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.

TRIM5α is a cytoplasmic antiviral effector induced by type I interferons (IFN-I) that has the potential to intercept incoming retroviruses by interacting with their capsid core, leading to uncoating induction and the partial degradation of core components. Most HIV-1 strains escape restriction by human TRIM5α due to a lack of interaction between TRIM5α and its viral molecular target. We previously showed, however, that two point mutations, R332G/R335G, in the capsid-binding region confer human TRIM5α with the capacity to target and strongly restrict HIV-1 upon the overexpression of the mutated protein. Here, we explored the possibility to introduce these two mutations in the endogenous human TRIM5 gene by CRISPR-Cas9-mediated gene editing. For this, we electroporated CRISPR ribonucleoproteins (RNPs) and the donor DNA into Jurkat T lymphocytic cells and isolated clones by limiting dilution. We analyzed 47 clones using specific PCR assays, and found that six clones (13%) contained at least one gene-edited allele. One clone (clone 6) had both alleles edited for R332G, but only one of the two alleles was edited for 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 IFN-β treatment, the inhibition of HIV-1 infection in clone 6 was significantly enhanced (~50-fold inhibition), whereas IFN-β treatment had no effect on TRIM5α overexpressed by retroviral transduction. Knockdown experiments confirmed that HIV-1 was inhibited by the edited TRIM5 gene products, whereas quantification of HIV-1 reverse transcription products confirmed 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, but our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.

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 over-expression of the mutated protein. Here, we used 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 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 editing of innate effector genes. Our results also emphasize the importance of biallelic modification in order to reach significant levels of inhibition by TRIM5α.