Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto, Japan.
Retrovirology. 2009 Dec 15;6:114. doi: 10.1186/1742-4690-6-114.
DNA double strand break (DSB) repair enzymes are thought to be necessary for retroviral infection, especially for the post-integration repair and circularization of viral cDNA. However, the detailed roles of DSB repair enzymes in retroviral infection remain to be elucidated.
A GFP reporter assay showed that the infectivity of an HIV-based vector decreased in ATM- and DNA-PKcs-deficient cells when compared with their complemented cells, while that of an MLV-based vector was diminished in Mre11- and DNA-PKcs-deficient cells. By using a method based on inverse- and Alu-PCR, we analyzed sequences around 3' HIV-1 integration sites in ATM-, Mre11- and NBS1- deficient cells. Increased abnormal junctions between the HIV-1 provirus and the host DNA were found in these mutant cell lines compared to the complemented cell lines and control MRC5SV cells. The abnormal junctions contained two types of insertions: 1) GT dinucleotides, which are normally removed by integrase during integration, and 2) inserted nucleotides of unknown origin. Artemis-deficient cells also showed such abnormalities. In Mre11-deficient cells, part of a primer binding site sequence was also detected. The 5' host-virus junctions in the mutant cells also contained these types of abnormal nucleotides. Moreover, the host-virus junctions of the MLV provirus showed similar abnormalities. These findings suggest that DSB repair enzymes play roles in the 3'-processing reaction and protection of the ends of viral DNA after reverse transcription. We also identified both 5' and 3' junctional sequences of the same provirus by inverse PCR and found that only the 3' junctions were abnormal with aberrant short repeats, indicating that the integration step was partially impaired in these cells. Furthermore, the conserved base preferences around HIV-1 integration sites were partially altered in ATM-deficient cells.
These results suggest that DSB repair enzymes are involved in multiple steps including integration and pre-integration steps during retroviral replication.
DNA 双链断裂 (DSB) 修复酶被认为是逆转录病毒感染所必需的，特别是对于病毒 cDNA 的整合后修复和环化。然而，DSB 修复酶在逆转录病毒感染中的详细作用仍有待阐明。
GFP 报告基因检测显示，与互补细胞相比，ATM 和 DNA-PKcs 缺陷细胞中基于 HIV 的载体的感染性降低，而基于 MLV 的载体的感染性在 Mre11 和 DNA-PKcs 缺陷细胞中降低。通过使用基于反向和 Alu-PCR 的方法，我们分析了 ATM、Mre11 和 NBS1 缺陷细胞中 HIV-1 整合位点周围的序列。与互补细胞系和对照 MRC5SV 细胞相比，在这些突变细胞系中发现 HIV-1 前病毒与宿主 DNA 之间的异常连接增加。异常连接包含两种类型的插入物：1）GT 二核苷酸，整合酶在整合过程中通常会将其切除，以及 2）未知来源的插入核苷酸。Artemis 缺陷细胞也表现出这种异常。在 Mre11 缺陷细胞中，也检测到部分引物结合位点序列。突变细胞中的 5' 宿主病毒连接也含有这些类型的异常核苷酸。此外，MLV 前病毒的宿主病毒连接也显示出类似的异常。这些发现表明 DSB 修复酶在逆转录后病毒 DNA 的 3' 加工反应和末端保护中发挥作用。我们还通过反向 PCR 鉴定了相同前病毒的 5' 和 3' 连接序列，发现只有 3' 连接异常，存在异常短重复，表明这些细胞中的整合步骤部分受损。此外，ATM 缺陷细胞中 HIV-1 整合位点周围的保守碱基偏好部分改变。
这些结果表明 DSB 修复酶参与包括整合和整合前步骤在内的多个步骤，在逆转录病毒复制过程中发挥作用。
