Department of Medical Biology, Université du Québec à Trois-Rivières, Trois-Rivières, QC G9A 5H7, Canada.
Viruses. 2020 Dec 25;13(1):24. doi: 10.3390/v13010024.
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.
三结构域蛋白 5 异构体 α（TRIM5α）是一种细胞质抗病毒效应物，可被 I 型干扰素（IFN-I）上调。我们之前曾表明，在突变蛋白过表达时，位于逆转录病毒衣壳结合区的两个点突变 R332G/R335G 赋予了人类 TRIM5α靶向和强烈限制 HIV-1 的能力。在这里，我们使用簇状规则间隔短回文重复（CRISPR）-Cas9 介导的同源定向修复（HDR）在人类 TRIM5 基因的内源性基因中引入这两个突变。我们发现 47 个分离的细胞克隆中有 6 个至少含有一个 HDR 编辑等位基因。一个克隆（克隆 6）两个等位基因都含有 R332G，但只有两个等位基因中的一个含有 R335G。在受到 HIV-1 载体的挑战时，与未修饰的细胞相比，克隆 6 的允许性明显降低，而具有单等位基因修饰的细胞克隆则只是稍微不允许。在用干扰素（IFN）-β处理后，HIV-1 感染在克隆 6 中的抑制显著增强（~40 倍抑制）。TRIM5α 敲低证实了编辑的 TRIM5 基因产物抑制了 HIV-1。对 HIV-1 逆转录产物的定量显示，抑制是通过预期的机制发生的。总之，我们证明了通过编辑先天效应基因来有效抑制病毒感染的可行性。我们的结果还强调了双等位基因修饰对于通过 TRIM5α 达到显著抑制水平的重要性。
