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Abstract The capsid-binding assay is an in vitro experiment used to determine whether cellular proteins interact with the HIV-1 core. In vitro assembled HIV-1 capsids recapitulate the surface of the HIV-1 core. The assay involves the incubation of in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes with the protein in question. Subsequently, the mixture is spun through a sucrose cushion using an ultracentrifuge, and the pellet is analyzed for the presence of the protein in question. Although this binding assay is reliable, it is labor intensive and does not contain washing steps. Here we have developed a simpler and faster assay to measure whether a cellular protein is binding to capsid. More importantly, this novel capsid-binding assay contains washing steps. In this assay, we took advantage of the HIV-1 capsid mutant A14C/E45C protein, which is stabilized by disulfide bonds, and is resistant to washing steps. We validated the reliability and specificity of this novel assay by testing the capsid binding ability of TRIMCyp, CPSF6 and MxB with their corresponding controls. Overall, this novel assay provides a reliable and fast methodology to search for novel capsid binders. Highlights • Novel reliable and fast assay to find new HIV-1 capsid binding proteins was developed. • It has washing steps which helps to remove proteins bound to capsid weakly or non specifically. • Binding of the protein of interest both to wild type and mutant capsid proteins can be tested. • The new assay is more sensitive, easier to perform and allows removing non-specific binding. [ABSTRACT FROM AUTHOR]

Highlights • omTRIMCyp induces NF-κB activation in a dose-dependent, but npmTRIMCyp does not. • Both omTRIMCyp and npmTRIMCyp can promote AP-1 and IFN-β activation. • CypA domain of omTRIMCyp plays an important role in NF-κB activation. • omTRIMCyp enhances IκBα and IRF3 phosphorylation, but npmTRIMCyp does not. Abstract TRIMCyp generated by retrotransposition of a cyclophilin A inserting into TRIM5 locus, has been identified in owl monkey and most of Old World monkeys (OWM). Owl monkey TRIMCyp (omTRIMCyp) inhibits HIV-1 infection by direct interaction with viral capsid and indirect innate immune induction, whereas most of TRIMCyps from OWM cannot inhibit HIV-1, and the impact of which on immunoregulation is largely unknown. Here we reported that omTRIMCyp induces NF-κB, AP-1 and IFN-β activation in a dose-dependent manner, while TRIMCyp from northern pig-tailed macaque (npmTRIMCyp) does not activate NF-κB and moderately enhances AP-1 and IFN-β activities. The Cyclophilin A (CypA) domain plays an important role in omTRIMCyp-mediated NF-κB activation, and RBCC domains have a synergetic effect. We further indicated the mechanism by which npmTRIMCyp unable to activate NF-κB is that npmTRIMCyp hardly phosphorylates IκBα, different from omTRIMCyp which dramatically induces IκBα phosphorylation. Ubiquitination activity of omTRIMCyp was greater than npmTRIMCyp, although both could be ubiquitylated. Given that npmTRIMCyp neither interacts with viral capsid resulting in susceptibility to HIV-1 infection, nor activates NF-κB that is indispensable to HIV-1 provirus transcription, we proposed a model that npmTRIMCyp may play an important role in HIV-1 infected northern pig-tailed macaque with latency. [ABSTRACT FROM AUTHOR]

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.

The origin of novel genes and their evolutionary fates are long-standing questions in evolutionary biology. These questions become more complicated for genes conserved across various lineages, such as TRIM5, an antiretroviral restriction factor and a retrovirus capsid sensor in immune signaling. TRIM5 has been subjected to numerous pathogenic challenges and undergone dynamic evolution, making it an excellent example for studying gene diversification. Previous studies among several species showed that TRIM5 gained genetic and functional novelty in a lineage-specific manner, either through gene duplication or a cyclophilin A retrotransposing into the TRIM5 locus, creating the gene fusion known as TRIM5-Cyclophilin A (TRIMCyp). To date, the general pattern of TRIM5 across the mammalian lineage remains elusive. In this study, we surveyed 36 mammalian genomes to verify a potentially novel TRIM5 pattern that uniquely seems to have occurred in tree shrews (Tupaia belangeri), and found that both gene duplication and retrotransposition worked jointly to form a specific TRIM5/TRIMCyp cluster not found among other mammals. Evolutionary analyses showed that tree shrew TRIMCyp (tsTRIMCyp) originated independently in comparison with previously reported TRIMCyps and underwent strong positive selection, whereas no signal of positive selection was detected for other tree shrew TRIM5 (tsTRIM5) genes. Functional assay results suggest a functional divergence between tsTRIMCyp and its closest paralog TRIM5-4, likely reflecting different fates under diverse evolutionary forces. These findings present a rare example of novel gene origination resulting from a combination of gene duplication, retrotransposition, and exon shuffling processes, providing a new paradigm to study genetic innovations and evolutionary fates of duplicated genes. [ABSTRACT FROM PUBLISHER]

