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.     

Cyclosporine increases human immunodeficiency virus type 1 vector transduction of primary mouse cells

Murine primary cells are poorly permissive to human immunodeficiency virus type 1 (HIV-1) vector infection. Retroviral infectivity is influenced by dominant inhibitors such as TRIM5alpha. Sensitivity to TRIM5alpha is altered by interactions between cyclophilin A and the HIV-1 capsid. Here we demonstrate that competitive inhibitors of cyclophilins, cyclosporine or the related Debio-025, stimulate HIV-1 vector transduction of primary murine cells, including bone marrow and macrophages, up to 20-fold. Unexpectedly, the infectivity of an HIV-1 mutant or a simian lentivirus that does not recruit cyclophilin A is also stimulated by these drugs. We propose that cyclosporine and related compounds will be useful tools for experimental infection of murine primary cells. It is possible that HIV-1 infection of murine cells is inhibited by dominant factors related to immunophilins.     

Naturally occurring capsid substitutions render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilin-resistant in Owl monkey cells

In this study, we asked if a naturally occurring HIV-1 variant exists that circumvents CypA dependence in human cells. To address this issue, we sought viruses for CypA independence using Debio-025, a cyclosporine A (CsA) analog that disrupts CypA-capsid interaction. Surprisingly, viral variants from the Main group replicate even in the presence of the drug. Sequencing analyses revealed that these viruses encode capsid substitutions within the CypA-binding site (V86P/H87Q/I91V/M96I). When we introduced these substitutions into viruses that normally rely on CypA for replication, these mutants no longer depended on CypA, suggesting that naturally occurring capsid substitutions obviate the need for CypA. This is the first demonstration that isolates from the Main group naturally develop CypA-independent strategies to replicate in human cells. Surprisingly, we found that these capsid substitutions render HIV-1 capable of infecting Owl monkey (OMK) cells that highly restrict HIV-1. OMK cell resistance to HIV-1 is mediated via TRIM-Cyp, which arose from a retrotransposition of CypA into the TRIM5 alpha gene. Interestingly, saturation experiments suggest that the Pro86/Gln87/Val91/Ile96 capsid core is "invisible" to TRIM-Cyp. This study demonstrates that specific capsid substitutions can release HIV-1 from both CypA dependence in human cells and TRIM-Cyp restriction in monkey cells.     

A single amino acid of the human immunodeficiency virus type 2 capsid affects its replication in the presence of cynomolgus monkey and human TRIM5alphas

Human immunodeficiency virus type 2 (HIV-2) strains vary widely in their abilities to grow in Old World monkey (OWM) cells such as those of cynomolgus monkeys (CM). We evaluated eight HIV-2 isolates for their sensitivities to CM TRIM5alpha, an anti-HIV factor in OWM cells. We found that different HIV-2 isolates showed differences in their sensitivities to CM TRIM5alpha. Sequence analysis showed that TRIM5alpha-sensitive viruses had proline at the 120th position of the capsid protein (CA), whereas TRIM5alpha-resistant viruses had either alanine or glutamine. Mutagenesis studies indicated that the single amino acid at the 120th position indeed affected the sensitivity of the virus to CM TRIM5alpha.     

Differential restriction of human immunodeficiency virus type 2 and simian immunodeficiency virus SIVmac by TRIM5alpha alleles

Primate lentiviruses have narrow host ranges, due in part to their sensitivities to mammalian intracellular antiviral factors such as APOBEC3G and TRIM5alpha. Despite the protection provided by this innate immune system, retroviruses are able to transfer between species where they can cause disease. This is true for sooty mangabey simian immunodeficiency virus, which has transferred to humans as HIV-2 and to rhesus macaques as SIVmac, where it causes AIDS. Here we examine the sensitivities of the closely related HIV-2 and SIVmac to restriction by TRIM5alpha. We show that rhesus TRIM5alpha can restrict HIV-2 but not the closely related SIVmac. SIVmac has not completely escaped TRIM5alpha, as shown by its sensitivity to distantly related TRIM5alpha from the New World squirrel monkey. Squirrel monkey TRIM5alpha blocks SIVmac infection after DNA synthesis and is not saturable with restriction-sensitive virus-like particles. We map the determinant for TRIM5alpha sensitivity to the structure in the capsid protein that recruits CypA into HIV-1 virions. We also make an SIV, mutated at this site, which bypasses restriction in all cells tested.     

