSTRACT
TRIM5α proteins are a potent barrier to the cross-species transmission of retroviruses. TRIM5α proteins exhibit an ability to self-associate at many levels, ultimately leading to the formation of protein assemblies with hexagonal symmetry in vitro and cytoplasmic assemblies when expressed in cells. However, the role of these assemblies in restriction, the determinants that mediate their formation, and the organization of TRIM5α molecules within these assemblies have remained unclear. Here we show that α-helical elements within the Linker2 region of rhesus macaque TRIM5α govern the ability to form cytoplasmic assemblies in cells and restrict HIV-1 infection. Mutations that reduce α-helix formation by the Linker2 region disrupt assembly and restriction. More importantly, mutations that enhance the α-helical content of the Linker2 region, relative to the wild-type protein, also exhibit an increased ability to form cytoplasmic assemblies and restrict HIV-1 infection. Molecular modeling of the TRIM5α dimer suggests a model in which α-helical elements within the Linker2 region dock to α-helices of the coiled-coil domain, likely establishing proper orientation and spacing of protein domains necessary for assembly and restriction. Collectively, these studies provide critical insight into the determinants governing TRIM5α assembly and restriction and demonstrate that the antiviral potency of TRIM5α proteins can be significantly increased without altering the affinity of SPRY/capsid binding.
IMPORTANCE Many members of the tripartite motif (TRIM) family of proteins act as restriction factors that directly inhibit viral infection and activate innate immune signaling pathways. Another common feature of TRIM proteins is the ability to form protein assemblies in the nucleus or the cytoplasm. However, the determinants in TRIM proteins required for assembly and the degree to which assembly affects TRIM protein function have been poorly understood. Here we show that alpha helices in the Linker2 (L2) region of rhesus TRIM5α govern assembly and restriction of HIV-1 infection. Helix-disrupting mutations disrupt the assembly and restriction of HIV-1, while helix-stabilizing mutations enhance assembly and restriction relative to the wild-type protein. Circular dichroism analysis suggests that that the formation of this helical structure is supported by intermolecular interactions with the coiled-coil (CC) domain in the CCL2 dimer. These studies reveal a novel mechanism by which the antiviral activity of TRIM5α proteins can be regulated and provide detailed insight into the assembly determinants of TRIM family proteins.
doi:10.1128/JVI.01134-14
PMCID: PMC4136267  PMID: 24872590
11.  In Silico Analysis of Functional Single Nucleotide Polymorphisms in the Human TRIM22 Gene 
PLoS ONE  2014;9(7):e101436.
Tripartite motif protein 22 (TRIM22) is an evolutionarily ancient protein that plays an integral role in the host innate immune response to viruses. The antiviral TRIM22 protein has been shown to inhibit the replication of a number of viruses, including HIV-1, hepatitis B, and influenza A. TRIM22 expression has also been associated with multiple sclerosis, cancer, and autoimmune disease. In this study, multiple in silico computational methods were used to identify non-synonymous or amino acid-changing SNPs (nsSNP) that are deleterious to TRIM22 structure and/or function. A sequence homology-based approach was adopted for screening nsSNPs in TRIM22, including six different in silico prediction algorithms and evolutionary conservation data from the ConSurf web server. In total, 14 high-risk nsSNPs were identified in TRIM22, most of which are located in a protein interaction module called the B30.2 domain. Additionally, 9 of the top high-risk nsSNPs altered the putative structure of TRIM22's B30.2 domain, particularly in the surface-exposed v2 and v3 regions. These same regions are critical for retroviral restriction by the closely-related TRIM5α protein. A number of putative structural and functional residues, including several sites that undergo post-translational modification, were also identified in TRIM22. This study is the first extensive in silico analysis of the highly polymorphic TRIM22 gene and will be a valuable resource for future targeted mechanistic and population-based studies.
doi:10.1371/journal.pone.0101436
PMCID: PMC4077803  PMID: 24983760
12.  Lentiviral Gene Therapy Against Human Immunodeficiency Virus Type 1, Using a Novel Human TRIM21-Cyclophilin A Restriction Factor 
Human Gene Therapy  2012;23(11):1176-1185.
Abstract
TRIM5α (tripartite motif-containing protein-5, isoform α)–cyclophilin A fusion proteins are anti-human immunodeficiency virus (HIV) restriction factors that have evolved in certain nonhuman primates over millions of years and protect against HIV and related viruses. Restriction by TRIM5αCypA is potent and highly resistant to viral escape by mutation and, in combination with a suitable gene delivery platform, offers the possibility of novel therapeutic approaches against HIV. Here we report that lentiviral vector delivery of human mimics of TRIM5α-cyclophilin A (TRIM5CypA) fusion proteins afforded robust and durable protection against HIV-1, but resulted in downregulation of host cell antiviral responses mediated by endogenous TRIM5α. We found that substitution of TRIM5α RING, B-box, and coiled-coil domains with similar domains from a related TRIM protein, TRIM21, produced a novel and equally potent inhibitor of HIV-1. Both TRIM5CypA and TRIM21CypA inhibited transduction by HIV-1-derived viral vectors and prevented propagation of replication-competent HIV-1 in human cell lines and in primary human T cells. Restriction factor-modified T cells exhibited preferential survival in the presence of wild-type HIV. Restriction was dependent on proteasomal degradation and was reversed in the presence of the cyclophilin inhibitor cyclosporin. Importantly, TRIM21CypA did not disturb endogenous TRIM5α-mediated restriction of gammaretroviral infection. Furthermore, endogenous TRIM21 antiviral activity was assessed by measuring inhibition of adenovirus–antibody complexes and was found to be preserved in all TRIMCypA-modified groups. We conclude that lentivirus-mediated expression of the novel chimeric restriction factor TRIM21CypA provides highly potent protection against HIV-1 without loss of normal innate immune TRIM activity.
