Human tripartite motif 5alpha domains responsible for retrovirus restriction activity and specificity

The tripartite motif 5alpha protein (TRIM5alpha) is one of several factors expressed by mammalian cells that inhibit retrovirus replication. Human TRIM5alpha (huTRIM5alpha) 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 huTRIM5alpha with its rhesus macaque counterpart (rhTRIM5alpha) endows it with the ability to potently inhibit HIV-1 infection. The B30.2/SPRY domain and an additional domain in huTRIM5alpha, 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 huTRIM5alpha multimerization. Truncated huTRIM5alpha 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 huTRIM5alpha and are dominant inhibitors of its N-MLV restricting activity, suggesting that homomultimerization of intact huTRIM5alpha monomers is necessary for N-MLV restriction. However, localization in large cytoplasmic bodies is not required for inhibition of N-MLV by huTRIM5alpha or for inhibition of HIV-1 by chimeric or rhTRIM5alpha.     

Species-specific variation in the B30.2(SPRY) domain of TRIM5alpha determines the potency of human immunodeficiency virus restriction

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 TRIM5alpha, a tripartite motif (TRIM) protein composed of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. Rhesus monkey TRIM5alpha (TRIM5alpha(rh)) more potently blocks HIV-1 infection than human TRIM5alpha (TRIM5alpha(hu)). Here, by studying chimeric TRIM5alpha proteins, we demonstrate that the major determinant of anti-HIV-1 potency is the B30.2(SPRY) domain. Analysis of species-specific variation in TRIM5alpha has identified three variable regions (v1, v2, and v3) within the B30.2 domain. The TRIM5alpha 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 TRIM5alpha(hu) B30.2 v1 region with the corresponding TRIM5alpha(rh) residues resulted in a TRIM5alpha molecule that restricted HIV-1 nearly as efficiently as wild-type TRIM5alpha(rh). Surprisingly, a single-amino-acid change in this region of TRIM5alpha(hu) allowed potent restriction of simian immunodeficiency virus, a phenotype not observed for either wild-type TRIM5alpha(hu) or TRIM5alpha(rh). Some of the chimeric TRIM5alpha 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 TRIM5alpha(rh) antiviral potency.     

灵长类动物中TRIMCyp融合基因模式及对逆转录病毒复制的限制作用

TRIM5-CypA融合基因(TRIMCyp)是一种独特的TRIM5基因形式。迄今已发现新大陆猴中包括鹰猴在内的夜猴属所有代表种,以及在北平顶猴、巽他平顶猴、食蟹猴、印度恒河猴和熊猴等旧大陆猴中均存在这种基因融合现象,但在新大陆猴与旧大陆猴中的TRIMCyp融合基因的基因融合模式和表达剪接方式不同。新大陆猴TRIMCyp融合基因是由CypA假基因的cDNA序列通过LINE-1逆转座子介导的逆转座方式插入至TRIM5α基因的第7和第8外显子之间的内含子中形成,而旧大陆猴TRIMCyp融合基因则是由CypA假基因的cDNA序列以相似的逆转座方式插入至TRIM5基因的3’非翻译区(untranslatedregions,UTR)形成。TRIMCyp融合基因在不同灵长类动物中的存在比例、基因型、TRIMCyp融合蛋白的表达以及对逆转录病毒的限制活性均有所差异。鹰猴和平顶猴的TRIMCyp融合基因研究较多,鹰猴TRIMCyp融合蛋白可能以与TRIM5α相似机制限制HIV-1的感染,而平顶猴TRIMCyp融合蛋白则丧失了限制HIV-1的作用。这两个功能截然不同的融合基因为TRIM5α作用机制研究提供了难得的实验材料,也为建立HIV-1感染的新型灵长类动物艾滋病模型奠定了科学依据。该文综述了TRIMCyp融合基因在灵长类动物中的分布、存在形式及其限制逆转录病毒复制的作用机制等方面的研究情况。     

Rhesus monkey TRIM5alpha restricts HIV-1 production through rapid degradation of viral Gag polyproteins

