Retroviral restriction factor TRIM5alpha is a trimer

The retrovirus restriction factor TRIM5alpha targets the viral capsid soon after entry. Here we show that the TRIM5alpha protein oligomerizes into trimers. The TRIM5alpha coiled-coil and B30.2(SPRY) domains make important contributions to the formation and/or stability of the trimers. A functionally defective TRIM5alpha mutant with the RING and B-box 2 domains deleted can form heterotrimers with wild-type TRIM5alpha, accounting for the observed dominant-negative activity of the mutant protein. Trimerization potentially allows TRIM5alpha to interact with threefold pseudosymmetrical structures on retroviral capsids.     

Interfering residues narrow the spectrum of MLV restriction by human TRIM5alpha

TRIM5alpha is a restriction factor that limits infection of human cells by so-called N- but not B- or NB-tropic strains of murine leukemia virus (MLV). Here, we performed a mutation-based functional analysis of TRIM5alpha-mediated MLV restriction. Our results reveal that changes at tyrosine(336) of human TRIM5alpha, within the variable region 1 of its C-terminal PRYSPRY domain, can expand its activity to B-MLV and to the NB-tropic Moloney MLV. Conversely, we demonstrate that the escape of MLV from restriction by wild-type or mutant forms of huTRIM5alpha can be achieved through interdependent changes at positions 82, 109, 110, and 117 of the viral capsid. Together, our results support a model in which TRIM5alpha-mediated retroviral restriction results from the direct binding of the antiviral PRYSPRY domain to the viral capsid, and can be prevented by interferences exerted by critical residues on either one of these two partners.     

Evolution of the antiretroviral restriction factor TRIMCyp in Old World primates

The retroviral restriction factor TRIMCyp, which is a fusion protein derived from the TRIM5 gene, blocks replication at a post-entry step. Among Old World primates, TRIMCyp has been found in four species of Asian macaques, but not in African monkeys. To further define the evolutionary origin of Old World TRIMCyp, we examined two species of baboons (genus Papio) and three additional macaque species, including M. sylvanus, which is the only macaque species found outside Asia, and represents the earliest diverging branch of the macaque lineage. None of four P. cynocephalus anubis, one P. hamadryas, and 36 M. sylvanus had either TRIMCyp mRNA or the genetic features required for its expression. M. sylvanus genomic sequences indicated that the lack of TRIMCyp in this species was not due to genetic homogeneity among specimens studied and revealed the existence of four TRIM5α alleles, all distinct from M. mulatta and Papio counterparts. Together with existing data on macaque evolution, our findings indicate that TRIMCyp evolved in the ancestors of Asian macaques approximately 5-6 million years before present (ybp), likely as a result of a retroviral threat. TRIMCyp then became fixed in the M. nemestrina lineage after it diverged from M. nigra, approximately 2 million ybp. The macaque lineage is unique among primates studied so far due to the presence and diversity of both TRIM5 and TRIMCyp restriction factors. Studies of these antiviral proteins may provide valuable information about natural antiviral mechanisms, and give further insight into the factors that shaped the evolution of macaque species.     

Color vision variations in Old and New World primates

It has long been recognized that there are significant individual variations in color vision among humans. Recently, even more widespread individual variation in color vision has been found to occur in members of several genera of New World monkeys. This article addresses the question of whether a representative genus of Old World monkeys, Macaca, expresses individual variations in color vision. The principal approach was to compare behavioral measurements of increment-threshold spectral sensitivity for large samples of squirrel monkeys (Saimiri sp.) and macaque monkeys (Macaca mulatta, M. fascicularis). We conclude that, if they occur at all, individual variations in color vision among macaque monkeys must be rare.     

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.     