Cyclophilin, TRIM5, and innate immunity to HIV-1

The peptidyl-prolyl isomerase cyclophilin A (CypA) binds a proline-rich loop on the surface of HIV-1 capsid (CA). This interaction increases HIV-1 infectivity in humans but promotes an anti-HIV-1 restriction activity in non-human primates. Efforts to understand these paradoxical effects of cyclophilin, along with more targeted approaches to uncover the genetic basis for HIV-1 restriction, led to the discovery of TRIM5 (tripartite motif protein 5), a CA-specific receptor for the retroviral core. The ensuing TRIM5 publication flurry established a paradigm of innate immunity in which the protein lattice of an invading retroviral core, rather than double-stranded RNA or lipopolysaccharide, is recognized by a multimeric, cytoplasmic receptor. CypA modulates HIV-1 virion core detection by this class of innate pattern recognition molecule, apparently by inducing subtle shifts in CA conformation.     

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.     

An active TRIM5 protein in rabbits indicates a common antiviral ancestor for mammalian TRIM5 proteins

The recent identification of antiretroviral tripartite motif-bearing restriction factors that protect against retroviral infection has revealed a novel branch of innate immunity. The factors target the retroviral capsid and inhibit infectivity soon after the capsid has entered the cytoplasm by an incompletely characterized mechanism. Restriction is species specific. For example, TRIM5alpha from Old World monkeys, but not humans, restricts human immunodeficiency virus type 1 infection. Here, we identify an antiviral TRIM5 molecule in rabbits that is closely related to antiviral TRIM5 of both primates and cattle. We demonstrate that the rabbit TRIM5 protein is active against divergent retroviruses and leads to a strong block to viral DNA synthesis and infectivity. Furthermore, we show that antiviral activity is directed against the viral capsid and that human TRIM5 proteins are dominant negative to restriction in rabbit cells. We propose that the sequence and restriction characteristics conserved between restriction factors from primates, cattle, and rabbits indicate that these factors have evolved from a common ancestor with antiretroviral properties.     

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。     

Cyclophilin A and TRIM5alpha independently regulate human immunodeficiency virus type 1 infectivity in human cells

Cyclophilin A (CypA), a cytoplasmic, human immunodeficiency virus type 1 (HIV-1) CA-binding protein, acts after virion membrane fusion with human cells to increase HIV-1 infectivity. HIV-1 CA is similarly greeted by CypA soon after entry into rhesus macaque or African green monkey cells, where, paradoxically, the interaction decreases HIV-1 infectivity by facilitating TRIM5alpha-mediated restriction. These observations conjure a model in which CA recognition by the human TRIM5alpha orthologue is precluded by CypA. Consistent with the model, selection of a human cell line for decreased restriction of the TRIM5alpha-sensitive, N-tropic murine leukemia virus (N-MLV) rendered HIV-1 transduction of these cells independent of CypA. Additionally, HIV-1 virus-like particles (VLPs) saturate N-MLV restriction activity, particularly when the CA-CypA interaction is disrupted. Here the effects of CypA and TRIM5alpha on HIV-1 restriction were examined directly. RNA interference was used to show that endogenous human TRIM5alpha does indeed restrict HIV-1, but the magnitude of this antiviral activity was not altered by disruption of the CA-CypA interaction or by elimination of CypA protein. Conversely, the stimulatory effect of CypA on HIV-1 infectivity was completely independent of human TRIM5alpha. Together with previous reports, these data suggest that CypA protects HIV-1 from an unknown antiviral activity in human cells. Additionally, target cell permissivity increased after loading with heterologous VLPs, consistent with a common saturable target that is epistatic to both TRIM5alpha and the putative CypA-regulated restriction factor.     