Chan and colleagues have constructed a modified TRIM5α-Cyclophilin A (TRIM5CypA) fusion protein that retains potent HIV-1-inhibitory ability without disturbing endogenous TRIM5α-mediated restriction of gammaretroviral infection. The TRIM5α RING, B-Box, and coiled-coil domains were substituted with similar domains from a related TRIM protein, TRIM21, and delivered by lentiviral vectors. Both TRIM5CypA and TRIM21CypA vectors inhibited transduction by HIV-1-derived viral vectors and prevented propagation of replication-competent HIV-1 in human cell lines and in primary human T cells.
doi:10.1089/hum.2012.083
PMCID: PMC3498886  PMID: 22909012
13.  Role of SUMO-1 and SUMO interacting motifs in rhesus TRIM5α-mediated restriction 
Retrovirology  2013;10:10.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1742-4690-10-10
PMCID: PMC3599732  PMID: 23369348
SUMO-1; SIM; rhTRIM5α; HIV-1
14.  The B30.2(SPRY) Domain of the Retroviral Restriction Factor TRIM5α Exhibits Lineage-Specific Length and Sequence Variation in Primates 
Journal of Virology  2005;79(10):6111-6121.
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.
doi:10.1128/JVI.79.10.6111-6121.2005
PMCID: PMC1091705  PMID: 15857996
15.  TRIM5α and Species Tropism of HIV/SIV 
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.
doi:10.3389/fmicb.2012.00013
PMCID: PMC3264904  PMID: 22291694
TRIM5α; TRIMCyp; HIV-1; HIV-2; SIV; rhesus monkey; cynomolgus monkey
16.  Role of Human TRIM5α in Intrinsic Immunity 
Human immunodeficiency virus (HIV) has a very narrow host range. HIV type 1 (HIV-1) does not infect Old World monkeys, such as the rhesus monkey (Rh). Rh TRIM5α was identified as a factor that confers resistance, intrinsic immunity, to HIV-1 infection. Unfortunately, human TRIM5α is almost powerless to restrict HIV-1. However, human TRIM5α potently restricts N-tropic murine leukemia viruses (MLV) but not B-tropic MLV, indicating that human TRIM5α represents the restriction factor previously designated as Ref1. African green monkey TRIM5α represents another restriction factor previously designated as Lv1, which restricts both HIV-1 and simian immunodeficiency virus isolated from macaque (SIVmac) infection. TRIM5 is a member of the tripartite motif family containing RING, B-box2, and coiled-coil domains. The RING domain is frequently found in E3 ubiquitin ligase, and TRIM5α is thought to degrade viral core via ubiquitin–proteasome-dependent and -independent pathways. The alpha isoform of TRIM5 has an additional C-terminal PRYSPRY domain, which is a determinant of species-specific retrovirus restriction by TRIM5α. On the other hand, the target regions of viral capsid protein (CA) are scattered on the surface of core. A single amino acid difference in the surface-exposed loop between α-helices 6 and 7 (L6/7) of HIV type 2 (HIV-2) CA affects viral sensitivity to human TRIM5α and was also shown to be associated with viral load in West African HIV-2 patients, indicating that human TRIM5α is a critical modulator of HIV-2 replication in vivo. Interestingly, L6/7 of CA corresponds to the MLV determinant of sensitivity to mouse factor Fv1, which potently restricts N-tropic MLV. In addition, human genetic polymorphisms also affect antiviral activity of human TRIM5α. Recently, human TRIM5α was shown to activate signaling pathways that lead to activation of NF-κB and AP-1 by interacting with TAK1 complex. TRIM5α is thus involved in control of viral infection in multiple ways.
doi:10.3389/fmicb.2012.00097
PMCID: PMC3304089  PMID: 22435067
Fv1; TRIM5α; TAB2; HIV-1; HIV-2; SIV; capsid; TRIMCyp
17.  A large new subset of TRIM genes highly diversified by duplication and positive selection in teleost fish 
BMC Biology  2009;7:7.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1741-7007-7-7
PMCID: PMC2657112  PMID: 19196451
18.  Gain-of-Sensitivity Mutations in a Trim5-Resistant Primary Isolate of Pathogenic SIV Identify Two Independent Conserved Determinants of Trim5α Specificity 
PLoS Pathogens  2013;9(5):e1003352.
