DDX3 RNA解螺旋脢及其交互作用蛋白於C型肝炎病毒複製所扮演之角色及作用機制之探討

Abstract

C 型肝炎病毒為輸血性非A 非B 型肝炎的主要肇因，其持續性潛伏感染可導致慢 性肝炎,肝硬化甚至肝癌的發生。目前為止，C 型肝炎病毒之生活史尚未完全釐清，然透 過部分基因組複製系統Con-1 及具感染力之全基因組複製病毒株JFH-1 之研究，已有了 長足的看見。研究指出，多種細胞因子可與病毒蛋白或病毒RNA 基因組之5’或3’端結 合而調節病毒之基因組複製，並為病毒複製所必須。而RNA 解螺旋酶DDX3 則為其中 之一。 多年來，本實驗室致力於闡明RNA 解螺旋酶DDX3 之生物功能，過去之研究成果 指出DDX3 廣泛地參與轉錄、轉譯、RNA 剪輯等細胞生理活動之調控，並影響細胞之 生長，為一新穎之抑癌基因。此外，該蛋白可與C 型肝炎病毒核心蛋白發生交互作用， 其ATP 水解及RNA 解螺旋酵素活性並因此而增強。目前，利用試管內及活體內結合實 驗，本實驗室亦發現C 型肝炎病毒非結構蛋白5B 可與DDX3 發生交互作用。由於研究 報告指出，DDX3 可與病毒RNA 基因組之3’端結合，因此，我們推測DDX3 可藉由與 病毒複製所必須之病毒蛋白發生交互作用並與病毒基因組結合而參與了病毒的基因組 複製。因此，為驗證此一假說並釐清DDX3 參與病毒複製的作用機轉，本計畫將進行下 述研究： 由於DDX3 為病毒複製所必須且其酵素活性受病毒蛋白之調控，故本實驗室首先 將釐清DDX3 之酵素活性對於C 型肝炎病毒複製之調控是否必須。我們將檢視DDX3 是否亦可與HCV RNA 基因組之5’端或反基因組之5’與3’非轉譯區域結合，並劃定這些 RNA 片段上之DDX3 結合區。我們亦將找出DDX3 之RNA 結合功能區，並利用定點 突變的方式破壞DDX3 的ATP 水解，RNA 解螺旋及RNA 結合活性，並偵測這些酵素 活性的喪失對病毒複製的影響，以瞭解DDX3 酵素活性對病毒複製的重要性。 接著，我們將進一步闡明DDX3 與病毒蛋白之交互作用對於病毒複製的影響。本 實驗室將檢視DDX3 與核心蛋白及非結構蛋白5B 之交互作用是否增強病毒之複製，並 偵測病毒蛋白對DDX3 酵素活性的影響，及DDX3 對非結構蛋白5B 之RNA依賴性RNA 聚合酶活性之調節，以明瞭DDX3 與病毒蛋白交互作用對病毒複製影響的作用機轉。 最後，我們將探討一個可與病毒RNA 基因組之3’端非轉譯區域結合之DDX3 交互 作用蛋白YB-1 於C 型肝炎病毒複製中扮演的角色。我們將探討細胞中YB-1 表現與否 對於病毒複製的影響。研究該蛋白與C 型肝炎病毒基因組與反基因組RNA 之結合，及 其RNA 結合特性對於病毒複製之調控是否必須。我們亦將檢視該蛋白是否與病毒蛋白 或病毒複製酵素複合物結合而參與了病毒的複製過程，並探討YB-1 與DDX3 之交互作 用對於病毒複製的影響，以進一步釐清宿主細胞因子影響病毒複製之作用機轉。 總而言之，透過本計畫，吾人將瞭解DDX3 影響C 型肝炎病毒複製之分子作用機 轉，並提供宿主細胞因子透過蛋白交互作用而介入病毒複製之模式，將有利於未來C 型 肝炎病毒治療方式之設計與開發。

Hepatitis C virus (HCV) is a leading cause of transfusion-associated non-A, non-B hepatitis. Long lasting HCV infection leads to hepatocellular carcinoma. The life cycle of HCV is not fully understood; however, the establishment of the subgenomic Con1 replicon and the discovery of replication and infection capable HCV strain, JFH-1, shed light on this issue. Studies using these two replicons indentify various cellular factors required for HCV replication, and the most important one is DDX3 RNA helicase that was originally isolated as HCV core-interacting protein by several laboratories including ours. Notably, our lab found out that this HCV core-DDX3 association enhances the ATPase and RNA helicase activity of DDX3. Our recent findings also indicate that DDX3 possesses pleiotropic functions, such as acting as transcriptional activator, a negative regulator for cap-dependent translation, and a putative tumor suppressor involved in cell growth control. Most interestingly, DDX3 together with several cellular factors including YB-1 are found to associate with the 3’-untranslated region (NTR) of HCV genome. More recently, our preliminary results also suggest that DDX3 physically interacts with the HCV RNA-dependent RNA polymerase (NS5B) which is responsible for initiating HCV replication at 3’-end of HCV genome. In view of these, it is likely that the HCV replication relies on the physical and functional interaction of HCV core and NS5B proteins with DDX3 and its associated cellular factor. Thus, in this proposal, efforts will be made to elaborate the detailed underlying mechanism by which DDX3 and its associated factors modulate HCV RNA replication. In the first part of this proposal, we will clarify whether the enzymatic activity of DDX3 is required for HCV replication. The RNA binding property of DDX3 and the DDX3 binding elements on 5’- or 3’-NTR of plus/minus-strand HCV RNA genome will be delineated. DDX3 mutants defective in enzymatic activities will be generated to examine whether RNA-binding, ATPase or helicase activity are required for the modulation activity on HCV replication. Secondly, effects of interaction between DDX3 and HCV viral products (core and NS5B) on HCV replication will be examined, and the underlying mechanisms account for these effects will be clarified by analyzing the reciprocal modulation of DDX3, core and NS5B on enzymatic activities of DDX3 or NS5B required for HCV replication. Finally, the role of DDX3 associated factor YB-1, which also interacts with HCV 3’-NTR, on HCV RNA replication will be addressed. We will characterize the RNA binding property of YB-1 on HCV genome, examine the effect of YB-1 on HCV replication, and clarify whether the RNA binding property conducts effects exerted by YB-1 on HCV. Additionally, we will explore the physical and functional interaction between YB-1 and HCV viral products or viral replication complex. The interplay between DDX3 and YB-1 on HCV replication will also be investigated. Taken together, approaches outlined above in this proposal will decipher how the HCV core and NS5B proteins target to RNA helicase DDX3 and its associated complex, and the detailed mechanism by which these interactions are associated with HCV replication. Hopefully, these studies will provide useful information for therapeutic designs against HCV infection.