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.     

TRIM5 alpha Drives SIVsmm Evolution in Rhesus Macaques

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 TRIM5^Q was significantly higher than in macaques expressing two restrictive TRIM5alpha alleles TRIM5^TFP/TFP or TRIM5^Cyp/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 TRIM5^TFP restriction and substitutions in the CypA binding-loop (GPLPA87-91) in capsid were associated with escape from TRIM5^Cyp. 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 TRIM^TFP 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.     

Retrovirus Restriction by TRIM5 Proteins Requires Recognition of Only a Small Fraction of Viral Capsid Subunits

The host restriction factors TRIM5α and TRIMCyp potently inhibit retrovirus infection by binding to the incoming retrovirus capsid. TRIM5 proteins are dimeric, and their association with the viral capsid appears to be enhanced by avidity effects owing to formation of higher-order oligomeric complexes. We examined the stoichiometric requirement for TRIM5 functional recognition by quantifying the efficiencies of restriction of HIV-1 and murine leukemia virus (MLV) particles containing various proportions of restriction-sensitive and -insensitive CA subunits. Both TRIMCyp and TRIM5α inhibited infection of retrovirus particles containing as little as 25% of the restriction-sensitive CA protein. Accordingly, we also observed efficient binding of TRIMCyp in vitro to capsid assemblies containing as little as one-fourth wild-type CA protein. Paradoxically, the ability of HIV-1 particles to abrogate TRIMCyp restriction in trans was more strongly dependent on the fraction of wild-type CA than was restriction of infection. Collectively, our results indicate that TRIM5 restriction factors bind to retroviral capsids in a highly cooperative manner and suggest that TRIM5 can engage a capsid lattice containing a minimum of three or fewer recognizable subunits per hexamer. Our study supports a model in which localized binding of TRIM5 to the viral capsid nucleates rapid polymerization of a TRIM5 lattice on the capsid surface.     

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.     

Interfering Residues Narrow the Spectrum of MLV Restriction by Human TRIM5α

TRIM5α 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 TRIM5α-mediated MLV restriction. Our results reveal that changes at tyrosine³³⁶ of human TRIM5α, 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 huTRIM5α 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 TRIM5α-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.     

TRIM5α Modulates Immunodeficiency Virus Control in Rhesus Monkeys

The cytoplasmic TRIM5α proteins of certain mammalian lineages efficiently recognize the incoming capsids of particular retroviruses and potently restrict infection in a species-specific manner. Successful retroviruses have evolved capsids that are less efficiently recognized by the TRIM5α proteins of the natural hosts. To address whether TRIM5α contributes to the outcome of retroviral infection in a susceptible host species, we investigated the impact of TRIM5 polymorphisms in rhesus monkeys on the course of a simian immunodeficiency virus (SIV) infection. Full-length TRIM5α cDNAs were derived from each of 79 outbred monkeys and sequenced. Associations were explored between the expression of particular TRIM5 alleles and both the permissiveness of cells to SIV infection in vitro and clinical sequelae of SIV infection in vivo. Natural variation in the TRIM5α B30.2(SPRY) domain influenced the efficiency of SIVmac capsid binding and the in vitro susceptibility of cells from the monkeys to SIVmac infection. We also show the importance in vivo of the interaction of SIVmac with different allelic forms of TRIM5, demonstrating that particular alleles are associated with as much as 1.3 median log difference in set-point viral loads in SIVmac-infected rhesus monkeys. Moreover, these allelic forms of TRIM5 were associated with the extent of loss of central memory (CM) CD4+ T cells and the rate of progression to AIDS in the infected monkeys. These findings demonstrate a central role for TRIM5α in limiting the replication of an immunodeficiency virus infection in a primate host.     

Assisted Evolution Enables HIV-1 to Overcome a High TRIM5α-Imposed Genetic Barrier to Rhesus Macaque Tropism

Diversification of antiretroviral factors during host evolution has erected formidable barriers to cross-species retrovirus transmission. This phenomenon likely protects humans from infection by many modern retroviruses, but it has also impaired the development of primate models of HIV-1 infection. Indeed, rhesus macaques are resistant to HIV-1, in part due to restriction imposed by the TRIM5α protein (rhTRIM5α). Initially, we attempted to derive rhTRIM5α-resistant HIV-1 strains using two strategies. First, HIV-1 was passaged in engineered human cells expressing rhTRIM5α. Second, a library of randomly mutagenized capsid protein (CA) sequences was screened for mutations that reduced rhTRIM5α sensitivity. Both approaches identified several individual mutations in CA that reduced rhTRIM5α sensitivity. However, neither approach yielded mutants that were fully resistant, perhaps because the locations of the mutations suggested that TRIM5α recognizes multiple determinants on the capsid surface. Moreover, even though additive effects of various CA mutations on HIV-1 resistance to rhTRIM5α were observed, combinations that gave full resistance were highly detrimental to fitness. Therefore, we employed an ‘assisted evolution’ approach in which individual CA mutations that reduced rhTRIM5α sensitivity without fitness penalties were randomly assorted in a library of viral clones containing synthetic CA sequences. Subsequent passage of the viral library in rhTRIM5α-expressing cells resulted in the selection of individual viral species that were fully fit and resistant to rhTRIM5α. These viruses encoded combinations of five mutations in CA that conferred complete or near complete resistance to the disruptive effects of rhTRIM5α on incoming viral cores, by abolishing recognition of the viral capsid. Importantly, HIV-1 variants encoding these CA substitutions and SIV_mac239 Vif replicated efficiently in primary rhesus macaque lymphocytes. These findings demonstrate that rhTRIM5α is difficult to but not impossible to evade, and doing so should facilitate the development of primate models of HIV-1 infection.     

