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TRIM5 is a RING domain E3 ubiquitin ligase with potent antiretroviral function. TRIM5 assembles into a hexagonal lattice on retroviral capsids, causing envelopment of the infectious core. Concomitantly, TRIM5 initiates innate immune signaling and orchestrates disassembly of the viral particle, yet how these antiviral responses are regulated by capsid recognition is unclear. We show that hexagonal assembly triggers N-terminal polyubiquitination of TRIM5 that collectively drives antiviral responses. In uninfected cells, N-terminal monoubiquitination triggers non-productive TRIM5 turnover. Upon TRIM5 assembly on virus, a trivalent RING arrangement allows elongation of N-terminally anchored K63-linked ubiquitin chains (N-K63-Ub). N-K63-Ub drives TRIM5 innate immune stimulation and proteasomal degradation. Inducing ubiquitination before TRIM5 assembly triggers premature degradation and ablates antiviral restriction. Conversely, driving N-K63 ubiquitination after TRIM5 assembly enhances innate immune signaling. Thus, the hexagonal geometry of TRIM5's antiviral lattice converts a capsid-binding protein into a multifunctional antiviral platform., (Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.)

TRIM5α is an antiviral, cytoplasmic, E3 ubiquitin (Ub) ligase that assembles on incoming retroviral capsids and induces their premature dissociation. It inhibits reverse transcription of the viral genome and can also synthesize unanchored polyubiquitin (polyUb) chains to stimulate innate immune responses. Here, we show that TRIM5α employs the E2 Ub-conjugating enzyme Ube2W to anchor the Lys63-linked polyUb chains in a process of TRIM5α auto-ubiquitination. Chain anchoring is initiated, in cells and in vitro, through Ube2W-catalyzed monoubiquitination of TRIM5α. This modification serves as a substrate for the elongation of anchored Lys63-linked polyUb chains, catalyzed by the heterodimeric E2 enzyme Ube2N/Ube2V2. Ube2W targets multiple TRIM5α internal lysines with Ub especially lysines 45 and 50, rather than modifying the N-terminal amino group, which is instead αN-acetylated in cells. E2 depletion or Ub mutation inhibits TRIM5α ubiquitination in cells and restores restricted viral reverse transcription, but not infection. Our data indicate that the stepwise formation of anchored Lys63-linked polyUb is a critical early step in the TRIM5α restriction mechanism and identify the E2 Ub-conjugating cofactors involved., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Restriction factors are a collection of antiviral proteins that form an important aspect of the innate immune system. Their constitutive expression allows immediate response to viral infection, ahead of other innate or adaptive immune responses. We review the molecular mechanism of restriction for four categories of restriction factors; TRIM5, tetherin, APOBEC3G and SAMHD1 and go on to consider how the TRIM5 and TRIMCyp proteins in particular, show promise for exploitation using gene therapy strategies. Such approaches could form an important alternative to current anti-HIV-1 drug regimens, especially if combined with strategies to eradicate HIV reservoirs. Autologous CD4+ T cells or their haematopoietic stem cell precursors engineered to express TRIMCyp restriction factors, and provided in a single therapeutic intervention could then be used to restore functional immunity with a pool of cells protected against HIV. We consider the challenges ahead and consider how early clinical phase testing may best be achieved.

Polymorphism in the TRIM5α/TRIMcyp gene, which interacts with the lentiviral capsid, has been shown to impact on simian immunodeficiency virus (SIV) replication in certain macaque species. Here, in the context of a live-attenuated SIV vaccine study conducted in Mauritian-origin cynomolgus macaques (MCM), we demonstrate upregulation of TRIM5α expression in multiple lymphoid tissues immediately following vaccination. Despite this, the restricted range of TRIM5α genotypes and lack of TRIMcyp variants had no or only limited impact on the replication kinetics in vivo of either the SIVmac viral vaccine or wild-type SIVsmE660 challenge. Additionally, there appeared to be no impact of TRIM5α genotype on the outcome of homologous or heterologous vaccination/challenge studies. The limited spectrum of TRIM5α polymorphism in MCM appears to minimize host bias to provide consistency of replication for SIVmac/SIVsm viruses in vivo, and therefore on vaccination and pathogenesis studies conducted in this species.

The TRIM5α restriction factor can protect some species of monkeys, but not humans, from HIV infection. It has also emerged that some monkeys have a cyclophilin A domain retrotransposed into the TRIM5 locus resulting in the expression of a TRIMCyp protein with anti-retroviral activity. A high degree of sequence variation in the primate TRIM5 gene has been reported that varies between populations of rhesus macaques, a widely used non-human primate model of HIV/AIDS, and recently shown to correlate with susceptibility to simian immunodeficiency viruses in this species. Cynomolgus macaques are also used widely in HIV research. A non-indigenous population on Mauritius has highly restricted genetic diversity compared with macaques from Indonesia. The relative allelic diversity of TRIM5α and TRIMCyp within these two sub-populations may impact on the susceptibility of the macaques to simian immunodeficiency virus thereby influencing the outcome of studies using these monkeys. We sought to establish the genetic diversity of these alleles in cynomolgus macaques. We identified seven TRIM5α alleles in Indonesian macaques, three of which are novel, but only three in the Mauritian-origin macaques. Strikingly, 87% of Indonesian, but none of the Mauritian macaques, possessed a retrotransposed Cyp domain. A splice acceptor site single-nucleotide polymorphism that allows formation of a TRIMCyp protein was absent for the TRIM5α alleles found in the Mauritian macaques. The level of allelic diversity reported here is greater than previously proposed for cynomolgus macaque species.

