Association of TRIMCyp and TRIM5α from assam macaques leads to a functional trade-off between HIV-1 and N-MLV inhibition

TRIM5α restricts retroviruses in a species-specific manner. Cyclophilin A was independently retrotransposed into the TRIM5 loci in different species, leading to the generation of antiviral TRIM5-cyclophilin A(TRIMCyp) proteins. Previously, we found that assam macaques express a TRIMCyp chimera(am TRIMCyp), along with a TRIM5α allelic protein(am TRIM5α). Herein,we investigated the antiviral activity of am TRIMCyp and am TRIM5α individually, as well as their interaction and joint effects.am TRIMCyp showed a divergent restriction pattern from am TRIM5α. Although both proteins potently restricted the replication of HIV-1, only am TRIM5α inhibited N-MLV. Remarkably, cellular anti-HIV-1 activity increased when am TRIMCyp and am TRIM5α were coexpressed, indicating a synergistic block of HIV-1 replication. Consistently, PMBCs from heterozygous am TRIM5α/TRIMCyp showed stronger resistance to HIV-1 infection than those from am TRIM5α/TRIM5α homozygotes. The anti-HIV-1 synergistic effect was dependent on the am TRIMCyp-am TRIM5α interaction. In contrast, am TRIMCyp completely abrogated the anti-N-MLVactivity mediated by am TRIM5α, showing a dominant-negative effect, indicating that the generation of am TRIMCyp was involved in the trade-off between divergent restriction activities. Our results provide a new paradigm to study functional trade-offs mediated by allelic proteins, a theoretical basis for utilizing animal models with various TRIM5 alleles, as well as novel HIV-1 gene therapy strategies.     

Homology-based identification of capsid determinants that protect HIV1 from human TRIM5α restriction

The tropism of retroviruses relies on their ability to exploit cellular factors for their replication as well as to avoid host-encoded inhibitory activities such as TRIM5α. N-tropic murine leukemia virus is sensitive to human TRIM5α (huTRIM5α) restriction, whereas human immunodeficiency virus type 1 (HIV1) escapes this antiviral factor. We previously revealed that mutation of four critical amino acid residues within the capsid can render murine leukemia virus resistant to huTRIM5α. Here, we exploit the high degree of conservation in the tertiary structure of retroviral capsids to map the corresponding positions on the HIV1 capsid. We then demonstrated that, when changes were introduced at some of these positions, HIV1 becomes sensitive to huTRIM5α restriction, a phenomenon reinforced by additionally mutating the nearby cyclophilin A binding loop of the viral protein. These results indicate that retroviruses have evolved similar mechanisms to escape TRIM5α restriction via the interference of structurally homologous determinants in the viral capsid.     

Inhibitory effect of human TRIM5α on HIV-1 production

Tripartite motif-containing 5 isoform-α (TRIM5α), a host restriction factor, blocks infection of some retroviruses at a post-entry, pre-integration stage in a species-specific manner. A recent report by Sakuma et al. describes a second antiretroviral activity of rhesus macaque TRIM5α, which blocks HIV-1 production through rapid degradation of HIV-1 Gag polyproteins. Here, we find that human TRIM5α limits HIV-1 production. Transient expression of TRIM5α decreased HIV-1 production, whereas knockdown of TRIM5α in human cells increased virion release. A single amino acid substitution (R437C) in the SPRY domain diminished the restriction effect. Moderate levels of human wild-type TRIM5α and a little amount of R437C mutant were incorporated into HIV-1 virions. The R437C mutant also lost restriction activity against N-tropic murine leukemia virus infection. However, the corresponding R to C mutation in rhesus macaque TRIM5α had no effect on the restriction ability. Our findings suggest human TRIM5α is an intrinsic immunity factor against HIV-1 infection. The importance of arginine at 437 aa in SPRY domain for the late restriction is species-specific.     

Pressure from TRIM5α Contributes to Control of HIV-1 Replication by Individuals Expressing Protective HLA-B Alleles

The expression of certain HLA class I alleles, including HLA-B*27 and HLA-B*57, is associated with better control of human immunodeficiency virus type 1 (HIV-1) infection, but the mechanisms responsible are not fully understood. We sought evidence that pressure from the human restriction factor TRIM5α (hTRIM5α) could contribute to viral control. The hTRIM5α sensitivity of viruses from both HLA-B*57-positive (HLA-B*57⁺) and HLA-B*27⁺ patients who spontaneously controlled viral replication, but not viruses from viremic patients expressing these alleles, was significantly greater than that of viruses from patients not expressing these protective HLA-B alleles. Overall, a significant negative correlation between hTRIM5α sensitivity and viral load was observed. In HLA-B*57⁺ patients, the T242N mutation in the HLA-B*57-restricted TW10 CD8⁺ T lymphocyte (CTL) epitope was strongly associated with hTRIM5α sensitivity. In HLA-B*27⁺ controllers, hTRIM5α sensitivity was associated with a significant reduction in emergence of key CTL mutations. In several patients, viral evolution to avoid hTRIM5α sensitivity was observed but could be associated with reduced viral replicative capacity. Thus, in individuals expressing protective HLA-B alleles, the combined pressures exerted by CTL, hTRIM5α, and capsid structural constraints can prevent viral escape both by impeding the selection of necessary resistance/compensatory mutations and forcing the selection of escape mutations that increase hTRIM5α sensitivity or impair viral replicative capacity.     

Diversity of TRIM5α and TRIMCyp sequences in cynomolgus macaques from different geographical origins

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.     

