Dyson Ossie F, Pagano Joseph S, Whitehurst Christopher B
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Virol. 2017 Sep 12;91(19). doi: 10.1128/JVI.00600-17. Print 2017 Oct 1.
Epstein-Barr virus (EBV) infection and lytic replication are known to induce a cellular DNA damage response. We previously showed that the virally encoded BPLF1 protein interacts with and regulates several members of the translesion synthesis (TLS) pathway, a DNA damage tolerance pathway, and that these cellular factors enhance viral infectivity. BPLF1 is a late lytic cycle gene, but the protein is also packaged in the viral tegument, indicating that BPLF1 may function both early and late during infection. The BPLF1 protein expresses deubiquitinating activity that is strictly conserved across the ; mutation of the active site cysteine results in a loss of enzymatic activity. Infection with an EBV BPLF1 knockout virus results in decreased EBV infectivity. Polymerase eta (Pol η), a specialized DNA repair polymerase, functions in TLS and allows for DNA replication complexes to bypass lesions in DNA. Here we report that BPLF1 interacts with Pol η and that Pol η protein levels are increased in the presence of functional BPLF1. BPLF1 promotes a nuclear relocalization of Pol η molecules which are focus-like in appearance, consistent with the localization observed when Pol η is recruited to sites of DNA damage. Knockdown of Pol η resulted in decreased production of infectious virus, and further, Pol η was found to bind to EBV DNA, suggesting that it may allow for bypass of damaged viral DNA during its replication. The results suggest a mechanism by which EBV recruits cellular repair factors, such as Pol η, to sites of viral DNA damage via BPLF1, thereby allowing for efficient viral DNA replication. Epstein-Barr virus is the causative agent of infectious mononucleosis and infects approximately 90% of the world's population. It causes lymphomas in individuals with acquired and innate immune disorders and is strongly associated with Hodgkin's lymphoma, Burkitt's lymphoma, diffuse large B-cell lymphomas, nasopharyngeal carcinoma (NPC), and lymphomas that develop in organ transplant recipients. Cellular DNA damage is a major determinant in the establishment of oncogenic processes and is well studied, but there are few studies of endogenous repair of viral DNA. This work evaluates how EBV's BPLF1 protein and its conserved deubiquitinating activity regulate the cellular DNA repair enzyme polymerase eta and recruit it to potential sites of viral damage and replication, resulting in enhanced production of infectious virus. These findings help to establish how EBV enlists and manipulates cellular DNA repair factors during the viral lytic cycle, contributing to efficient infectious virion production.
已知爱泼斯坦-巴尔病毒（EBV）感染和裂解复制可诱导细胞DNA损伤反应。我们之前表明，病毒编码的BPLF1蛋白与跨损伤合成（TLS）途径（一种DNA损伤耐受途径）的几个成员相互作用并对其进行调节，并且这些细胞因子可增强病毒感染性。BPLF1是一个晚期裂解周期基因，但该蛋白也被包装在病毒被膜中，这表明BPLF1可能在感染的早期和晚期均发挥作用。BPLF1蛋白具有去泛素化活性，该活性在整个……中严格保守；活性位点半胱氨酸的突变会导致酶活性丧失。用EBV BPLF1基因敲除病毒感染会导致EBV感染性降低。聚合酶η（Pol η）是一种特殊的DNA修复聚合酶，在TLS中发挥作用，并使DNA复制复合物能够绕过DNA中的损伤。在此我们报告，BPLF1与Pol η相互作用，并且在功能性BPLF1存在的情况下，Pol η蛋白水平会升高。BPLF1促进Pol η分子的核重新定位，这些分子外观呈焦点样，这与Pol η被招募到DNA损伤位点时观察到的定位一致。敲低Pol η会导致感染性病毒产生减少，此外，发现Pol η与EBV DNA结合，这表明它可能在病毒复制过程中允许绕过受损的病毒DNA。这些结果提示了一种机制，通过该机制EBV经由BPLF1将细胞修复因子（如Pol η）招募到病毒DNA损伤位点，从而实现高效的病毒DNA复制。爱泼斯坦-巴尔病毒是传染性单核细胞增多症的病原体，感染了世界上约90%的人口。它在获得性和先天性免疫紊乱的个体中引发淋巴瘤，并且与霍奇金淋巴瘤、伯基特淋巴瘤、弥漫性大B细胞淋巴瘤、鼻咽癌（NPC）以及器官移植受者中发生的淋巴瘤密切相关。细胞DNA损伤是致癌过程发生的一个主要决定因素，并且已得到充分研究，但关于病毒DNA的内源性修复的研究很少。这项工作评估了EBV的BPLF1蛋白及其保守的去泛素化活性如何调节细胞DNA修复酶聚合酶η，并将其招募到病毒损伤和复制的潜在位点，从而导致感染性病毒产生增加。这些发现有助于确定EBV在病毒裂解周期中如何招募和操纵细胞DNA修复因子，从而促进高效感染性病毒粒子的产生。
