Knockdown of DNA ligase IV/XRCC4 by RNA interference inhibits herpes simplex virus type I DNA replication
Knockdown of DNA ligase IV/XRCC4 by RNA interference inhibits herpes simplex virus type I DNA replication
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DOI：
10.1074/jbc.m611834200
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发表时间：
2007-04-13
影响因子：
4.8
通讯作者：
Elias, Per
中科院分区：
文献类型：
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作者：
Muylaert, Isabella;Elias, Per
单纯疱疹病毒具有线性双链DNA基因组，其具有切割和包装复制的DNA所需的直接重复的末端序列。在受感染的细胞中，线性基因组迅速变得无穷无尽。目前正在讨论的问题是，无限的基因组是支持滚环复制的环，还是通过线性基因组重组形成多联体而产生的。在这里，我们已经研究了哺乳动物DNA连接酶在单纯疱疹病毒，I型（HSV-1）的生命周期中的作用，采用RNA干扰（RNAi）在人类1BR.3.N成纤维细胞。我们发现RNAi介导的DNA连接酶IV及其辅因子XRCC 4的敲低导致病毒产量降低百倍，小空斑表型和DNA合成减少。该效应是特异性的，因为针对DNA连接酶I或DNA连接酶III的RNAi不能减少HSV-1复制。此外，针对DNA连接酶IV和XRCC 4的RNA干扰不影响腺病毒的复制。此外，人类DNA连接酶IV缺陷细胞中的HSV-1高度多重感染揭示了感染性病毒产生的显着延迟。最后，我们证明了通过RNAi介导的DNA连接酶IV和XRCC 4的耗竭抑制了无限基因组的形成。我们的研究结果表明，DNA连接酶IV/XRCC 4在疱疹病毒的复制周期中起着重要作用，并且可能是在生产性感染早期形成无限基因组所必需的。
Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IV-deficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection.