DeSmet Marsha, Kanginakudru Sriramana, Rietz Anne, Wu Wai-Hong, Roden Richard, Androphy Elliot J
Department of Dermatology, Indiana University School of Medicine, Indianapolis, Indiana, United States of America.
Department of Pathology, Johns Hopkins University, Baltimore, Maryland, United States of America.
PLoS Pathog. 2016 Oct 4;12(10):e1005934. doi: 10.1371/journal.ppat.1005934. eCollection 2016 Oct.
The origin recognition complex (ORC) coordinates a series of events that lead to initiation of DNA strand duplication. As a nuclear double stranded DNA plasmid, the papillomavirus (PV) genome resembles a mini-chromosome in infected cells. To initiate its replication, the viral E2 protein binds to and recruits the E1 DNA helicase at the viral origin. PV genome replication program exhibits three stages: initial amplification from a single genome upon infection to a few copies per cell, a cell cycle linked maintenance phase, and a differentiation dependent late stage where the genome is amplified to thousands of copies. Involvement of ORC or other pre-replication complex (pre-RC) factors has not been described. We report that human PV (HPV) and bovine PV (BPV-1) E2 proteins bind to ORC2, however, ORC2 was not detected at the viral origin. Depletion of ORC2 enhanced PV replication in a transient replication model and in keratinocytes stably maintaining viral episomes, while there was no effect on copy number in a cell line with integrated HPV genomes. Consistent with this, occupancy of E1 and E2 at the viral origin increased following ORC2 silencing. These data imply that ORC2 is not necessary for activation of the PV origin by E1 and E2 but instead suppresses E2 replicative function. Furthermore, we observed that over-expression of HPV E2 decreased ORC2 occupation at two known mammalian origins of replication, suggesting that E2 restricts pre-ORC assembly that could otherwise compete for host replication complexes necessary for viral genome amplification. We infer that the ORC2 complex with E2 restricts viral replication in the maintenance phase of the viral replication program and that elevated levels of E2 that occur during the differentiation dependent amplification stage subvert ORC loading and hence DNA synthesis at cellular origins.
起始识别复合物（ORC）协调一系列导致DNA链复制起始的事件。作为一种核双链DNA质粒，乳头瘤病毒（PV）基因组在受感染细胞中类似于一个微型染色体。为了启动其复制，病毒E2蛋白在病毒起源处结合并募集E1 DNA解旋酶。PV基因组复制程序呈现三个阶段：感染后从单个基因组初始扩增至每个细胞几个拷贝，一个与细胞周期相关的维持阶段，以及一个依赖分化的后期阶段，在此阶段基因组扩增至数千个拷贝。尚未描述ORC或其他复制前复合物（pre-RC）因子的参与情况。我们报告称，人乳头瘤病毒（HPV）和牛乳头瘤病毒（BPV-1）的E2蛋白与ORC2结合，然而，在病毒起源处未检测到ORC2。在瞬时复制模型和稳定维持病毒附加体的角质形成细胞中，ORC2的缺失增强了PV复制，而对整合有HPV基因组的细胞系中的拷贝数没有影响。与此一致的是，ORC2沉默后，E1和E2在病毒起源处的占据增加。这些数据表明，ORC2对于E1和E2激活PV起源并非必需，而是抑制E2的复制功能。此外，我们观察到HPV E2的过表达降低了ORC2在两个已知的哺乳动物复制起点的占据，这表明E2限制了复制前复合物的组装，否则其可能会竞争病毒基因组扩增所需的宿主复制复合物。我们推断ORC2与E2的复合物在病毒复制程序的维持阶段限制病毒复制，并且在依赖分化的扩增阶段发生的E2水平升高会破坏ORC的装载，从而破坏细胞起源处的DNA合成。
