登革病毒聚合酶(RdRp)活性平台開發暨抗登革病毒藥物篩選之研究

A Cell-based Reporter Assay for Inhibitor Screening of Dengue Virus RNA-dependent RNA polymerase and anti-Dengue Virus Agent Screening

吳宇軒(Yu-Hsuan Wu)

指導教授 ： 李景欽

高雄醫學大學/生命科學院/生物科技學系碩士班/碩士(2014年)

摘要

登革病毒是一種藉由蚊子叮咬所傳播的病原體，每年世界上有五千到一億人口被感染。受到登革病毒感染嚴重者會引發登革出血熱及休克症候群，提高感染的死亡率。目前尚未有有效治療登革熱的藥物或者可預防登革病毒感染的疫苗，因此研發新療法是非常重要的。登革病毒NS5蛋白具有RNA聚合酶(RNA-dependent RNA polymerase)活性，與病毒RNA複製息息相關，因此是個具有潛力的抗病毒藥物標的。以往偵測NS5蛋白RdRp活性，只能使用試管內同位素標定方法，無法得知藥物是否能順利進入細胞內以及藥物對細胞的毒性。為了克服此難題，本研究針對登革病毒NS5蛋白RNA聚合酶(RNA-dependent RNA polymerase)活性建立了一個可同時反應藥物所造成之細胞毒性及抑制RdRp活性狀況的雙報導基因系統，此系統建構有renilla luciferase及firefly luciferase兩個冷光報導基因，其中renilla luciferase為正股，而firefly luciferase則為反股，兩邊被反股的登革病毒的5'及3'非轉譯區域所包夾，renilla luciferase活性代表細胞毒性，而fierfly luciferase活性則代表了RdRp活性。藉由這個系統我們發現了一個核苷酸類似物NA01具有抑制登革病毒RdRp活性的效果。本研究進一步於登革病毒次基因體複製子細胞株及登革病毒感染系統中證實了NA01同樣可抑制登革病毒RNA複製，確定了NA01對於病毒複製活性抑制的效果。為了進一步了解NA01對於RdRp抑制的情形，我們使用報導基因系統來測定NA01對RdRp的抑制常數(Ki=11.4 μM)。另外，我們也發現了NA01在小鼠體內同樣可抑制登革病毒的複製。我們將複製子細胞株植入免疫缺陷小鼠皮下並經由腹腔注射給予NA01，結果發現接受NA01注射的小鼠體內病毒複製量下降。由這些結果我們可知NA01是一個非常具有潛力之抗登革病毒藥物。

關鍵字

登革病毒 ； 核酸依賴核酸聚合酶

並列摘要

Dengue virus (DENV) is an arthropod-borne pathogen, which infects 50-100 million people in the world. DENV infection can result in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), which are risk factors leading to death. Nowadays there are no any drugs or vaccine can efficiently inhibit or prevent dengue infection, so investigation of new therapies is one of the important things for the scientists. DENV nonstructural protein 5 (NS5) contains RNA-dependent-RNA polymerase (RdRp) activity, which is a potential target for anti-dengue agents. However, the commonly used method for analyzing polymerase inhibitor screening is base on the scintillation counting of isotope-labeling RNA product synthesized. This method can not analyze the cell cytotoxicity of compounds. To overcome this liminitation, , we construct a biscistronic reporter plasmid to simultaneously measure cellular toxicity and anti-RdRp activity of compound.The reporter plasmid contains positive strand Renilla gene (RLuc) and negative strand firefly gene (FLuc), which flanked by negative strand DENV 5' and 3' untranslated regions (UTR). RLuc is regulated by host polymerase, which stands of the cell cytotoxicity. FLuc is regulated by DENV RdRp, which reflects the RdRp activity. Using RdRp reporter system, we identified a nucleoside analogue, NA01, to inhibit DENV RNA replication and protein synthesis through inhibition of RdRp activity without cytotoxicity in DENV replicon cells. Besides, NA01 could reduce DENV replication in viral infectious system (strain 16681). We also determined the inhibition constant of NA01 to RdRp is 11.4 μM. Notably, we highlighted the anti-dengue activity in the xenotransplanted NU/NU mice. NU/NU mice were xenotransplanted with DENV subgenomic reporter replicon cells and followed by intraperitoneal injection (I.P.) of NA01. The bioluminescence imaging analysis revealed that NA01could inhibit DENV replication in vivo. Altogether, the result indicated that NA01 can serve as a potential anti-DENV reagent.

並列關鍵字

Dengue virus ； RNA-dependent-RNA polymerase

參考文獻

參考文獻

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