Mammalian cells have developed diverse strategies to restrict retroviral infection. Retroviruses have therefore evolved to counteract such restriction factors, in order to colonize their hosts. Tripartite motif-containing 5 isoform-alpha (TRIM5alpha) protein from rhesus monkey (TRIM5alpharh) restricts human immunodeficiency virus type 1 (HIV-1) infection at a postentry, preintegration stage in the viral life cycle, by recognizing the incoming capsid and promoting its premature disassembly. TRIM5alpha comprises an RBCC (RING, B-box 2 and coiled-coil motifs) domain and a B30.2(SPRY) domain. Sequences in the B30.2(SPRY) domain dictate the potency and specificity of the restriction. As TRIM5alpharh targets incoming mature HIV-1 capsid, but not precursor Gag, it was assumed that TRIM5alpharh did not affect HIV-1 production. Here we provide evidence that TRIM5alpharh, but not its human ortholog (TRIM5alphahu), blocks HIV-1 production through rapid degradation of HIV-1 Gag polyproteins. The specificity for this restriction is determined by sequences in the RBCC domain. Our observations suggest that TRIM5alpharh interacts with HIV-1 Gag during or before Gag assembly through a mechanism distinct from the well-characterized postentry restriction. This finding demonstrates a cellular factor blocking HIV-1 production by actively degrading a viral protein. Further understanding of this previously unknown restriction mechanism may reveal new targets for future anti-HIV-1 therapy.     

Structural and functional insights into the B30.2/SPRY domain

The B30.2/SPRY domain is present in approximately 700 eukaryotic (approximately 150 human) proteins, including medically important proteins such as TRIM5alpha and Pyrin. Nonetheless, the functional role of this modular domain remained unclear. Here, we report the crystal structure of an SPRY-SOCS box family protein GUSTAVUS in complex with Elongins B and C, revealing a highly distorted two-layered beta-sandwich core structure of its B30.2/SPRY domain. Ensuing studies identified one end of the beta-sandwich as the surface interacting with an RNA helicase VASA with a 40 nM dissociation constant. The sequence variation in TRIM5alpha responsible for HIV-1 restriction and most of the mutations in Pyrin causing familial Mediterranean fever map on this surface, implicating the corresponding region in many B30.2/SPRY domains as the ligand-binding site. The amino acids lining the binding surface are highly variable among the B30.2/SPRY domains, suggesting that these domains are protein-interacting modules, which recognize a specific individual partner protein rather than a consensus sequence motif.     

Retrovirus restriction by TRIM5alpha variants from Old World and New World primates

The TRIM5alpha proteins of humans and some Old World monkeys have been shown to block infection of particular retroviruses following virus entry into the host cell. Infection of most New World monkey cells by the simian immunodeficiency virus of macaques (SIVmac) is restricted at a similar point. Here we examine the antiretroviral activity of TRIM5alpha orthologs from humans, apes, Old World monkeys, and New World monkeys. Chimpanzee and orangutan TRIM5alpha proteins functionally resembled human TRIM5alpha, potently restricting infection by N-tropic murine leukemia virus (N-MLV) and moderately restricting human immunodeficiency virus type 1 (HIV-1) infection. Notably, TRIM5alpha proteins from several New World monkey species restricted infection by SIVmac and the SIV of African green monkeys, SIVagm. Spider monkey TRIM5alpha, which has an expanded B30.2 domain v3 region due to a tandem triplication, potently blocked infection by a range of retroviruses, including SIVmac, SIVagm, HIV-1, and N-MLV. Tandem duplications in the TRIM5alpha B30.2 domain v1 region of African green monkeys are also associated with broader antiretroviral activity. Thus, variation in TRIM5alpha proteins among primate species accounts for the observed patterns of postentry restrictions in cells from these animals. The TRIM5alpha proteins of some monkey species exhibit dramatic lengthening of particular B30.2 variable regions and an expanded range of susceptible retroviruses.     

