建立表現FLAG-RPB9細胞株研究RNA 聚合酶II 對於D型肝炎複製所扮演的角色

Establishment of FLAG-RPB9 expressing cell line to study Hepatitis Delta Virus replication by cellular RNA polymerase II

李澤峰(Ze-Fong Li)

指導教授 ： 陳培哲

國立臺灣大學/醫學院/微生物學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202102614

摘要

D肝炎病毒是B肝炎病毒的衛星病毒，根據過去統計資料，全球約有1500~2000萬人感染B型肝炎和D型肝炎病。根據2020年的綜合分析，目前全球人口有約10.6% 的B型肝炎帶原者 (約6200~7200百萬人) 同時感染B及D型肝炎病毒。D型肝炎病毒感染分成兩種形式，其一是B肝炎病毒和D肝炎病毒同時共同感染，另一種為患有慢性B肝病毒感染患者再被D肝病毒感染。受到HBV和HDV感染可能會導致最嚴重的肝炎疾病 - 猛爆性肝炎。D肝病毒帶有1.7 kb的Genomic RNA 並會產生一種病毒蛋白-delta抗原，delta抗原 (HDAg)，由於該蛋白不具有聚合酶活性。因此，D型肝炎病毒必須利用部分宿主酵素來完成其生命週期。目前關於 D肝炎病毒複製中涉及的宿主酵素有兩種主要理論。其中一派認為RNA 聚合酶 II 參與了基因組和反基因組 RNA 複製，因為這兩個過程都對低劑量的 RNA 聚合酶 II 敏感抑製劑 α-鵝膏菌素敏感，而另一派認為 RNA聚合酶II以外的聚合酶可能參與基因組 RNA 的合成，因為α-鵝膏菌素在高濃度下無法阻斷反基因組 RNA 的合成。迄今為止，是否有 RNA 聚合酶 II以外的聚合酶否參與 D肝炎病毒的複製仍存有爭議。除了抑制劑實驗結果外，將RNA聚合酶II上的次單位以小分子干擾RNA減少表現後會使D型肝炎病毒感染率顯著下降。另一個研究指出，部分純化的RNA聚合酶II可以利用D型肝炎病毒的RNA在體外進行轉錄作用。基於前人研究，因此我們假設 RNA 聚合酶 II 參與了 D肝炎病毒的複製過程。為了驗證該假說，我們試圖透過建立在 RPB9（RNA 聚合酶 II 的一個次單位）上過量表達FLAG- RPB9的海拉細胞 (HeLa cell) 作為純化 RNA 聚合酶II，並分析 RNA 聚合酶 II 是否可以在體外轉錄D肝炎病毒的RNA。本研究提供RNA 聚合酶II純化所需的細胞株，為未來解決RNA 聚合酶II參與在D肝病毒複製過程中的爭議。

關鍵字

D型肝炎病毒 ； 小型Delta 抗原 ； RNA 聚合酶 II ； D肝病毒複製

並列摘要

Hepatitis Delta Virus (HDV) is the satellite virus of Hepatitis B Virus (HBV). It was thought there are 15-20 million people worldwide coinfected with HBV and HDV. According to recent meta-analysis in 2020, there are approximate 10.6% of HBsAg-positive patients, or 62-72 million people worldwide infected with Hepatitis B Virus (HBV) and Hepatitis Delta Virus (HDV). HDV infection occurs in two distinct patterns, either simultaneously with HBV (coinfection) or in patients with established chronic HBV infection (superinfection). Infection with HBV and HDV could lead to fulminant hepatitis, which is the most severe form of liver hepatitis. HDV virion carries a 1.7 kb genomic RNA that encodes one viral protein - delta antigen (HDAg), which doesn’t possess polymerase activity. Therefore, the virion must utilize host enzyme to fulfill its life cycle. There are two main theories regarding host factors involved in HDV replication. One assumed that RNA polymerase II participated in both genomic and antigenomic RNA synthesis, as both processes was sensitive to low dosage of RNA polymerase II sensitive inhibitor α-amanitin. The other group argue that a polymerase other than RNA polymerase II might involve in antigenomic RNA synthesis, as α-amanitin was unable to completely block antigenomic RNA synthesis even at high concentration. To date, whether RNA polymerase II is solely responsible for HDV replication is still controversial. In addition to inhibitor experiment, previous study shows that HDV infection rate reduce significantly after transfection of siRNA targeting RNA polymerase II, suggesting that RNA polymerase II participate in replication process. Another study show that partially purified RNA polymerase II can transcribe HDV RNA template in vitro. Therefore, we hypothesize that RNA polymerase II is involved in HDV replication process. To verify our hypothesis, we have established a HeLa cell line that overexpress FLAG tag on RPB9 for FLAG-tag purification of RNA polymerase II. With functional RNA polymerase II purified, we could further use HDV RNA as template to conduct in vitro transcription and provide direct biochemical evidences that RNA polymerase II is involved in HDV replication. My study provides the cell line for RNA polymerase II purification, that could further resolve the dispute of RNA polymerase II involvement in HDV replication.

並列關鍵字

Hepatitis Delta Virus ； Small Delta Antigen ； RNA polymerase II ； HDV replication

參考文獻

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