Evidence that stable retroviral transduction and cell survival following DNA integration depend on components of the nonhomologous end joining repair pathway
Evidence that stable retroviral transduction and cell survival following DNA integration depend on components of the nonhomologous end joining repair pathway
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DOI：
10.1128/jvi.78.16.8573-8581.2004
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发表时间：
2004-08-01
影响因子：
5.4
通讯作者：
Skalka, AM
中科院分区：
文献类型：
--
作者：
Daniel, R;Greger, JG;Skalka, AM
我们以前曾报道过几条证据支持细胞DNA修复系统在完成逆转录病毒DNA整合过程中的作用。在病毒DNA整合到宿主DNA中的过程中，如果不能修复中间体，似乎会引发大部分感染细胞的生长停滞或死亡。参与非同源末端连接（NHEJ）途径的细胞蛋白（DNA-PKCS）和损伤信号激酶（ATM和ATR）参与了这一过程。然而，一些研究表明，NHEJ蛋白可能不是完成慢病毒DNA整合所必需的。在这里，我们提供了额外的证据表明，NHEJ蛋白是稳定转导所需的人类免疫缺陷1型（HIV-1）为基础的载体。我们用两种不同的报告基因进行的分析表明，与对照细胞的数量相比，稳定转导的DNA-PKCS缺陷型scid成纤维细胞的数量减少了80%至90%。此外，转导效率可以恢复到野生型scid细胞中的水平，补充功能DNA-PKCS基因。在感染Xrcc 4和连接酶IV缺陷型细胞后，基于HIV-1的载体的稳定转导效率也降低，这意味着NHEJ修复途径的这些组分的作用。最后，我们表明，连接酶IV缺陷的细胞被杀死的整合酶，但不是整合酶缺陷的HIV-1载体感染。在这项研究中提出的结果进一步支持NHEJ DNA修复途径在完成逆转录病毒DNA整合过程中的一般作用。
We have previously reported several lines of evidence that support a role for cellular DNA repair systems in completion of the retroviral DNA integration process. Failure to repair an intermediate in the process of integrating viral DNA into host DNA appears to trigger growth arrest or death of a large percentage of infected cells. Cellular proteins involved in the nonhomologous end joining (NHEJ) pathway (DNA-PKCS) and the damage-signaling kinases (ATM and ATR) have been implicated in this process. However, some studies have suggested that NHEJ proteins may not be required for the completion of lentiviral DNA integration. Here we provide additional evidence that NHEJ proteins are required for stable transduction by human immunodeficiency type 1 (HIV-1)-based vectors. Our analyses with two different reporters show that the number of stably transduced DNA-PKCS-deficient scid fibroblasts was reduced by 80 to 90% compared to the number of control cells. Furthermore, transduction efficiency can be restored to wild-type levels in scid cells that are complemented with a functional DNA-PKCS gene. The efficiency of stable transduction by an HIV-1-based vector is also reduced upon infection of Xrcc4 and ligase IV-deficient cells, implying a role for these components of the NHEJ repair pathway. Finally, we show that cells deficient in ligase IV are killed by infection with an integrase-competent but not an integrase-deficient HIV-1 vector. Results presented in this study lend further support to a general role for the NHEJ DNA repair pathway in completion of the retroviral DNA integration process.