Highlights: [•] TRIMe7-CypA expresses in rhesus and pig-tailed, but not long-tailed macaques. [•] TRIMe7-CypA does not show the restriction to a HIV-GFP report virus in vitro. [•] It acts as a negative modulator to TRIM5α likely by competitive inhibition. [ABSTRACT FROM AUTHOR]

A recent study showed that the frequency of an antiretroviral factor TRIM5 gene-derived isoform, TRIMCyp, in cynomolgus macaques (Macaca fascicularis) varies widely according to the particular habitat examined. However, whether the findings actually reflect the prevalence of TRIMCyp in wild cynomolgus macaques is still uncertain because the previous data were obtained with captive monkeys in breeding and rearing facilities. Here, we characterized the TRIM5 gene in cynomolgus macaques captured in the wild, and found that the frequency of the TRIMCyp allele was comparable to those in captive monkeys. This suggests that the previous results with captive monkeys do indeed reflect the natural allele frequency and that breeding and rearing facilities may not affect the frequency of TRIM5 alleles. Interestingly, the prevalence of a minor haplotype of TRIMCyp in wild macaques from the Philippines was significantly lower than in captive ones, suggesting that it is advantageous for wild monkeys to possess the major haplotype of TRIMCyp. Overall, our results add to our understanding of the geographic and genetic prevalence of cynomolgus macaque TRIMCyp.

Background: Many species of non-human primates in Africa are naturally infected by simian immunodeficiency viruses (SIV) and humans stand at the forefront of exposure to these viruses in Sub-Saharan Africa. Cross-species transmission and adaptation of SIV to humans have given rise to human immunodeficiency viruses (HIV-1 and HIV-2) on twelve accountable, independent occasions. However, the determinants contributing to a simian-to-human lasting transmission are not fully understood. Following entry, viral cores are released into the cytoplasm and become the principal target of host cellular factors. Here, we evaluated cellular factors likely to be involved in potential new SIV cross-species transmissions. We investigated the interactions of capsids from naturally circulating SIV isolates with both HIV-1 restricting (i.e. TRIM5 proteins) and facilitating (i.e. cyclophilin A and nucleopore-associated Nup358/RanBP2 and Nup153) factors in single-round infectivity assays that reproduce early stages of the viral life-cycle. Results: We show that human TRIM5α is unlikely to prevent cross-species transmission of any SIV we tested and observed that the SIV CA-CypA interaction is a widespread but not a universal feature. Moreover, entry in the nucleus of different SIV appeared to follow pathways that do not necessarily recruit Nup358/RanBP2 or Nup153, and this regardless of their interaction with CypA. Nevertheless, we found that, like HIV-1, human-adapted HIV-2 infection was dependent on Nup358/RanBP2 and Nup153 interactions for optimal infection. Furthermore, we found that, unlike HIV CA, SIV CA did not require a direct interaction with the Cyp-like domain of Nup358/RanBP2 to carry out successful infection. Conclusions: Circulating SIV present a variety of phenotypes with regard to CA-interacting restricting or facilitating factors. Altogether, we unveiled unidentified pathways for SIV CA, which could also be exploited by HIV in different cellular contexts, to drive entry into the nucleus. Our findings warrant a closer evaluation of other potential defenses against circulating SIV. [ABSTRACT FROM AUTHOR]