Unique spectrum of activity of prosimian TRIM5alpha against exogenous and endogenous retroviruses

Lentiviruses, the genus of retrovirus that includes HIV-1, rarely endogenize. Some lemurs uniquely possess an endogenous lentivirus called PSIV ("prosimian immunodeficiency virus"). Thus, lemurs provide the opportunity to study the activity of host defense factors, such as TRIM5α, in the setting of germ line invasion. We characterized the activities of TRIM5α proteins from two distant lemurs against exogenous retroviruses and a chimeric PSIV. TRIM5α from gray mouse lemur, which carries PSIV in its genome, exhibited the narrowest restriction activity. One allelic variant of gray mouse lemur TRIM5α restricted only N-tropic murine leukemia virus (N-MLV), while a second variant restricted N-MLV and, uniquely, B-tropic MLV (B-MLV); both variants poorly blocked PSIV. In contrast, TRIM5α from ring-tailed lemur, which does not contain PSIV in its genome, revealed one of the broadest antiviral activities reported to date against lentiviruses, including PSIV. Investigation into the antiviral specificity of ring-tailed lemur TRIM5α demonstrated a major contribution of a 32-amino-acid expansion in variable region 2 (v2) of the B30.2/SPRY domain to the breadth of restriction. Data on lemur TRIM5α and the prediction of ancestral simian sequences hint at an evolutionary scenario where antiretroviral specificity is prominently defined by the lineage-specific expansion of the variable loops of B30.2/SPRY.     

The TRIM5alpha B-box 2 domain promotes cooperative binding to the retroviral capsid by mediating higher-order self-association

The retroviral restriction factor, TRIM5α, blocks infection of a spectrum of retroviruses soon after virus entry into the cell. TRIM5α consists of RING, B-box 2, coiled-coil, and B30.2(SPRY) domains. The B-box 2 domain is essential for retrovirus restriction by TRIM5α, but its specific function is unknown. We show here that the B-box 2 domain mediates higher-order self-association of TRIM5α_rh oligomers. This self-association increases the efficiency of TRIM5α binding to the retroviral capsid, thus potentiating restriction of retroviral infection. The contribution of the B-box 2 domain to cooperative TRIM5α association with the retroviral capsid explains the conditional nature of the restriction phenotype exhibited by some B-box 2 TRIM5α mutants; the potentiation of capsid binding that results from B-box 2-mediated self-association is essential for restriction when B30.2(SPRY) domain-mediated interactions with the retroviral capsid are weak. Thus, B-box 2-dependent higher-order self-association and B30.2(SPRY)-dependent capsid binding represent complementary mechanisms whereby sufficiently dense arrays of capsid-bound TRIM5α proteins can be achieved.     

Removal of arginine 332 allows human TRIM5alpha to bind human immunodeficiency virus capsids and to restrict infection

Human TRIM5alpha (TRIM5alpha(hu)) only modestly inhibits human immunodeficiency virus type 1 (HIV-1) and does not inhibit simian immunodeficiency virus (SIV(mac)). Alteration of arginine 332 in the TRIM5alpha(hu) B30.2 domain to proline, the residue found in rhesus monkey TRIM5alpha, has been shown to create a potent restricting factor for both HIV-1 and SIV(mac.) Here we demonstrate that the potentiation of HIV-1 inhibition results from the removal of a positively charged residue at position 332 of TRIM5alpha(hu.) The increase in restricting activity correlated with an increase in the ability of TRIM5alpha(hu) mutants lacking arginine 332 to bind HIV-1 capsid complexes. A change in the cyclophilin A-binding loop of the HIV-1 capsid decreased TRIM5alpha(hu) R332P binding and allowed escape from restriction. The ability of TRIM5alpha(hu) to restrict SIV(mac) could be disrupted by the presence of any charged residue at position 332. Thus, charged residues in the v1 region of the TRIM5alpha(hu) B30.2 domain can modulate capsid binding and restriction potency. Therapeutic strategies designed to neutralize arginine 332 of TRIM5alpha(hu) might potentiate the innate resistance of human cells to HIV-1 infection.     

Functional replacement of the RING, B-box 2, and coiled-coil domains of tripartite motif 5alpha (TRIM5alpha) by heterologous TRIM domains

Tripartite motif 5alpha (TRIM5alpha) restricts some retroviruses, including human immunodeficiency virus type 1 (HIV-1), from infecting the cells of particular species. TRIM5alpha 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 TRIM5alpha (TRIM5alpha(rh)). The RING, B-box 2, and CC domains of the paralogous TRIM6 and TRIM34 proteins functionally replaced the corresponding TRIM5alpha(rh) 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 TRIM5alpha(rh) domains.     