The Second-Site Mutation in the Herpes Simplex Virus Recombinants Lacking the γ134.5 Genes Precludes Shutoff of Protein Synthesis by Blocking the Phosphorylation of eIF-2α

In cells infected with the herpes simplex virus 1 (HSV-1) recombinant R3616 lacking both copies of the γ₁34.5 gene, the double-stranded protein kinase R (PKR) is activated, eIF-2α is phosphorylated, and protein synthesis is shut off. Although PKR is also activated in cells infected with the wild-type virus, the product of the γ₁34.5 gene, infected-cell protein 34.5 (ICP34.5), binds protein phosphatase 1α and redirects it to dephosphorylate eIF-2α, thus enabling sustained protein synthesis. Serial passage in human cells of a mutant lacking the γ₁34.5 gene yields second-site, compensatory mutants lacking various domains of the α47 gene situated next to the U_S11 gene (I. Mohr and Y. Gluzman, EMBO J. 15:4759–4766, 1996). We report the construction of two recombinant viruses: R5103, lacking the γ₁34.5, U_S8, -9, -10, and -11, and α47 (U_S12) genes; and R5104, derived from R5103 and carrying a chimeric DNA fragment containing the U_S10 gene and the promoter of the α47 gene fused to the coding domain of the U_S11 gene. R5104 exhibited a protein synthesis profile similar to that of wild-type virus, whereas protein synthesis was shut off in cells infected with R5103 virus. Studies on the wild-type parent and mutant viruses showed the following: (i) PKR was activated in cells infected with parent or mutant virus but not in mock-infected cells, consistent with earlier studies; (ii) lysates of R3616, R5103, and R5104 virus-infected cells lacked the phosphatase activity specific for eIF-2α characteristic of wild-type virus-infected cells; and (iii) lysates of R3616 and R5103, which lacked the second-site compensatory mutation, contained an activity which phosphorylated eIF-2α in vitro, whereas lysates of mock-infected cells or cells infected with HSV-1(F) or R5104 did not phosphorylate eIF-2α. We conclude that in contrast to wild-type virus-infected cells, which preclude the shutoff of protein synthesis by causing rapid dephosphorylation of eIF-2α, in cells infected with γ₁34.5⁻ virus carrying the compensatory mutation, eIF-2α is not phosphorylated. The activity made apparent by the second-site mutation may represent a more ancient mechanism evolved to preclude the shutoff of protein synthesis.     

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.     

Identification of postentry restrictions to Mason-Pfizer monkey virus infection in New World monkey cells

TRIM5α has been shown to be a major postentry determinant of the host range for gammaretroviruses and lentiviruses and, more recently, spumaviruses. However, the restrictive potential of TRIM5α against other retroviruses has been largely unexplored. We sought to determine whether or not Mason-Pfizer monkey virus (M-PMV), a prototype betaretrovirus isolated from rhesus macaques, was sensitive to restriction by TRIM5α. Cell lines from both Old World and New World primate species were screened for their susceptibility to infection by vesicular stomatitis virus G protein pseudotyped M-PMV. All of the cell lines tested that were established from Old World primates were found to be susceptible to M-PMV infection. However, fibroblasts established from three New World monkey species specifically resisted infection by this virus. Exogenously expressing TRIM5α from either tamarin or squirrel monkeys in permissive cell lines resulted in a block to M-PMV infection. Restriction in the resistant cell line of spider monkey origin was determined to occur at a postentry stage. However, spider monkey TRIM5α expression in permissive cells failed to restrict M-PMV infection, and interference with endogenous TRIM5α in the spider monkey fibroblasts failed to relieve the block to infectivity. Our results demonstrate that TRIM5α specificity extends to betaretroviruses and suggest that New World monkeys have evolved additional mechanisms to restrict the infection of at least one primate betaretrovirus.     

Costameric integrin and sarcoglycan protein levels are altered in a Drosophila model for Limb-girdle muscular dystrophy type 2H

Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of βPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and β-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration.     

The Carboxyl-Terminus of Human Immunodeficiency Virus Type 2 Circulating Recombinant form 01_AB Capsid Protein Affects Sensitivity to Human TRIM5α

Human immunodeficiency virus (HIV) type 2 shows limited geographical distribution compared with HIV type 1. Although 8 genetic groups of HIV type 2 (HIV-2) have been described, recombinant viruses between these groups are rarely observed. Recently, three HIV-2 patients in Japan were described with rapidly progressive, acquired immunodeficiency. These patients were infected with an A/B inter-group recombinant designated CRF01_AB. Here, we characterize the capsid protein (CA) encoded by the viruses from these patients. HIV-2 CRF01_AB CA showed unique amino acid sequence almost equally distinct from group A and group B viruses. Notably, HIV-2 CRF01_AB CA showed potent resistance to human TRIM5α. In addition to the previously identified amino acid position 119 in the N-terminal domain of CA, we found that HIV-2 CRF01_AB-specific amino acid substitutions in the C-terminal domain also were necessary for resistance to human TRIM5α. These results indicate that retroviruses can evade TRIM5α by substitution at residues within the C-terminal domain of CA.