The B30.2(SPRY) domain of the retroviral restriction factor TRIM5alpha exhibits lineage-specific length and sequence variation in primates

Tripartite motif (TRIM) proteins are composed of RING, B-box 2, and coiled coil domains. Some TRIM proteins, such as TRIM5alpha, also possess a carboxy-terminal B30.2(SPRY) domain and localize to cytoplasmic bodies. TRIM5alpha 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 TRIM5alpha was analyzed by amplifying, cloning, and sequencing nonhuman primate TRIM5 orthologs. Lineage-specific expansion and sequential duplication occurred in the TRIM5alpha 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 TRIM5alpha 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 TRIM5alpha in innate immunity against retroviruses.     

Evidence for restriction of ancient primate gammaretroviruses by APOBEC3 but not TRIM5alpha proteins

Because of evolutionary pressures imposed through episodic colonization by retroviruses, many mammals express factors, such as TRIM5alpha and APOBEC3 proteins, that directly restrict retroviral replication. TRIM5 and APOBEC restriction factors are most often studied in the context of modern primate lentiviruses, but it is likely that ancient retroviruses imposed the selective pressure that is evident in primate TRIM5 and APOBEC3 genes. Moreover, these antiretroviral factors have been shown to act against a variety of retroviruses, including gammaretroviruses. Endogenous retroviruses can provide a 'fossil record' of extinct retroviruses and perhaps evidence of ancient TRIM5 and APOBEC3 antiviral activity. Here, we investigate whether TRIM5 and APOBEC3 proteins restricted the replication of two groups of gammaretroviruses that were endogenized in the past few million years. These endogenous retroviruses appear quite widespread in the genomes of old world primates but failed to colonize the human germline. Our analyses suggest that TRIM5alpha proteins did not pose a major barrier to the cross-species transmission of these two families of gammaretroviruses, and did not contribute to their extinction. However, we uncovered extensive evidence for inactivation of ancient gammaretroviruses through the action of APOBEC3 cytidine deaminases. Interestingly, the identities of the cytidine deaminases responsible for inactivation appear to have varied in both a virus and host species-dependent manner. Overall, sequence analyses and reconstitution of ancient retroviruses from remnants that have been preserved in the genomes of modern organisms offer the opportunity to probe and potentially explain the evolutionary history of host defenses against retroviruses.     

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.     

Modulation of retroviral restriction and proteasome inhibitor-resistant turnover by changes in the TRIM5alpha B-box 2 domain

An intact B-box 2 domain is essential for the antiretroviral activity of TRIM5alpha. We modeled the structure of the B-box 2 domain of TRIM5alpha based on the existing three-dimensional structure of the B-box 2 domain of human TRIM29. Using this model, we altered the residues predicted to be exposed on the surface of this globular structure. Most of the alanine substitutions in these residues exerted little effect on the antiretroviral activity of human TRIM5alphahu or rhesus monkey TRIM5alpharh. However, alteration of arginine 119 of TRIM5alphahu or the corresponding arginine 121 of TRIM5alpharh diminished the abilities of the proteins to restrict retroviral infection without affecting trimerization or recognition of the viral capsid. The abilities of these functionally defective TRIM5alpha proteins to accelerate the uncoating of the targeted retroviral capsid were abolished. Removal of the positively charged side chain from B-box 2 arginines 119/120/121 resulted in diminished proteasome-independent turnover of TRIM5alpha and the related restriction factor TRIMCyp. However, testing of an array of mutants revealed that the rapid turnover and retroviral restriction functions of this B-box 2 region are separable.     

Cyclophilin A renders human immunodeficiency virus type 1 sensitive to Old World monkey but not human TRIM5 alpha antiviral activity

