C型肝炎病毒（HCV）是引發慢性肝炎、肝硬化及肝癌的一個主要成因。HCV的非結構蛋白5B（NS5B）可以轉譯出一個以RNA為模版進行複製的RNA聚合酶（RdRp），但是這樣的聚合酶在病毒複製的過程中卻缺乏了校正的功能。由於NS5B蛋白的特性，導致HCV具有很高的基因歧異度，並可發現在慢性感染HCV的病人體中有許多類種（Quasispecies）的存在。根據這些發現，科學家們試著去探討在C型肝炎病毒基因體上出現的變異點以及這些變異點對病毒致病力與抗病毒治療的一些影響。在此研究中，我們從Los Alomas HCV資料庫下載了225條存在於病人體中的HCV全長序列，並利用生物資訊軟體Weka其中的關連法則（Association rule）尋找發生在HCV基因體的非轉譯區（5’UTR and 3’UTR）及非結構蛋白2、3、5B（NS2, NS3, NS5B）其中的共同演化現象（co-evolutionary effects）。從結果中，我們找出了133組共同演化配對（co-evolutionary pair）。為了探討這些共同變異配對對於C型肝炎病毒複製能力的影響，我們研究了存在於5’UTR核苷酸第243位點由鳥嘌呤（Guanine）變成腺嘌呤（Adenine）配對NS2氨基酸第76位點由異白胺酸（Isoleucine）轉變為纈胺酸（Valine）的交互影響，並利用HCV次基因複製子（Subgenomic replicon）pFKI341Pi Luc/NS2-3’/ET來進行研究。從結果中，我們發現到單獨出現非結構蛋白2（NS2）其氨基酸第76個位置由異白胺酸（Isoleucine）轉變為纈胺酸（Valine）時，subgenomic replicon的複製能力會顯著降低。然而，假如同時再置換位於5’UTR核苷酸第243位點由鳥糞嘌呤（Guanine）變成腺嘌呤（Adenine），則會回復其複製能力。除此之外，單獨置換5’UTR核苷酸第243位點由鳥糞嘌呤（Guanine）為腺嘌呤（Adenine），對於subgenomic replicons pFKI341Pi Luc/NS2-3’/ET和pFKI341Pi Luc/NS3-3’/ET的複製能力沒有顯著影響。從置換5’UTR核苷酸第243位點為胞嘧啶（Cytosine）及胸腺嘧啶（Thymidine）的結果中，我們發現，將5’UTR第243位點置換成胞嘧啶（Cytosine）會降低一半subgenomic replicon正常的複製能力; 將5’UTR第243位點置換成胸腺嘧啶（Thymidine），subgenomic replicon 的複製能力則會幾乎消失殆盡。為了研究5’UTR 核苷酸第243位點和NS2氨基酸第76位點的交互作用，我們利用了RNA免疫沈澱法將會產生交互作用的RNA從表現有FKI341Pi Luc/NS3-3’/ET及NS2蛋白的細胞株中粹取出來。由結果可知，帶有氨基酸第76位點由異白胺酸（Isoleucine）轉變為纈胺酸（Valine）的NS2蛋白，會和HCV subgenomic replicons的RNAs有交互作用。由此可知，結合帶有氨基酸第76位點由異白胺酸（Isoleucine）轉變為纈胺酸（Valine）的NS2蛋白可能會阻斷HCV RNAs的正常折疊而降低其複製的能力。接下來，我們更建構了帶有螢火蟲冷光酵素基因（Firefly luciferae gene）及新酵素磷酸轉移酶基因（Neomycin phosphotransferase gene）融合蛋白（Fusion protein）的subgenomic replicons，並將此融合蛋白命名為Feo 蛋白，再藉由這種subgenomic replicons來研究適應性突變（Adaptive mutation）在細胞培養中的產生情形。經由篩選後，我們放大並比對包含5’UTR到NS2的序列。然而，我們並沒有發現到有符合co-evolutionary pair的adaptive mutations出現。總結來說，5’UTR第243位點由鳥嘌呤（Guanine）變成腺嘌呤（Adenine）配對NS2氨基酸第76位點由異白胺酸（Isoleucine）轉變為纈胺酸（Valine）的共同演化配對可能是一組和複製相關並存在感染HCV的病人體中的共同演化配對。

Hepatitis C virus (HCV) is one of the major etiologies to cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The nonstructural (NS) protein 5B of HCV is an RNA-dependent RNA polymerase lacking full proofreading activity during replication. Because of the characteristics of NS5B, HCV demonstrates great genetic diversity and viral quasispecies in individuals chronically infected with HCV. Consequently, numerous efforts have been put to investigate the emerging of mutations appearing on HCV genome, as well as the effects of these mutations toward viral virulence and efficacy of antiviral therapeutics. In this study, by utilizing the Weka bioinformatic software, we used the association rule to discern “co-evolutionary effects” between the untranslated regions (5’UTR and 3’UTR) and the NS proteins (NS2, NS3, NS5B) of 225 full-length genomic sequences from patient samples retrieved from Los Alamos HCV database. The results showed that there were 133 co-evolutionary pairs predicted by the association rule. To investigate whether the putative co-evolutionary pairs play roles in HCV replication, we investigated further the mutual effects between 5’UTR ntG243A and NS2 aaI76V in the HCV pFKI341Pi Luc/NS2-3’/ET subgenomic replicon. The experiments demonstrated that the single mutation of NS2 aaI76V significantly impaired HCV replication activity. However, double mutations of 5’UTR ntG243A and NS2 aaI76V could restore the replication activity, while, the single mutation of 5’UTR ntG243A had slight or no impact on the activities of replicons pFKI341Pi Luc/NS2-3’/ET and pFKI341Pi Luc/NS3-3’/ET, respectively. In addition, the replacement of 5’UTR ntG243 to C reduced HCV replication to half and the substitution of 5’UTR ntG243 to T fully eliminated the replication activity. To investigate whether interaction exists between 5’UTR nt243 and NS2 aa76, RNA-immunoprecipitation assay was conducted to pull down RNAs from cHuh7/Rep Pi Feo/NS3-3’/ET with ectopic expressed NS2 proteins. The result indicated that only NS2-I76V mutant protein could interact with HCV replicon RNA, suggesting that the replication process might be down-regulated by the binding of NS2-I76V protein to HCV RNAs. Furthermore, we constructed subgenomic replicons with a fusion protein of firefly luciferase and neomycin phosphotransferase (Feo) to study the adaptive mutations generated in cultivated cells. After selection, we amplified and sequenced the segment spanning 5’UTR to NS2. However, co-evolutionary pair between 5’UTR nt243 and NS2 aa76 was undetectable under the in vitro selection. In conclusion, 5’UTR ntG243A and NS2 aaI76V might be one of the replication-dependent co-evolutionary events, which existed in HCV from patient samples.

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