Inhibitory effect of human TRIM5α on HIV-1 production

Tripartite motif-containing 5 isoform-α (TRIM5α), a host restriction factor, blocks infection of some retroviruses at a post-entry, pre-integration stage in a species-specific manner. A recent report by Sakuma et al. describes a second antiretroviral activity of rhesus macaque TRIM5α, which blocks HIV-1 production through rapid degradation of HIV-1 Gag polyproteins. Here, we find that human TRIM5α limits HIV-1 production. Transient expression of TRIM5α decreased HIV-1 production, whereas knockdown of TRIM5α in human cells increased virion release. A single amino acid substitution (R437C) in the SPRY domain diminished the restriction effect. Moderate levels of human wild-type TRIM5α and a little amount of R437C mutant were incorporated into HIV-1 virions. The R437C mutant also lost restriction activity against N-tropic murine leukemia virus infection. However, the corresponding R to C mutation in rhesus macaque TRIM5α had no effect on the restriction ability. Our findings suggest human TRIM5α is an intrinsic immunity factor against HIV-1 infection. The importance of arginine at 437 aa in SPRY domain for the late restriction is species-specific.     

The contribution of RING and B-box 2 domains to retroviral restriction mediated by monkey TRIM5alpha

TRIM5alpha is a cytoplasmic protein that mediates a post-entry block to infection by some retroviruses. TRIM5alpha contains a tripartite motif (TRIM), which includes RING, B-box 2, and coiled-coil domains, and a C-terminal B30.2 (SPRY) domain. We investigated the contribution of the RING and B-box 2 domains to the antiretroviral activity of rhesus monkey TRIM5alpha (TRIM5alpharh), which potently restricts infection by human immunodeficiency virus, type 1 (HIV-1) and simian immunodeficiency virus of African green monkeys (SIVagm). Disruption of the RING domain caused mislocalization of TRIM5alpharh so that the cytoplasmic level of the protein was decreased compared with that of the wild-type protein. Nonetheless, partial ability to restrict HIV-1 and SIVagm was retained by the RING domain mutants. By contrast, although TRIM5alpharh mutants with disrupted B-box 2 domains were efficiently expressed and correctly localized to the cytoplasm, antiretroviral activity was absent. The B-box 2 mutants colocalized and associated with wild-type TRIM5alpharh and exerted dominant-negative effects on the antiretroviral activity of the wild-type protein. Taken together with other data, these results indicate that functionally defective TRIM5alpharh molecules that retain a coiled coil can act as dominant-negative inhibitors of wild-type TRIM5alpharh function. The RING domain of TRIM5alpharh is not absolutely required for retrovirus restriction but can influence cytoplasmic levels of the protein and thus indirectly alter function. The B-box 2 domain, by contrast, appears to be essential for efficient retrovirus restriction.     

Variable prevalence and functional diversity of the antiretroviral restriction factor TRIMCyp in Macaca fascicularis

The retroviral restriction factor TRIMCyp, derived from the TRIM5 gene, blocks replication at a postentry step. TRIMCyp has so far been found in four species of Asian macaques, Macaca fascicularis, M. mulatta, M. nemestrina, and M. leonina. M. fascicularis is commonly used as a model for AIDS research, but TRIMCyp has not been analyzed in detail in this species. We analyzed the prevalence of TRIMCyp in samples from Indonesia, Indochina, the Philippines, and Mauritius. We found that TRIMCyp is present at a higher frequency in Indonesian than in Indochinese M. fascicularis macaques and is also present in samples from the Philippines. TRIMCyp is absent in Mauritian M. fascicularis macaques. We then analyzed the restriction specificity of TRIMCyp derived from three animals of Indonesian origin. One allele, like the prototypic TRIMCyp alleles described for M. mulatta and M. nemestrina, restricts human immunodeficiency virus type 2 (HIV-2) and feline immunodeficiency virus (FIV) but not HIV-1. The others restrict HIV-1 and FIV but not HIV-2. Mutagenesis studies confirmed that polymorphisms at amino acid residues 369 and 446 in TRIMCyp (or residues 66 and 143 in the cyclophilin A [CypA] domain) confer restriction specificity. Additionally, we identified a polymorphism in the coiled-coil domain that appears to affect TRIMCyp expression or stability. Taken together, these data show that M. fascicularis has the most diverse array of TRIM5 restriction factors described for any primate species to date. These findings are relevant to our understanding of the evolution of retroviral restriction factors and the use of M. fascicularis models in AIDS research.