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Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1.

ABSTRACT: Tripartite-motif-containing protein 5 isoform ? (TRIM5?) is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5? the capacity to target and strongly restrict HIV-1 upon overexpression of the mutated protein. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (HDR) to introduce these two mutations in the endogenous human TRIM5 gene. We found 6 out of 47 isolated cell clones containing at least one HDR-edited allele. One clone (clone 6) had both alleles containing R332G, but only one of the two alleles containing R335G. Upon challenge with an HIV-1 vector, clone 6 was significantly less permissive compared to unmodified cells, whereas the cell clones with monoallelic modifications were only slightly less permissive. Following interferon (IFN)-? treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5? knockdown confirmed that HIV-1 was inhibited by the edited TRIM5 gene products. Quantification of HIV-1 reverse transcription products showed that inhibition occurred through the expected mechanism. In conclusion, we demonstrate the feasibility of potently inhibiting a viral infection through the editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5?.

SUBMITTER: Desaulniers K

PROVIDER: S-EPMC7824555 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1.

Désaulniers Kevin K Ortiz Levine L Dufour Caroline C Claudel Alix A Plourde Mélodie B MB Merindol Natacha N Berthoux Lionel L

Viruses 20201225 1

Tripartite-motif-containing protein 5 isoform α (TRIM5α) is a cytoplasmic antiretroviral effector upregulated by type I interferons (IFN-I). We previously showed that two points mutations, R332G/R335G, in the retroviral capsid-binding region confer human TRIM5α the capacity to target and strongly restrict HIV-1 upon overexpression of the mutated protein. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair (HDR) to introduce thes ...[more]

PMID: 33375604

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Project description:Current HIV-1 gene therapy approaches aim at stopping the viral life cycle at its earliest steps, such as entry or immediate postentry events. Among the most widely adopted strategies are CCR5 downregulation/knockout and the use of broadly neutralizing antibodies. However, the long-term efficacy and side effects are still unclear. TRIM5? is an interferon-stimulated restriction factor that can intercept incoming retroviruses within one hour of cytosolic entry and potently inhibit the infectivity of restriction-sensitive viruses. The human TRIM5? (TRIM5?hu) generally does not efficiently target HIV-1, but point mutations in its capsid-binding domain can confer anti-HIV-1 activity. Although the mechanisms by which TRIM5?hu mutants inhibit HIV-1 are relatively well understood, their characterization as potential transgenes for gene therapy is lacking. Additionally, previous reports of general immune activation by overexpression of TRIM5? have hindered its broad adoption as a potential transgene. Here we demonstrate the ability of the R332G-R335G TRIM5?hu mutant to efficiently restrict highly divergent HIV-1 strains, including Group O, as well as clinical isolates bearing cytotoxic T lymphocyte escape mutations. R332G-R335G TRIM5?hu efficiently protected human lymphocytes against HIV-1 infection, even when expressed at relatively low levels following lentiviral transduction. Most importantly, under these conditions Rhesus macaque TRIM5? (TRIM5?Rh) and TRIM5?hu (wild-type or mutated) had no major effects on the NF-?B pathway. Transgenic TRIM5? did not modulate the kinetics of I?B?, JunB, and TNFAIP3 expression following TNF-? treatment. Finally, we show that human lymphocytes expressing R332G-R335G TRIM5?hu have clear survival advantages over unmodified parental cells in the presence of pathogenic, replication-competent HIV-1. These results support the relevance of R332G-R335G and other mutants of TRIM5?hu as candidate effectors for HIV-1 gene therapy.

