The Wistar Institute, 3601 Spruce St., Philadelphia, PA 19104-4265, USA.
J Virol. 2011 Jun;85(12):5853-63. doi: 10.1128/JVI.02425-10. Epub 2011 Apr 13.
The Epstein-Barr virus (EBV) genome is maintained as an extrachromosomal episome during latent infection of B lymphocytes. Episomal maintenance is conferred by the interaction of the EBV-encoded nuclear antigen 1 (EBNA1) with a tandem array of high-affinity binding sites, referred to as the family of repeats (FR), located within the viral origin of plasmid replication (OriP). How this nucleoprotein array confers episomal maintenance is not completely understood. Previous studies have shown that DNA replication forks pause and terminate with high frequency at OriP. We now show that cellular DNA replication fork pausing and protection factors Timeless (Tim) and Tipin (Timeless-interacting protein) accumulate at OriP during S phase of the cell cycle. Depletion of Tim inhibits OriP-dependent DNA replication and causes a complete loss of the closed-circular form of EBV episomes in latently infected B lymphocytes. Tim depletion also led to the accumulation of double-strand breaks at the OriP region. These findings demonstrate that Tim is essential for sustaining the episomal forms of EBV DNA in latently infected cells and suggest that DNA replication fork protection is integrally linked to the mechanism of plasmid maintenance.
EB 病毒（EBV）基因组在 B 淋巴细胞的潜伏感染期间作为染色体外的附加体得以维持。EBV 编码的核抗原 1（EBNA1）与位于病毒质粒复制起点（OriP）内的高亲和力结合位点的串联阵列（称为重复家族，FR）的相互作用赋予了附加体的维持。这种核蛋白阵列如何赋予附加体的维持尚不完全清楚。先前的研究表明，DNA 复制叉在 OriP 处频繁暂停和终止。我们现在表明，在细胞周期的 S 期，细胞 DNA 复制叉暂停和保护因子 Timeless（Tim）和 Tipin（Timeless 相互作用蛋白）在 OriP 处积累。Tim 的耗竭抑制 OriP 依赖性 DNA 复制，并导致潜伏感染的 B 淋巴细胞中 EBV 附加体的闭环形式完全丧失。Tim 的耗竭也导致 OriP 区域双链断裂的积累。这些发现表明，Tim 对于维持潜伏感染细胞中 EBV DNA 的附加体形式是必不可少的，并表明 DNA 复制叉保护与质粒维持的机制紧密相关。
