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.     

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.     

Association of TRIMCyp and TRIM5α from assam macaques leads to a functional trade-off between HIV-1 and N-MLV inhibition

TRIM5α restricts retroviruses in a species-specific manner. Cyclophilin A was independently retrotransposed into the TRIM5 loci in different species, leading to the generation of antiviral TRIM5-cyclophilin A(TRIMCyp) proteins. Previously, we found that assam macaques express a TRIMCyp chimera(am TRIMCyp), along with a TRIM5α allelic protein(am TRIM5α). Herein,we investigated the antiviral activity of am TRIMCyp and am TRIM5α individually, as well as their interaction and joint effects.am TRIMCyp showed a divergent restriction pattern from am TRIM5α. Although both proteins potently restricted the replication of HIV-1, only am TRIM5α inhibited N-MLV. Remarkably, cellular anti-HIV-1 activity increased when am TRIMCyp and am TRIM5α were coexpressed, indicating a synergistic block of HIV-1 replication. Consistently, PMBCs from heterozygous am TRIM5α/TRIMCyp showed stronger resistance to HIV-1 infection than those from am TRIM5α/TRIM5α homozygotes. The anti-HIV-1 synergistic effect was dependent on the am TRIMCyp-am TRIM5α interaction. In contrast, am TRIMCyp completely abrogated the anti-N-MLVactivity mediated by am TRIM5α, showing a dominant-negative effect, indicating that the generation of am TRIMCyp was involved in the trade-off between divergent restriction activities. Our results provide a new paradigm to study functional trade-offs mediated by allelic proteins, a theoretical basis for utilizing animal models with various TRIM5 alleles, as well as novel HIV-1 gene therapy strategies.     

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.     

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.     

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。     

Arsenic counteracts human immunodeficiency virus type 1 restriction by various TRIM5 orthologues in a cell type-dependent manner

Arsenic trioxide (As(2)O(3)) increased human immunodeficiency virus type 1 (HIV-1) infectivity when particular Homo sapiens and Cercopithecus aethiops cell lines were used as targets. Knockdown of human TRIM5alpha by RNA interference eliminated the As(2)O(3) effect, demonstrating that the drug acts by modulating the activity of this retroviral restriction factor. In contrast, HIV-1 infectivity in target cell lines from other primate species (Cercopithecus tantalus, Macaca mulatta, and Aotus trivirgatus) was not increased by As(2)O(3), despite the potent TRIM5-dependent HIV-1 restriction activity that these cells exhibit. To determine if As(2)O(3) responsiveness is characteristic of particular TRIM5 orthologues and not others, TRIM5 cDNAs from these five primate species were transduced into cat fibroblasts, which lack endogenous HIV-1 restriction activity and, therefore, responsiveness to As(2)O(3). In this context, the HIV-1 restriction activity conferred by all TRIM5 orthologues was largely eliminated by As(2)O(3). The effect of As(2)O(3) on HIV-1 restriction is thus shared by different TRIM5 orthologues but dependent on factors specific to the cell line in which TRIM5 is expressed.     

Two surface-exposed elements of the B30.2/SPRY domain as potency determinants of N-tropic murine leukemia virus restriction by human TRIM5alpha

Human TRIM5alpha (TRIM5alpha(hu)) potently restricts N-tropic (N-MLV), but not B-tropic, murine leukemia virus in a manner dependent upon residue 110 of the viral capsid. Rhesus monkey TRIM5alpha (TRIM5alpha(rh)) inhibits N-MLV only weakly. The study of human-monkey TRIM5alpha chimerae revealed that both the v1 and v3 variable regions of the B30.2/SPRY domain contain potency determinants for N-MLV restriction. These variable regions are predicted to be surface-exposed elements on one face of the B30.2 domain. Acidic residues in v3 complement basic residue 110 of the N-MLV capsid. The results support recognition of the retroviral capsid by the TRIM5alpha B30.2 domain.     

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.     

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.     

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.