Abstract
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 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)-β treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (~40-fold inhibition). TRIM5α 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 TRIM5α.
Highlights
Academic Editor: Sébastien NisoleReceived: 6 December 2020Accepted: 22 December 2020Published: 25 December 2020Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.license (https://creativecommons.org/Individuals infected with the human immunodeficiency virus type 1 (HIV-1) are treated with combination antiretroviral therapy
Our results demonstrate that successfully Tripartite-motif-containing protein protein 55 (TRIM5)-edited cells have decreased permissiveness to HIV-1 and that restriction is stimulated by IFN-I as expected
We had designed three gRNAs leading to Cas9-mediated DNA cuts in the vicinity of Arg332 and Arg335 in TRIM5 [60]. gRNA1 induces the cut closest to the desired mutations, just upstream to the Arg332 triplet (Figure 1), and was the one used in this study
Summary
Academic Editor: Sébastien NisoleReceived: 6 December 2020Accepted: 22 December 2020Published: 25 December 2020Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.license (https://creativecommons.org/Individuals infected with the human immunodeficiency virus type 1 (HIV-1) are treated with combination antiretroviral therapy (cART). Despite successfully reducing viral loads to undetectable levels in a large fraction of the treated patients [1], drugs administered under cART have significant side effects [2], complicating adherence. They are not curative, as they do not target latently integrated HIV-1 that constitute the main reservoir [3], and are inefficient in some anatomical sanctuaries such as the central nervous system [4]. Zinc finger nucleases (ZFNs) have been designed to knock out the CCR5 gene, and phase I clinical trials demonstrated that patient-derived ZFN-engineered CCR5-knockout T cells were effective in controlling HIV-1 viral loads in some patients following treatment interruption [6]. Results from a patient in whom CXCR4-tropic HIV-1 rebounded the following transplantation with
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