Retroviral capsid recognition by Trim5 blocks productive infection. Rhesus macaques harbor three functionally distinct Trim5 alleles: Trim5αQ, Trim5αTFP and Trim5CypA. Despite the high degree of amino acid identity between Trim5αQ and Trim5αTFP alleles, the Q/TFP polymorphism results in the differential restriction of some primate lentiviruses, suggesting these alleles differ in how they engage these capsids. Simian immunodeficiency virus of rhesus macaques (SIVmac) evolved to resist all three alleles. Thus, SIVmac provides a unique opportunity to study a virus in the context of the Trim5 repertoire that drove its evolution in vivo. We exploited the evolved rhesus Trim5α resistance of this capsid to identify gain-of-sensitivity mutations that distinguish targets between the Trim5αQ and Trim5αTFP alleles. While both alleles recognize the capsid surface, Trim5αQ and Trim5αTFP alleles differed in their ability to restrict a panel of capsid chimeras and single amino acid substitutions. When mapped onto the structure of the SIVmac239 capsid N-terminal domain, single amino acid substitutions affecting both alleles mapped to the β-hairpin. Given that none of the substitutions affected Trim5αQ alone, and the fact that the β-hairpin is conserved among retroviral capsids, we propose that the β-hairpin is a molecular pattern widely exploited by Trim5α proteins. Mutations specifically affecting rhesus Trim5αTFP (without affecting Trim5αQ) surround a site of conservation unique to primate lentiviruses, overlapping the CPSF6 binding site. We believe targeting this site is an evolutionary innovation driven specifically by the emergence of primate lentiviruses in Africa during the last 12 million years. This modularity in targeting may be a general feature of Trim5 evolution, permitting different regions of the PRYSPRY domain to evolve independent interactions with capsid.
Author Summary
TRIM5α is an intrinsic immunity protein that blocks retrovirus infection through a specific interaction with the viral capsid. Uniquely among primates, rhesus macaques harbor three functionally distinct kinds of Trim5 alleles: rhTrim5αTFP, rhTrim5αQ and rhTrim5CypA. SIVmac239, a simian immunodeficiency virus that causes AIDS in rhesus macaques, is resistant to all three, whereas its relative, the human AIDS virus HIV-1, is inhibited by rhTrim5αTFP and rhTrim5αQ alleles. We exploited this difference between these two retroviruses to figure out how Trim5α proteins recognize viral capsids. By combining mutagenesis, structural biology and evolutionary data we determined that both rhTrim5αTFP and rhTrim5αQ recognize a conserved structure common to all retroviral capsids. However, we also found evidence suggesting that rhTrim5αTFP evolved to recognize an additional target that is specifically conserved among primate immunodeficiency viruses. Molecular evolutionary analysis indicates that this expanded function appeared in a common ancestor of modern African monkeys sometime between 9–12 million years ago, and that it thereafter continued to be modified by strong evolutionary pressure. Our results provide insight into the evolutionary flexibility of Trim5α-capsid interactions, and support the notion that viruses related to modern HIV and SIV have been present in Africa for millions of years.
doi:10.1371/journal.ppat.1003352
PMCID: PMC3649984  PMID: 23675300
19.  Novel TRIM5 Isoforms Expressed by Macaca nemestrina▿  
Journal of Virology  2007;81(22):12210-12217.
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.
doi:10.1128/JVI.02499-06
PMCID: PMC2168977  PMID: 17804491
20.  TRIM5 alpha Drives SIVsmm Evolution in Rhesus Macaques 
PLoS Pathogens  2013;9(8):e1003577.
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.
Author Summary
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.
doi:10.1371/journal.ppat.1003577
PMCID: PMC3749954  PMID: 23990789
21.  Role of TRIM5α RING Domain E3 Ubiquitin Ligase Activity in Capsid Disassembly, Reverse Transcription Blockade, and Restriction of Simian Immunodeficiency Virus▿† 
Journal of Virology  2011;85(16):8116-8132.
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.
doi:10.1128/JVI.00341-11
PMCID: PMC3147946  PMID: 21680520
22.  Functional Replacement of the RING, B-Box 2, and Coiled-Coil Domains of Tripartite Motif 5α (TRIM5α) by Heterologous TRIM Domains 
Journal of Virology  2006;80(13):6198-6206.
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.
doi:10.1128/JVI.00283-06
PMCID: PMC1488960  PMID: 16775307
23.  The Effect of Trim5 Polymorphisms on the Clinical Course of HIV-1 Infection 
PLoS Pathogens  2008;4(2):e18.
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.
Author Summary
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.
doi:10.1371/journal.ppat.0040018
PMCID: PMC2222955  PMID: 18248091
24.  Biochemical and Biophysical Characterization of a Chimeric TRIM21-TRIM5α Protein▿  
Journal of Virology  2008;82(23):11669-11681.
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.
doi:10.1128/JVI.01559-08
PMCID: PMC2583655  PMID: 18799572
25.  Enterovirus 71 induces degradation of TRIM38, a potential E3 ubiquitin ligase 
Virology Journal  2011;8:61.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1743-422X-8-61
PMCID: PMC3048563  PMID: 21306652

Results 1-25 (780563)