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.     

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.     

Cyclophilin interactions with incoming human immunodeficiency virus type 1 capsids with opposing effects on infectivity in human cells

Cyclophilin A (CypA) is a peptidyl-prolyl isomerase that binds to the capsid protein (CA) of human immunodeficiency virus type 1 (HIV-1) and by doing so facilitates HIV-1 replication. Although CypA is incorporated into HIV-1 virions by virtue of CypA-Gag interactions that occur during virion assembly, in this study we show that the CypA-CA interaction that occurs following the entry of the viral capsid into target cells is the major determinant of CypA's effects on HIV-1 replication. Specifically, by using normal and CypA-deficient Jurkat cells, we demonstrate that the presence of CypA in the target and not the virus-producing cell enhances HIV-1 infectivity. Moreover, disruption of the CypA-CA interaction with cyclosporine A (CsA) inhibits HIV-1 infectivity only if the target cell expresses CypA. The effect of CsA on HIV-1 infection of human cells varies according to which particular cell line is used as a target, and CA mutations that confer CsA resistance and dependence exert their effects only if target cells, and not if virus-producing cells, are treated with CsA. The differential effects of CsA on HIV-1 infection in different human cells appear not to be caused by polymorphisms in the recently described retrovirus restriction factor TRIM5alpha. We speculate that CypA and/or CypA-related proteins affect the fate of incoming HIV-1 capsid either directly or by modulating interactions with unidentified host cell factors.     

Disruption of human TRIM5alpha antiviral activity by nonhuman primate orthologues

TRIM5 is a determinant of species-specific differences in susceptibility to infection by retroviruses bearing particular capsids. Human immunodeficiency virus type 1 (HIV-1) infection is blocked by the alpha isoform of macaque TRIM5 (TRIM5alpha(rh)) or by the product of the owl monkey TRIM5-cyclophilin A gene fusion (TRIMCyp). Human TRIM5alpha potently restricts specific strains of murine leukemia virus (N-MLV) but has only a modest effect on HIV-1. The amino termini of TRIM5 orthologues are highly conserved and possess a coiled-coil domain that promotes homomultimerization. Here we show that heterologous expression of TRIM5alpha(rh) or TRIMCyp in human cells interferes with the anti-N-MLV activity of endogenous human TRIM5alpha (TRIM5alpha(hu)). Deletion of the cyclophilin domain from TRIMCyp has no effect on heteromultimerization or colocalization with TRIM5alpha(hu) but prevents interference with anti-N-MLV activity. These data demonstrate that TRIM5 orthologues form heteromultimers and indicate that C-terminal extensions alter virus recognition by multimers of these proteins.     

Apparent effect of rabbit endogenous lentivirus type K acquisition on retrovirus restriction by lagomorph Trim5αs

To test the hypothesis that rabbit endogenous lentivirus type K (RELIK) could play a role in shaping the evolution of TRIM5α, the susceptibility of viruses containing the RELIK capsid (CA) to TRIM5 restriction was evaluated. RELIK CA-containing viruses were susceptible to the TRIM5αs from Old World monkeys but were unaffected by most ape or New World monkey factors. TRIM5αs from various lagomorph species were also isolated and tested for anti-retroviral activity. The TRIM5αs from both cottontail rabbit and pika restrict a range of retroviruses, including HIV-1, HIV-2, FIV, EIAV and N-MLV. TRIM5αs from the European and cottontail rabbit, which have previously been found to contain RELIK, also restricted RELIK CA-containing viruses, whereas a weaker restriction was observed with chimeric TRIM5α containing the B30.2 domain from the pika, which lacks RELIK. Taken together, these results could suggest that the pika had not been exposed to exogenous RELIK and that endogenized RELIK might exert a selective pressure on lagomorph TRIM5α.