EB病毒核抗原蛋白EBNA2藉由與細胞蛋白MDC1交互作用而調控DNA雙股斷裂修復

Abstract

Epstein-Barr virus (EBV) 為一種致癌性的皰疹病毒，與多種人類惡性腫瘤有密切關聯，並能在體外實驗中使B細胞永生化為lymphoblastoid cell lines (LCLs)。Epstein-Barr virus nuclear antigen 2 (EBNA2) 為使B細胞永生化及轉型必要的一個轉活化蛋白 (transactivator)，並能活化許多宿主細胞致癌基因 (oncogene) 的表現，進而造成細胞過度增生 (hyperproliferation) 以及DNA雙股斷裂 (DNA double-strand breaks, DSBs)。先前我們實驗室發現EBNA2能促使U2-OS和Hep-2細胞形成多倍體 (polyploidy) 和微核 (micronuclei)。我們也發現EBNA2能使DSB產生後，mediator of DNA damage checkpoint protein 1 (MDC1) 焦點的形成以及53BP1焦點維持的能力衰減，暗示著EBNA2對於DSB修復有負向的影響。在本研究中，我們發現EBNA2能防止γ-H2AX與MDC1 tBRCT功能域進行交互作用，從而抑制MDC1在DSB處形成焦點。此外，MDC1焦點形成被抑制使得U2-OS細胞對於游離輻射有較高的感受性，以及表現較低的非同源性末端接合 (non-homologous end-joining, NHEJ) 效率。然而，在表現EBNA2的U2-OS細胞中，同源重組 (homologous recombination, HR) 的效率卻被增強，且這個增強可能是由於EBNA2和MDC1交互作用所造成。總結來說，本研究闡述了當細胞產生DSB時，EBNA2調控細胞DSB修復系統的新功能和作用機制，而此功能與機制有可能與EBV潛伏感染時維持病毒基因體穩定和促進宿主細胞腫瘤生成有所關聯。

Epstein-Barr virus (EBV) is an oncogenic herpesvirus closely linked to several human malignancies and can immortalize primary B cells into lymphoblastoid cell lines (LCLs) in vitro. Epstein-Barr virus nuclear antigen 2 (EBNA2), a transactivator which is necessary for B-cell immortalization and transformation, can up-regulate several host oncogenes and cause hyperproliferation and DNA double-strand breaks (DSBs). Previously, our laboratory found that EBNA2 could induce polyploidy and micronuclei formation in U2-OS and Hep-2 cells. We also found that EBNA2 could attenuate the foci formation of mediator of DNA damage checkpoint protein 1 (MDC1) and the foci retention of 53BP1 after DSBs induction, suggesting that EBNA2 might have a negative effect on DSB repair. In this study, we showed that EBNA2 could prevent γ-H2AX from interacting with the tBRCT domain of MDC1, thus inhibiting MDC1 foci formation at DSB sites. Furthermore, the inhibition of MDC1 foci formation led to higher sensitivity of U2-OS cells to irradiation treatment and lower non-homologous end-joining (NHEJ) efficiency in U2-OS cells. However, the HR efficiency in EBNA2 expressed U2-OS cells was enhanced and this enhancement was caused by the interaction between EBNA2 and MDC1. Together, these studies elucidate a novel function and mechanism by which EBNA2 modulates the DSB repair system in cells after DSBs formation, which might be responsible for maintaining viral genomic stability and promoting tumorigenesis of host cells during EBV latent infection.