The tripartite motif protein (TRIM)5α/CypA fusion protein TRIMCyp in Old World monkeys is generally considered unable to restrict HIV-1 replication. Monkeys with TRIMCyp can serve as a unique animal model for studies of HIV-1 infection. The present study investigated the distribution and expression status of TRIMCyp in four species of macaques originating from China and its borderlands: pigtail macaques ( Macaca nemestrina), rhesus macaques ( Macaca mulatta), long-tailed macaques ( Macaca fascicularis), and Tibetan macaques ( Macaca thibetana). The results revealed that the frequencies of the TRIMCyp genotype were significantly different among different species and even within different populations of the same species. Interestingly, the TRIMCyp genotype was more prevalent among macaques originating from Yunnan and surrounding regions than those from other regions of China. Importantly, TRIMCyp individuals were first identified in Chinese M. mulatta originating from Yunnan, although multiple earlier studies failed to find CypA retrotransposition in this subspecies. Furthermore, TRIMe7-CypA, one of the splicing isoforms of the TRIMCyp transcript was expressed in M. nemestrina and M. mulatta but not M. fascicularis. The intra- and interspecies polymorphisms in the deduced TRIMCyp amino acid sequences of these macaques were also analyzed. Taken together, the data in this study provide important information about the genomic background of TRIMCyp among major species of Chinese macaques. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Gene therapy approaches to the treatment of HIV infection have targeted both viral gene expression and the cellular factors that are essential for virus replication. However, significant concerns have been raised regarding the potential toxic effects of such therapies, the emergence of resistant viral variants and unforeseen biological consequences such as enhanced susceptibility to unrelated pathogens. Novel restriction factors formed by the fusion of the tripartite motif protein (TRIM5) and cyclophilin A (CypA), or “TRIMCyps”, offer an effective antiviral defence strategy with a very low potential for toxicity. In order to investigate the potential therapeutic utility of TRIMCyps in gene therapy for AIDS, a synthetic fusion protein between feline TRIM5 and feline CypA was generated and transduced into cells susceptible to infection with feline immunodeficiency virus (FIV). The synthetic feline TRIMCyp was highly efficient at preventing infection with both HIV and FIV and the cells resisted productive infection with FIV from either the domestic cat or the puma. Feline TRIMCyp and FIV infection of the cat offers a unique opportunity to evaluate TRIMCyp-based approaches to genetic therapy for HIV infection and the treatment of AIDS. [Copyright &y& Elsevier]

Abstract: Retroviral restriction factor research is explaining long-standing lentiviral mysteries. Asking why a particular retrovirus cannot complete a critical part of its life cycle in cells of a particular species has been the starting point for numerous discoveries, including heretofore elusive functions of HIV-1 accessory genes. The potential for therapeutic application is substantial. Analyzing the feline immunodeficiency virus (FIV) life cycle has been instrumental and the source of some surprising observations in this field. FIV is restricted in cells of various primates by several restriction factors including APOBEC3 proteins and, uniquely, TRIM proteins from both Old and New World monkeys. In contrast, the feline genome does not encode functional TRIM5alpha or TRIMCyp proteins and HIV-1 is primarily blocked in feline cells by APOBEC3 proteins. These can be overcome by inserting FIV vif or even SIVmac vif into HIV-1. The domestic cat and its lentivirus are positioned to offer strategic research opportunities as the field moves forward. [Copyright &y& Elsevier]

Abstract: Recent studies have extended the rapidly developing retroviral restriction factor field to cells of carnivore species. Carnivoran genomes, and the domestic cat genome in particular, are revealing intriguing properties vis-à-vis the primate and feline lentiviruses, not only with respect to their repertoires of virus-blocking restriction factors but also replication-enabling dependency factors. Therapeutic application of restriction factors is envisioned for human immunodeficiency virus (HIV) disease and the feline immunodeficiency virus (FIV) model has promise for testing important hypotheses at the basic and translational level. Feline cell-tropic HIV-1 clones have also been generated by a strategy of restriction factor evasion. We review progress in this area in the context of what is known about retroviral restriction factors such as TRIM5α, TRIMCyp, APOBEC3 proteins and BST-2/Tetherin. [Copyright &y& Elsevier]

