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Preclinical Assessment of Mutant Human TRIM5? as an Anti-HIV-1 Transgene.

ABSTRACT: 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.

SUBMITTER: Jung U

PROVIDER: S-EPMC4630796 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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?.

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Preclinical Assessment of Mutant Human TRIM5? as an Anti-HIV-1 Transgene.

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