Project description:UnlabelledTripartite motif-containing protein 5 (TRIM5) restricts human immunodeficiency virus type 1 (HIV-1) in a species-specific manner by uncoating viral particles while activating early innate responses. Although the contribution of TRIM5 proteins to cellular immunity has not yet been studied, their interactions with the incoming viral capsid and the cellular proteasome led us to hypothesize a role for them. Here, we investigate whether the expression of two nonhuman TRIM5 orthologs, rhesus TRIM5α (RhT5) and TRIM-cyclophilin A (TCyp), both of which are potent restrictors of HIV-1, could enhance immune recognition of infected cells by CD8(+) T cells. We illustrate how TRIM5 restriction improves CD8(+) T-cell-mediated HIV-1 inhibition. Moreover, when TRIM5 activity was blocked by the nonimmunosuppressive analog of cyclosporine (CsA), sarcosine-3(4-methylbenzoate)-CsA (SmBz-CsA), we found a significant reduction in CD107a/MIP-1β expression in HIV-1-specific CD8(+) T cells. This finding underscores the direct link between TRIM5 restriction and activation of CD8(+) T-cell responses. Interestingly, cells expressing RhT5 induced stronger CD8(+) T-cell responses through the specific recognition of the HIV-1 capsid by the immune system. The underlying mechanism of this process may involve TRIM5-specific capsid recruitment to cellular proteasomes and increase peptide availability for loading and presentation of HLA class I antigens. In summary, we identified a novel function for nonhuman TRIM5 variants in cellular immunity. We hypothesize that TRIM5 can couple innate viral sensing and CD8(+) T-cell activation to increase species barriers against retrovirus infection.ImportanceNew therapeutics to tackle HIV-1 infection should aim to combine rapid innate viral sensing and cellular immune recognition. Such strategies could prevent seeding of the viral reservoir and the immune damage that occurs during acute infection. The nonhuman TRIM5 variants, rhesus TRIM5α (RhT5) and TRIM-cyclophilin A (TCyp), are attractive candidates owing to their potency in sensing HIV-1 and blocking its activity. Here, we show that expression of RhT5 and TCyp in HIV-1-infected cells improves CD8(+) T-cell-mediated inhibition through the direct activation of HIV-1-specific CD8(+) T-cell responses. We found that the potency in CD8(+) activation was stronger for RhT5 variants and capsid-specific CD8(+) T cells in a mechanism that relies on TRIM5-dependent particle recruitment to cellular proteasomes. This novel mechanism couples innate viral sensing with cellular immunity in a single protein and could be exploited to develop innovative therapeutics for control of HIV-1 infection.

Project description:BackgroundBecause uncoating of the capsid is linked to reverse transcription, modifications that delay this process lead to the persistence in the cytoplasm of capsids susceptible to recognition by the human restriction factor TRIM5? (hTRIM5?). It is unknown, however, if increasing the time available for capsid-hTRIM5? interactions would actually render viruses more sensitive to hTRIM5?.ResultsViral sensitivity to hTRIM5? was evaluated by comparing their replication in human U373-X4 cells in which hTRIM5? activity had or had not been inhibited by overexpression of human TRIM5?. No differences were observed comparing wild-type HIV-1 and variants carrying mutations in reverse transcriptase or the central polypurine tract that delayed the completion of reverse transcription. In addition, the effect of delaying the onset of reverse transcription for several hours by treating target cells with nevirapine was evaluated using viral isolates with different sensitivities to hTRIM5?. Delaying reverse transcription led to a time-dependent loss in viral infectivity that was increased by inhibiting capsid-cyclophilin A interactions, but did not result in increased viral sensitivity to hTRIM5?, regardless of their intrinsic sensitivity to this restriction factor.ConclusionsConsistent with prior studies, the HIV-1 capsid can be targeted for destruction by hTRIM5?, but different strains display considerable variability in their sensitivity to this restriction factor. Capsids can also be lost more slowly through a TRIM5?-independent process that is accelerated when capsid-cyclophilin A interactions are inhibited, an effect that may reflect changes in the intrinsic stability of the capsid. Blocking the onset or delaying reverse transcription does not, however, increase viral sensitivity to hTRIM5?, indicating that the recognition of the capsids by hTRIM5? is completed rapidly following entry into the cytoplasm, as previously observed for the simian restriction factors TRIM-Cyp and rhesus TRIM5?.

Dataset Information

Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1.

Publications

Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1.

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