Abstract: In owl monkeys, the typical retroviral restriction factor of primates, TRIM5α, is replaced by TRIMCyp. TRIMCyp consists of the TRIM5 RING, B-box 2 and coiled-coil domains, as well as the intervening linker regions, fused with cyclophilin A. TRIMCyp restricts infection of retroviruses, such as human immunodeficiency virus (HIV-1) and feline immunodeficiency virus (FIV), with capsids that can bind cyclophilin A. The TRIM5 coiled coil promotes the trimerization of TRIMCyp. Here we show that cyclophilin A that is oligomeric as a result of fusion with a heterologous multimer exhibits substantial antiretroviral activity. The addition of the TRIM5 RING, B-box 2 and Linker 2 to oligomeric cyclophilin A generated a protein with antiretroviral activity approaching that of wild-type TRIMCyp. Multimerization increased the binding of cyclophilin A to the HIV-1 capsid, promoting accelerated uncoating of the capsid and restriction of infection. [Copyright &y& Elsevier]

Abstract: The retroviral restriction factors, TRIM5α and TRIMCyp, consist of RING and B-box 2 domains separated by a coiled coil from carboxy-terminal domains. These carboxy-terminal domains (the B30.2(SPRY) domain in TRIM5α and the cyclophilin A domain in TRIMCyp) recognize the retroviral capsid. Here we show that some B-box 2 changes in TRIM5α, but not in TRIMCyp, resulted in decreased human immunodeficiency virus (HIV-1) capsid binding. The phenotypic effects of these B-box 2 changes on the restriction of retroviral infection depended on the potency of restriction and the affinity of the TRIM5α interaction with the viral capsid, two properties specified by the B30.2(SPRY) domain. Thus, some alterations in the TRIM5α B-box 2 domain apparently affect the orientation or conformation of the B30.2(SPRY) domain, influencing capsid recognition. [Copyright &y& Elsevier]

Abstract: TRIM5α and TRIMCyp are retroviral restriction factors that, like other members of the tripartite motif (TRIM) family, contain RING, B-box 2 and coiled-coil domains. We found that both proteins are rapidly turned over, with half-lives of 50–60 min. Polyubiquitylation and rapid degradation of TRIM5α depended upon intact RING and B-box 2 domains. A chimera consisting of monkey TRIM5α with a RING domain of human TRIM21 exhibited a half-life of 210 min, yet potently restricted human immunodeficiency virus; therefore, rapid turnover of TRIM5α is not required for its antiretroviral activity. TRIM5α forms cytoplasmic bodies that contain other polyubiquitylated proteins, heat shock proteins and dynein, and thus resemble aggresome precursors. Consistent with this interpretation, proteasomal inhibitors triggered the formation of TRIM5αrh-containing aggresomes in a microtubule-dependent manner. Thus, TRIM5α levels in the cell are maintained by continuous synthesis and rapid proteasome-mediated degradation, imbalances in which result in the formation of pre-aggresomal cytoplasmic bodies. [Copyright &y& Elsevier]

The HIV-1 capsid is an ordered protein shell that houses the viral genome during early infection. Its expansive surface consists of an ordered and interfacing array of capsid protein hexamers and pentamers that are recognized by numerous cellular proteins. Many of these proteins recognize specific, assembled capsid interfaces not present in unassembled capsid subunits. We used protein-engineering tools to capture diverse capsid assembly intermediates. We built a repertoire of capsid assemblies (ranging from two to 42 capsid protein molecules) that recreate the various surfaces in infectious capsids. These assemblies reveal unique capsid-targeting mechanisms for each of the anti-HIV factors, TRIMCyp, MxB, and TRIM5α, linked to inhibition of virus uncoating and nuclear entry, as well as the HIV-1 cofactor FEZ1 that facilitates virus intracellular trafficking. This capsid assembly repertoire enables elucidation of capsid recognition modes by known capsid-interacting factors, identification of new capsid-interacting factors, and potentially, development of capsid-targeting therapeutics., (Published by Elsevier Inc.)

A recent study showed that the frequency of an antiretroviral factor TRIM5 gene-derived isoform, TRIMCyp, in cynomolgus macaques (Macaca fascicularis) varies widely according to the particular habitat examined. However, whether the findings actually reflect the prevalence of TRIMCyp in wild cynomolgus macaques is still uncertain because the previous data were obtained with captive monkeys in breeding and rearing facilities. Here, we characterized the TRIM5 gene in cynomolgus macaques captured in the wild, and found that the frequency of the TRIMCyp allele was comparable to those in captive monkeys. This suggests that the previous results with captive monkeys do indeed reflect the natural allele frequency and that breeding and rearing facilities may not affect the frequency of TRIM5 alleles. Interestingly, the prevalence of a minor haplotype of TRIMCyp in wild macaques from the Philippines was significantly lower than in captive ones, suggesting that it is advantageous for wild monkeys to possess the major haplotype of TRIMCyp. Overall, our results add to our understanding of the geographic and genetic prevalence of cynomolgus macaque TRIMCyp.