Cytoplasmic Body Component TRIM5α Requires Lipid-enriched Microdomains for Efficient HIV-1 Restriction

TRIM5α is a member of the tripartite motif (TRIM) family of proteins and affects both early and late phases of the retroviral life cycle. Although TRIM5α multimerizes to form cytoplasmic bodies, which are thought to play an important role in viral restriction, the identity of TRIM5α-containing cytoplasmic bodies remains elusive. To better understand TRIM5α cytoplasmic body constituents and the cellular proteins that could be involved in the TRIM5α-mediated antiviral activities, we sought TRIM5α-binding factors. We identified a lipid microdomain protein flotillin-1/Reggie-2 as an interacting partner of TRIM5α via co-immunoprecipitation. Immunohistochemistry studies confirmed the co-localization of rhesus monkey TRIM5α (TRIM5αrh) cytoplasmic bodies with flotillin-1/Reggie-2. Caveolin-1, another lipid microdomain-associated protein, also co-localized with TRIM5α cytoplasmic bodies. Intriguingly, disruption of cellular cholesterol by cyclodextrin perturbed TRIM5α cytoplasmic body formation. Furthermore, lipid starvation partially relieved the endogenous post-entry restriction of HIV-1 infection, which could be subsequently restored by lipid repletion. These observations indicate the involvement of cellular lipids in TRIM5α-mediated antiviral activities. Given that many viruses utilize cellular lipid microdomains for viral entry and assembly, it is plausible that lipid-enriched domains provide microenvironments where TRIM5α recognizes retroviral components.     

Rhesus Monkey TRIM5α Has Distinct HIV-1 Restriction Activity Among Different Mammalian Cell Lines

Rhesus monkey TRIM5α (TRIM5α_rh), a member of the tripartite motif (TRIM) family, was identified as the main restriction factor responsible for resistance of old world monkey cells to HIV-1 infection. However, the precise mechanism of HIV-1 infection inhibition by TRIM5α remains elusive and appears to be related to some cellular cofactors. Here we reported that TRIM5α_rh can significantly reduce the infection efficiency of VSV-G pseudotyped HIV-1/MA-YFP virus in human epithelial carcinoma (HeLa) cells, moderately reduce in porcine kidney (PK-15) cells and have no effect on the pseudotyped virus infection in Madin-Darby canine kidney (MDCK) cells. Furthermore, we found that the different HIV-1 restriction activities have no relation with the intracellular localization of TRIM5α_rh. These results indicate that the cellular environment is very important for the efficient anti-HIV-1 activity of TRIM5α_rh. We speculate that some unknown factors required for HIV-1 infection inhibition activity are adequately expressed in HeLa cells, inadequately expressed in PK-15 cells and absent in MDCK cells.     

TRIM5α associates with proteasomal subunits in cells while in complex with HIV-1 virions

Background

The HIV-1 p6 Gag protein regulates the final abscission step of nascent virions from the cell membrane by the action of two late assembly (L-) domains. Although p6 is located within one of the most polymorphic regions of the HIV-1 gag gene, the 52 amino acid peptide binds at least to two cellular budding factors (Tsg101 and ALIX), is a substrate for phosphorylation, ubiquitination, and sumoylation, and mediates the incorporation of the HIV-1 accessory protein Vpr into viral particles. As expected, known functional domains mostly overlap with several conserved residues in p6. In this study, we investigated the importance of the highly conserved serine residue at position 40, which until now has not been assigned to any known function of p6.

Results

Consistently with previous data, we found that mutation of Ser-40 has no effect on ALIX mediated rescue of HIV-1 L-domain mutants. However, the only feasible S40F mutation that preserves the overlapping pol open reading frame (ORF) reduces virus replication in T-cell lines and in human lymphocyte tissue cultivated ex vivo. Most intriguingly, L-domain mediated virus release is not dependent on the integrity of Ser-40. However, the S40F mutation significantly reduces the specific infectivity of released virions. Further, it was observed that mutation of Ser-40 selectively interferes with the cleavage between capsid (CA) and the spacer peptide SP1 in Gag, without affecting cleavage of other Gag products. This deficiency in processing of CA, in consequence, led to an irregular morphology of the virus core and the formation of an electron dense extra core structure. Moreover, the defects induced by the S40F mutation in p6 can be rescued by the A1V mutation in SP1 that generally enhances processing of the CA-SP1 cleavage site.

Conclusions

Overall, these data support a so far unrecognized function of p6 mediated by Ser-40 that occurs independently of the L-domain function, but selectively affects CA maturation and virus core formation, and consequently the infectivity of released virions.     

Secretion of MIP-1β and MIP-1α by CD8(+) T-lymphocytes correlates with HIV-1 inhibition independent of coreceptor usage

CD8⁺ T-lymphocytes can utilize noncytolytic mechanisms to suppress HIV-1 replication through the secretion of soluble factors. The secretion of MIP-1β, MIP-1α, IP-10, MIG, IL-1α, and interferon gamma correlated most strongly with soluble noncytolytic suppression (p < 0.0001). Since the noncytolytic response is impaired by histone hyperacetylation, we examined the ability of histone hyperacetylation to alter the expression of immune-related genes. MIP-1α and IP-10 were also among the genes that were down-regulated by histone hyperacetylation. We define a multifactorial cytokine profile of CD8⁺ T-lymphocytes capable of mediating noncytolytic suppression of CXCR4-tropic HIV-1 replication.