TRIM5alpha is an important mediator of antiretroviral innate immunity influencing species-specific retroviral replication. Here we investigate the role of the peptidyl prolyl isomerase enzyme cyclophilin A in TRIM5alpha antiviral activity. Cyclophilin A is recruited into nascent human immunodeficiency virus type 1 (HIV-1) virions as well as incoming HIV-1 capsids, where it isomerizes an exposed proline residue. Here we show that cyclophilin A renders HIV-1 sensitive to restriction by TRIM5alpha in cells from Old World monkeys, African green monkey and rhesus macaque. Inhibition of cyclophilin A activity with cyclosporine A, or reducing cyclophilin A expression with small interfering RNA, rescues TRIM5alpha-restricted HIV-1 infectivity. The effect of cyclosporine A on HIV-1 infectivity is dependent on TRIM5alpha expression, and expression of simian TRIM5alpha in permissive feline cells renders them able to restrict HIV-1 in a cyclosporine A-sensitive way. We use an HIV-1 cyclophilin A binding mutant (CA G89V) to show that cyclophilin A has different roles in restriction by Old World monkey TRIM5alpha and owl monkey TRIM-Cyp. TRIM-Cyp, but not TRIM5alpha, recruits its tripartite motif to HIV-1 capsid via cyclophilin A and, therefore, HIV-1 G89V is insensitive to TRIM-Cyp but sensitive to TRIM5alpha. We propose that cyclophilin A isomerization of a proline residue in the TRIM5alpha sensitivity determinant of the HIV-1 capsid sensitizes it to restriction by Old World monkey TRIM5alpha. In humans, where HIV-1 has adapted to bypass TRIM5alpha activity, the effects of cyclosporine A are independent of TRIM5alpha. We speculate that cyclophilin A alters HIV-1 sensitivity to a TRIM5alpha-independent innate immune pathway in human cells.     

Antiretroviral activity of ancestral TRIM5alpha

The antiretroviral protein TRIM5α is known to have evolved different restriction capacities against various retroviruses, driven by positive Darwinian selection. However, how these different specificities have evolved in the primate lineages is not fully understood. Here we used ancestral protein resurrection to estimate the evolution of antiviral restriction specificities of TRIM5α on the primate lineage leading to humans. We used TRIM5α coding sequences from 24 primates for the reconstruction of ancestral TRIM5α sequences using maximum-likelihood and Bayesian approaches. Ancestral sequences were transduced into HeLa and CRFK cells. Stable cell lines were generated and used to test restriction of a panel of extant retroviruses (human immunodeficiency virus type 1 [HIV-1] and HIV-2, simian immunodeficiency virus [SIV] variants SIV_mac and SIV_agm, and murine leukemia virus [MLV] variants N-MLV and B-MLV). The resurrected TRIM5α variant from the common ancestor of Old World primates (Old World monkeys and apes, ~25 million years before present) was effective against present day HIV-1. In contrast to the HIV-1 restriction pattern, we show that the restriction efficacy against other retroviruses, such as a murine oncoretrovirus (N-MLV), is higher for more recent resurrected hominoid variants. Ancestral TRIM5α variants have generally limited efficacy against HIV-2, SIV_agm, and SIV_mac. Our study sheds new light on the evolution of the intrinsic antiviral defense machinery and illustrates the utility of functional evolutionary reconstruction for characterizing recently emerged protein differences.     

Mode of transmission affects the sensitivity of human immunodeficiency virus type 1 to restriction by rhesus TRIM5alpha

Rhesus TRIM5α (rhTRIM5α), but not human TRIM5α (huTRIM5α), potently inhibits human immunodeficiency virus (HIV) infection and is thus a potentially valuable therapeutic tool. Primary human CD4 T cells engineered to express rhTRIM5α were highly resistant to cell-free HIV type 1 (HIV-1) infection. However, when cocultured with unmodified T cells, rhTRIM5α-expressing cells became highly permissive to HIV-1 infection. Physical separation of rhTRIM5α-expressing cells and unmodified cells revealed that rhTRIM5α efficiently restricts cell-free but not cell-associated HIV transmission. Furthermore, we observed that HIV-infected human cells could infect rhesus CD4 T cells by cell-to-cell contact, but the infection was self-limiting. Subsequently, we noted that a spreading infection ensued when HIV-1-infected rhTRIM5α-expressing human cells were cultured with huTRIM5α- but not rhTRIM5α-expressing cells. Our results suggest that cell-associated HIV transmission in humans is blocked only when both donor and recipient cells express rhTRIM5α. These studies further define the role of rhTRIM5α in cell-free and cell-associated HIV transmission and delineate the utility of rhTRIM5α in anti-HIV therapy.