BackgroundHIV-1 integration results in genomic DNA gaps that are repaired by cellular DNA repair pathways. This step of the lentiviral life cycle remains poorly understood despite its crucial importance for successful replication. We and others reported that Ku70 protein of the non-homologous end joining pathway (NHEJ) directly binds HIV-1 integrase (IN). Here, we studied the importance of this interaction for post-integrational gap repair and the recruitment of NHEJ factors in this process.ResultsWe engineered HIV-based pseudovirus with mutant IN defective in Ku70 binding and generated heterozygous Ku70, Ku80 and DNA-PKcs human knockout (KO) cells using CRISPR/Cas9. KO of either of these proteins or inhibition of DNA-PKcs catalytic activity substantially decreased the infectivity of HIV-1 with native IN but not with the mutant one. We used a recently developed qPCR assay for the measurement of gap repair efficiency to show that HIV-1 with mutant IN was defective in DNA post-integrational repair, whereas the wild type virus displayed such a defect only when NHEJ system was disrupted in any way. This effect was present in CRISPR/Cas9 modified 293T cells, in Jurkat and CEM lymphoid lines and in primary human PBMCs.ConclusionsOur data provide evidence that IN recruits DNA-PK to the site of HIV-1 post-integrational repair due to Ku70 binding—a novel finding that explains the involvement of DNA-PK despite the absence of free double stranded DNA breaks. In addition, our data clearly indicate the importance of interactions between HIV-1 IN and Ku70 in HIV-1 replication at the post-integrational repair step.

中文翻译：

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由于 Ku70 与 HIV-1 整合酶结合，NHEJ 通路参与了整合后的 DNA 修复

背景HIV-1 整合导致基因组DNA 缺口被细胞DNA 修复途径修复。尽管慢病毒生命周期的这一步骤对于成功复制至关重要，但它仍然知之甚少。我们和其他人报道了非同源末端连接途径 (NHEJ) 的 Ku70 蛋白直接结合 HIV-1 整合酶 (IN)。在这里，我们研究了这种相互作用对于整合后间隙修复和 NHEJ 因子在该过程中的招募的重要性。 结果我们设计了具有 Ku70 结合缺陷的突变体 IN 的基于 HIV 的假病毒，并产生了杂合的 Ku70、Ku80 和 DNA-PKcs 人类敲除(KO) 使用 CRISPR/Cas9 的细胞。这些蛋白质中的任何一种的 KO 或 DNA-PKcs 催化活性的抑制显着降低了 HIV-1 对天然 IN 的感染性，但对突变体则没有。我们使用最近开发的 qPCR 测定来测量间隙修复效率，以表明具有突变体 IN 的 HIV-1 在 DNA 后整合修复中存在缺陷，而野生型病毒仅在 NHEJ 系统以任何方式被破坏时才显示出这种缺陷. 这种效应存在于 CRISPR/Cas9 修饰的 293T 细胞、Jurkat 和 CEM 淋巴系以及原代人 PBMC 中。结论我们的数据提供证据表明，由于 Ku70 结合，IN 将 DNA-PK 募集到 HIV-1 整合后修复部位——一项新发现解释了 DNA-PK 的参与，尽管没有游离的双链 DNA 断裂。此外，我们的数据清楚地表明了 HIV-1 IN 和 Ku70 在整合后修复步骤中 HIV-1 复制中相互作用的重要性。