D型肝炎病毒於表現FLAG-RPB9細胞系中的複製

Abstract

D型肝炎病毒與B型肝炎病毒共感染肝臟細胞會引起最嚴重的病毒性肝炎，而因為我們對於D型肝炎病毒還有許多的未知，因此這對於發展D型肝炎的治療是一大挑戰。其中，我們對於D型肝炎病毒在宿主細胞內的複製機制較有興趣，因為其複製過程需要宿主RNA聚合酶的參與，而這與其他已知的RNA病毒複製不盡相同。先前已有許多報告研究發現哺乳動物細胞中的三種依DNA指引的RNA聚合酶都可能參與D型肝炎病毒的複製，在我們的研究中，我們認為並假設主要參與其複製的蛋白為RNA聚合酶II，我們的目標是純化出於轉染D型肝炎病毒RNA的HeLa細胞中的RNA聚合酶複製聚合體，並在細胞外以此聚合體進行D型肝炎病毒RNA的複製，以驗證我們的假說。 我們先前已成功建立了帶有FLAG-RPB9的HeLa細胞株，並在此細胞中以轉染D型肝炎病毒cDNA的方式證明此細胞株可以支持D型肝炎病毒的RNA複製。在本次的研究中，我們以轉染D型肝炎病毒RNA的方式在帶有FLAG-RPB9的HeLa細胞株建立了D型肝炎病毒RNA的轉染系統，接下來在此系統中進行細胞質、細胞核質及細胞核仁的分離，發現D型肝炎病毒的病毒抗原及RNA在複製過程中主要出現在細胞核質，同時此位置也為RNA聚合酶II的作用位置。隨著轉染後天數的增加，在轉染D型肝炎病毒RNA的細胞模型中，D型肝炎病毒的genomic及anti-genomic RNA都會隨時間增加，且於轉染D型肝炎病毒 anti-genomic RNA的細胞中發現D型肝炎病毒的genomic及anti-genomic RNA出現到細胞質中。在螢光免疫染色的結果中，我們也發現D型肝炎病毒抗原在細胞核質的分布會隨著轉染後天數的增加而增加。除此之外，在我們的D型肝炎病毒RNA轉染細胞模型中加入2 μg/ml的ɑ-amanitin能夠抑制D型肝炎病毒RNA的複製。 總結來說，我們在這個研究中建立了D型肝炎病毒RNA於FLAG-RPB9表現的HeLa細胞的轉染系統模型，並發現在D型肝炎病毒複製過程中新合成的D型肝炎病毒RNA主要位在細胞核質，同時為RNA聚合酶II的主要位置，且RNA聚合酶II抑制劑— ɑ-amanitin —能夠抑制新的D型肝炎病毒RNA合成，間接證明了RNA聚合酶II於D型肝炎病毒複製系統中的重要角色。接下來，我們將藉由分離此RNA轉染模型的細胞核進行細胞外轉錄實驗，觀察其對D型肝炎病毒RNA的複製，再進一步利用蔗糖梯度離心及免疫共沉澱的方式純化出RNA聚合酶II的複製聚合體進行細胞外轉錄，以證明RNA聚合酶II於D型肝炎病毒RNA的複製過程中的角色。

Hepatitis Delta Virus (HDV), which infects liver cells alongside Hepatitis B Virus (HBV), results in the most severe form of viral hepatitis. The limited understanding of HDV presents significant challenges in developing an effective cure. We are particularly interested in HDV replication because it uniquely utilizes the host's polymerase, unlike other known RNA viruses. Previous reports have indicated that three types of DNA-dependent RNA polymerases in mammalian cells might be involved in HDV replication. We hypothesized that RNA polymerase II is primarily responsible for this replication. Our study aims to purify the RNA polymerase II replication complex in an HDV transfected cell model to investigate its role in transcribing HDV RNA in vitro. We previously developed a FLAG-RPB9 expressing HeLa cell line, which successfully transcribes HDV RNA when transfected with HDV cDNA. In this study, we performed HDV RNA and mRNA co-transfection in this cell line to achieve effective HDV RNA transcription. We then conducted subcellular fractionation in HDV transfected HeLa cells and found that the newly synthesized HDV antigen and RNAs were primarily located in the nucleoplasm, where RNA polymerase II is localized. Over time, both HDV genomic and anti-genomic RNAs appeared in the cytoplasm in HDV anti-genomic RNA transfected HeLa cells. We also performed immunofluorescence imaging and observed that HDAg gradually appeared in the nucleoplasm during HDV RNA replication. Additionally, we discovered that the synthesis of both HDV genomic and anti-genomic RNAs were inhibited by 2 μg/ml ɑ-amanitin treatment in HDV RNA transfected HeLa cells. To summarize, we established an HDV RNA transfected HeLa cell model and demonstrated that newly synthesized HDV RNA predominantly appears in the nucleoplasm, which is the major localization of RNAPII. The ɑ-amanitin treatment effectively inhibited HDV RNA replication. Our next steps involve extracting the nucleus and conducting a nuclear run-on assay to confirm HDV RNA replication. We will then perform sucrose gradient centrifugation and co-immunoprecipitation to further purify the RNA polymerase II replication complex from the HDV RNA transfected FLAG-RPB9 HeLa cells, enabling us to investigate HDV RNA replication in vitro.