Human immunodeficiency virus (HIV) has a very narrow host range. HIV type 1 (HIV-1) does not infect Old World monkeys, such as the rhesus monkey (Rh). Rh TRIM5α was identified as a factor that confers resistance, intrinsic immunity, to HIV-1 infection. Unfortunately, human TRIM5α is almost powerless to restrict HIV-1. However, human TRIM5α potently restricts N-tropic murine leukemia viruses (MLV) but not B-tropic MLV, indicating that human TRIM5α represents the restriction factor previously designated as Ref1. African green monkey TRIM5α represents another restriction factor previously designated as Lv1, which restricts both HIV-1 and simian immunodeficiency virus isolated from macaque (SIVmac) infection. TRIM5 is a member of the tripartite motif family containing RING, B-box2, and coiled-coil domains. The RING domain is frequently found in E3 ubiquitin ligase, and TRIM5α is thought to degrade viral core via ubiquitin-proteasome-dependent and -independent pathways. The alpha isoform of TRIM5 has an additional C-terminal PRYSPRY domain, which is a determinant of species-specific retrovirus restriction by TRIM5α. On the other hand, the target regions of viral capsid protein (CA) are scattered on the surface of core. A single amino acid difference in the surface-exposed loop between α-helices 6 and 7 (L6/7) of HIV type 2 (HIV-2) CA affects viral sensitivity to human TRIM5α and was also shown to be associated with viral load in West African HIV-2 patients, indicating that human TRIM5α is a critical modulator of HIV-2 replication in vivo. Interestingly, L6/7 of CA corresponds to the MLV determinant of sensitivity to mouse factor Fv1, which potently restricts N-tropic MLV. In addition, human genetic polymorphisms also affect antiviral activity of human TRIM5α. Recently, human TRIM5α was shown to activate signaling pathways that lead to activation of NF-κB and AP-1 by interacting with TAK1 complex. TRIM5α is thus involved in control of viral infection in multiple ways.

Human immunodeficiency virus type 1 (HIV-1) infects humans and chimpanzees but not old world monkeys (OWMs) such as the rhesus monkey (Rh) and cynomolgus monkey (CM). HIV-1 efficiently enters cells of OWMs but encounters a block before reverse transcription. This narrow host range is attributed to a barrier in the host cell. In 2004, the screening of a Rh cDNA library identified tripartite motif 5α (TRIM5α) as a cellular antiviral factor. TRIM5α is one of splicing variants produced by TRIM5 gene and TRIM5 proteins are members of the TRIM family containing RING, B-box 2, and coiled-coil domains. The RING domain is frequently found in E3 ubiquitin ligase and TRIM5α is degraded via the ubiquitin-proteasome-dependent pathway. Among TRIM5 splicing variants, TRIM5α alone has an additional C-terminal PRYSPRY (B30.2) domain. Previous studies have shown that sequence variation in variable regions of the PRYSPRY domain among different monkey species affects species-specific retrovirus infection, while amino acid sequence differences in the viral capsid protein determine viral sensitivity to restriction. TRIM5α recognizes the multimerized capsid proteins (viral core) of an incoming virus by its PRYSPRY domain and is thus believed to control retroviral infection. There are significant intraspecies variations in the Rh-TRIM5 gene. It has also been reported that some Rh and CM individuals have retrotransposed cyclophilin A open reading frame in the TRIM5 gene, which produces TRIM5-cyclophilin A fusion protein (TRIMCyp). TRIMCyp, which was originally identified as an anti-HIV-1 factor of New World owl monkeys, is an interesting example of the gain of a new function by retrotransposition. As different TRIM5 genotypes of Rh showed different levels of simian immunodeficiency virus replication in vivo, the TRIM5 genotyping is thought to be important in acquired immunodeficiency syndrome monkey models.