TRIM5α proteins recruit and restrict incoming cytoplasmic retroviruses. Primate TRIM5α sequence diversity underlies species-specific restriction and is likely caused by selective pressure from ancient pathogenic infections. Here we show that TRIM5α from the European brown hare restricts diverse retroviruses. Furthermore, it differs significantly in sequence from TRIM5α from the closely related rabbit, suggesting evolutionary changes in the last 12 million years since these species diverged. We propose that, like primates, lagomorphs have been subject to selective pressure from TRIM5-sensitive viruses, possibly related to the endogenous lentivirus RELIK found in both rabbits and hares.

TRIM5alpha mediates a potent retroviral restriction phenotype in diverse mammalian species. Here, we identify a TRIM5 transcript in cat cells with a truncated B30.2 capsid binding domain and ablated restrictive function which, remarkably, is conserved across the Feliformia. Cat TRIM5 displayed no restriction activity, but ectopic expression conferred a dominant negative effect against human TRIM5alpha. Our findings explain the absence of retroviral restriction in cat cells and suggest that disruption of the TRIM5 locus has arisen independently at least twice in the Carnivora, with implications concerning the evolution of the host and pathogen in this taxon.

The potential risk of cross-species transmission of porcine endogenous retroviruses (PERV) to humans has slowed the development of xenotransplantation, using pigs as organ donors. Here, we show that PERVs are insensitive to restriction by divergent TRIM5alpha molecules despite the fact that they strongly restrict a variety of divergent lentiviruses. We also show that the human PERV A/C recombinant clone 14/220 reverse transcribes with increased efficiency in human cells, leading to significantly higher infectivity. We conclude that xenotransplantation studies should consider the danger of highly infectious TRIM5alpha-insensitive human-tropic PERV recombinants.

Pathogenic viral infections have exerted selection pressure on their hosts to evolve cellular antiviral inhibitors referred to as restriction factors. Examples of such molecules are APOBEC3G, APOBEC3F and TRIM5alpha. APOBEC3G and APOBEC3F are cytidine deaminases that are able to strongly inhibit retroviral replication by at least two mechanisms. They are counteracted by the lentiviral Vif protein. TRIM5alpha binds to sensitive, incoming retroviruses via its C-terminal PRY/SPRY domain and rapidly recruits them to the proteasome before significant viral DNA synthesis can occur. Both of these proteins robustly block retroviral replication in a species-specific way. It remains an open but important question as to whether innate restriction factors such as these can be harnessed to inhibit HIV-1 replication in humans.

Dendritic cells are central to the early events of human immunodeficiency virus type 1 (HIV-1) transmission, but HIV-1 infects dendritic cells inefficiently in vitro compared to activated CD4(+) T cells. There is a strong postentry restriction of HIV-1 infection in dendritic cells, partly mediated by the cellular restriction factor APOBEC3G. Here, we reveal that arsenic trioxide markedly increases HIV infection of immature and mature dendritic cells as well as blood-derived myeloid dendritic cells in an APOBEC3G- and TRIM5alpha-independent way. Our data suggest the presence of powerful, arsenic-sensitive antiviral activities in primary human immune cells of the dendritic cell lineage.

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.

The control of retroviral infection by antiviral factors referred to as restriction factors has become an exciting area in infectious disease research. TRIM5alpha has emerged as an important restriction factor impacting on retroviral replication including HIV-1 replication in primates. TRIM5alpha has a tripartite motif comprising RING, B-Box and coiled coil domains. The antiviral alpha splice variant additionally encodes a B30.2 domain which is recruited to incoming viral cores and determines antiviral specificity. TRIM5 is ubiquitinylated and rapidly turned over by the proteasome in a RING dependent way. Protecting restricted virus from degradation, by inhibiting the proteasome, rescues DNA synthesis, but not infectivity, indicating that restriction of infectivity by TRIM5alpha does not depend on the proteasome but the early block to DNA synthesis is likely to be mediated by rapid degradation of the restricted cores. The peptidyl prolyl isomerase enzyme cyclophilin A isomerises a peptide bond on the surface of the HIV-1 capsid and impacts on sensitivity to restriction by TRIM5alpha from Old World monkeys. This suggests that TRIM5alpha from Old World monkeys might have a preference for a particular capsid isomer and suggests a role for cyclophilin A in innate immunity in general. Whether there are more human antiviral TRIMs remains uncertain although the evidence for TRIM19's (PML) antiviral properties continues to grow. A TRIM5-like molecule with broad antiviral activity in cattle suggests that TRIM mediated innate immunity might be common in mammals. Certainly the continued study of restriction of viral infectivity by antiviral host factors will remain of interest to a broad audience and impact on a variety of areas including development of animal models for infection, development of viral vectors for gene therapy and the search for novel antiviral drug targets.

Lv1/TRIM5alpha (tripartite motif 5alpha) has recently emerged as an important factor influencing species-specific permissivity to retroviral infection in a range of primates, including humans. Old World monkey TRIM5alpha blocks human immunodeficiency virus type 1 (HIV-1) infectivity, and the human and New World monkey TRIM5alpha proteins are inactive against HIV-1 but active against divergent murine (N-tropic murine leukemia virus [MLV-N]) and simian (simian immunodeficiency virus from rhesus macaque [SIVmac]) retroviruses, respectively. Here we demonstrate antiviral activity of the first nonprimate TRIM protein, from cattle, active against divergent retroviruses, including HIV-1. The number of closely related human TRIM sequences makes assignment of the bovine sequence as a TRIM5alpha ortholog uncertain, and we therefore refer to it as bovine Lv1. Bovine Lv1 is closely related to primate TRIM5alpha proteins in the N-terminal RING and B-box 2 domains but significantly less homologous in the C-terminal B30.2 domain, particularly in the region shown to influence antiviral specificity. Intriguingly, some viruses restricted by bovine Lv1, including HIV-1 and MLV-N, are unable to synthesize viral DNA by reverse transcription, whereas restricted HIV-2 makes normal amounts of DNA. The data support the conclusion that TRIM protein-mediated restriction of retroviral infection is a more common attribute of mammals than previously appreciated.

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.

The restriction factors Fv1 and TRIM5alpha provide dominant blocks to retroviral infection, targeting incoming capsids at a postentry, preintegration step. They both restrict N-tropic murine leukemia virus with similar specificity yet act at different points in the viral life cycle. TRIM5alpha-restricted virus is usually unable to reverse transcribe, whereas Fv1-restricted virus reverse transcribes normally. Here we investigate the relationship between these two restriction factors by expressing Fv1 alleles in human cells. We demonstrate that Fv1 is able to compete with TRIM5alpha for virus before reverse transcription. In human cells expressing Fv1(b), N-tropic restricted virus becomes less infectious but reverse transcribes more efficiently, indicating competition between the two antiviral molecules and protection of the virus from TRIM5alpha by Fv1. Our findings suggest that, like TRIM5alpha, Fv1 interacts with virus before reverse transcription, but the consequences of this interaction are not realized until a later stage of the life cycle. We also demonstrate that Fv1 is functionally independent of TRIM5alpha when expressed in human cells.

It is of great interest to understand the molecular details of the pathways that constitute species barriers to viral infection. The tripartite motif protein TRIM5alpha has emerged as an important mediator of species-specific retroviral replication and innate immunity. This review considers the role of TRIM5alpha as an antiviral protein in mammals. The methods used to identify species-specific restriction to retroviral infection, and the identification of TRIM5alpha itself, are outlined. TRIM5alpha mediates an early postentry block to sensitive retroviral infection, usually before viral DNA synthesis. Results from mutational analysis of TRIM5alpha and their contribution to a mechanistic model for TRIM5alpha antiviral activity are discussed. The antiviral role of other TRIM proteins is considered, as is the role of TRIM5alpha cytoplasmic bodies.

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.

The rhesus macaque tripartite motif containing protein TRIM5alpha specifically restricts HIV-1 infection at an early post-entry step before reverse transcription [Stremlau, M., Owens, C. M., Perron, M. J., Kiessling, M., Autissier, P. & Sodroski, J. (2004) Nature 427, 848-853]. Here, we show that the human and African green monkey (AGM) TRIM5alpha genes encode Ref1 and Lv1 antiretroviral activities, respectively. Expression of TRIM5alpha in permissive cat cells renders them resistant to restriction-sensitive murine leukemia virus but not closely related insensitive virus. Disruption of TRIM5alpha expression in human and AGM cells with small interfering RNA rescues infectivity of restricted virus without affecting unrestricted virus. We also demonstrate that the activity of the murine restriction factor Fv1 depends on TRIM5alpha expression when Fv1 is expressed in human cells. Furthermore, a drug that modifies the behavior of the related promyelocytic leukemia protein PML specifically rescues infection by viruses restricted by human TRIM5alpha. Alignment of the TRIM5alpha proteins from rhesus macaque and AGM indicates an 18-aa insertion. We speculate that this insertion may contribute to the broader specificity of the AGM TRIM5alpha restriction as compared with the human and rhesus macaque proteins.