人類 MIOS 蛋白質在 EB 病毒溶裂期的促進

Abstract

EB 病毒 (Epstein-Barr virus, EBV) 是第一個在人類被證實具有致癌性的病毒，感染全球超過九成的人口。EB 病毒的生活史可分成潛伏期 (latency) 與溶裂期 (lytic cycle)，在溶裂期時，病毒的極早期基因 BRLF1 與 BZLF1 所表現的兩個轉錄促進因子 Rta 與 Zta，會進一步活化病毒複製相關的基因表現，促進病毒顆粒產生。此外先前研究已經證實 Rta 能夠促進雙基因 BRLF1-BZLF1 mRNA 中的 Zta 被有效的轉譯。本實驗室先前以免疫沉澱及質譜儀分析發現了一個在病毒進入溶裂期時會與 Rta 結合的人類細胞蛋白質，稱為 meiosis regulator for oocyte development (MIOS)，是與哺乳動物雷帕黴素靶蛋白 （mammalian target of rapamycin, mTOR） 活化相關的上游蛋白質。MIOS 為 GATOR2 (GAP activity towards Rags 2) 複合物中的一員，能夠透過 Rags 蛋白質活化下游的 mTORC1，且在細胞週期與胚胎發育皆扮演了重要的角色。本研究以免疫共沉澱分析以及 GST pull-down assay 證實 Rta 會以其錄活化區 (transactivation domain) 與 MIOS 結合。本研究也發現 MIOS 會透過活化轉譯促進 Rta 蛋白質的表現。亦利用先前所建構的雙基因表現質體 pCMV-RZLuc 進行 luciferase reporter assay，證實了 MIOS 會進一步促進 Rta 活化下游 ORF 的轉譯。此外也進一步以帶有 EB 病毒潛伏的 HEK293(2089) 與P3HR1 細胞證實了 MIOS 會加速 EB 病毒進入溶裂期。綜合以上結果，本研究證實了 MIOS 會與 Rta 結合並促進其蛋白質的表現， MIOS 也能夠促進 EB 病毒進入溶裂期，顯示 MIOS 在 EB 病毒的溶裂期扮演了非常重要的角色。

The Epstein-Barr virus (EBV) is the first virus proven to be oncogenic in humans, infecting over 90% of the global population. The life cycle of EBV can be divided into two stages, latency and the lytic cycle. During the lytic cycle, the virus expresses two transcriptional activators, Rta and Zta, which are encoded by the immediate-early genes BRLF1 and BZLF1, respectively. These two proteins further activate early and late genes, leading to the production of viral particles. In our laboratory, through immunoprecipitation and mass spectrometry analysis, a cellular protein, named as meiosis regulator for oocyte development (MIOS) was found to interact with Rta. MIOS is an upstream protein associated with the activation of the mammalian target of rapamycin complex 1 (mTORC1), which is a central regulator of mammalian metabolism and physiology. MIOS is a member of the GATOR2 complex and plays important roles in the cell cycle and embryonic development. This study used immunoprecipitation and GST pull-down assays to confirm the interaction between Rta and MIOS. Furthermore, MIOS binds to the transactivation domain of Rta. Additionally, MIOS enhances the expression of the Rta protein through translational activation. By using luciferase reporter assay, this study found that MIOS promotes the translation of downstream open reading frames (ORFs) activated by Rta. Remarkly, this study indicated that MIOS accelerates the transition of EBV into the lytic cycle through experiments conducted in HEK293(2089) and P3HR1 cells carrying latent EBV. Taken together, these results demonstrate that MIOS interacts with Rta and enhances its protein expression. Furthermore, MIOS promotes the lytic transition of EBV, indicating its crucial role in EBV life cycle.