Timeless-Dependent DNA Replication-Coupled Recombination Promotes Kaposi's Sarcoma-Associated Herpesvirus Episome Maintenance and Terminal Repeat Stability
Timeless-Dependent DNA Replication-Coupled Recombination Promotes Kaposi's Sarcoma-Associated Herpesvirus Episome Maintenance and Terminal Repeat Stability
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DOI：
10.1128/jvi.02211-12
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发表时间：
2013-04-01
影响因子：
5.4
通讯作者：
Lieberman, Paul M.
中科院分区：
文献类型：
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作者：
Dheekollu, Jayaraju;Chen, Horng-Shen;Lieberman, Paul M.
卡波西肉瘤相关疱疹病毒 (KSHV) 在潜伏感染的胸腔积液淋巴瘤 (PEL) 细胞中以稳定的游离体形式维持。附加体的维持是通过 KSHV 编码的 LANA 蛋白与病毒末端重复序列 (TR) 的结合来实现的。在这里，我们表明 KSHV TR 中的 DNA 复制与 DNA 重组相结合，并部分通过细胞复制叉保护因子 Timeless (Tim) 和 Tipin 介导。我们通过二维 (2D) 琼脂糖凝胶电泳表明，在未受干扰的细胞周期中，复制叉自然停止并在 TR 处形成重组样结构。染色质免疫沉淀 (ChIP) 分析显示，Tim 和 Tipin 在 S 期期间以 LANA 依赖性方式在 KSHV TR 处选择性富集。 Tim 耗竭抑制了 LANA 依赖性 TR DNA 复制，并导致潜伏感染的 PEL 细胞中 KSHV 附加体丢失。 Tim 耗尽导致重组结构异常积累并在 TR 处抑制 MCM 解旋酶。 Tim 耗竭不会诱导 KSHV 裂解周期或细胞凋亡。我们提出，KSHV 附加体的维持需要在病毒末端重复上有 Tim 辅助的复制叉保护，并且在 TR 处形成 Tim 依赖性重组样结构，以促进 DNA 重复稳定性和病毒基因组维持。
Kaposi's Sarcoma-associated herpesvirus (KSHV) is maintained as a stable episome in latently infected pleural effusion lymphoma (PEL) cells. Episome maintenance is conferred by the binding of the KSHV-encoded LANA protein to the viral terminal repeats (TR). Here, we show that DNA replication in the KSHV TR is coupled with DNA recombination and mediated in part through the cellular replication fork protection factors Timeless (Tim) and Tipin. We show by two-dimensional (2D) agarose gel electrophoresis that replication forks naturally stall and form recombination-like structures at the TR during an unperturbed cell cycle. Chromatin immunoprecipitation (ChIP) assays revealed that Tim and Tipin are selectively enriched at the KSHV TR during S phase and in a LANA-dependent manner. Tim depletion inhibited LANA-dependent TR DNA replication and caused the loss of KSHV episomes from latently infected PEL cells. Tim depletion resulted in the aberrant accumulation of recombination structures and arrested MCM helicase at TR. Tim depletion did not induce the KSHV lytic cycle or apoptotic cell death. We propose that KSHV episome maintenance requires Tim-assisted replication fork protection at the viral terminal repeats and that Tim-dependent recombination-like structures form at TR to promote DNA repeat stability and viral genome maintenance.