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

Tripartite-motif-containing protein 5 isoform alpha (TRIM5 alpha) 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 alpha 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)-beta treatment, inhibition of HIV-1 infection in clone 6 was significantly enhanced (similar to 40-fold inhibition). TRIM5 alpha 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 alpha.

Automated summary

Introducing two mutations into the endogenous human TRIM5 gene using CRISPR-Cas9-mediated HDR significantly enhances TRIM5 alpha's ability to restrict HIV-1. Biallelic modification is crucial for achieving significant levels of inhibition by TRIM5 alpha.