人類RNA聚合酶I在D型肝炎病毒反基因股RNA合成中扮演之角色

Abstract

D型肝炎病毒 (hepatitis delta virus；HDV)為目前已知可感染人類的最小RNA病毒，且病毒RNA約有70%之分子內互補以形成類桿狀的雙股結構特性。由於本身不具備病毒複製所需的RNA-dependent RNA聚合酶 (RdRp)，因此D型肝炎病毒被認為具有改變宿主DNA-dependent RNA聚合酶功能以完成病毒RNA複製的能力。研究普遍認為宿主RNA聚合酶II會參與病毒RNA合成，包含由病毒反基因股RNA合成基因股RNA以及由病毒基因股RNA合成mRNA此二階段。然而，由病毒基因股RNA合成反基因股RNA的階段究竟為何種宿主RNA聚合酶參與，仍存有爭議。本實驗室先前的研究結果暗示宿主RNAP I可能在D型肝炎病毒生命週期扮演某種角色。而本研究主要探討宿主RNAP I是否參與在病毒生命週期中的病毒反基因股RNA之複製階段，又其扮演的角色為何。本研究利用轉染系統讓病毒RNA在細胞內進行複製，發現對病毒RNA複製重要的小型delta抗原以及病毒基因股RNA皆能與宿主RNAP I的最大次單元RPA194產生交互作用。進一步以較貼近真實情況的病毒感染系統進行探討，發現在宿主RPA194表現量降低的環境，細胞內的病毒反基因股RNA合成明顯降低。本研究結果顯示宿主RNAP I可能在病毒反基因股RNA合成中扮演重要角色。另外，以不同應用軟體分析在病毒基因股RNA上是否有類似於宿主rRNA合成之調控區域，結果發現HDV基因股RNA含有類似於CTCF之cis-element (nt 317~321)。進一步突變此區域，發現突變型病毒RNA會讓細胞內病毒反基因股和基因股RNA的合成量明顯下降，顯示此區域在病毒RNA複製可能具有重要意義。然而，宿主RNAP I與突變型1 (MT1)病毒基因股RNA的結合能力不降反升，而宿主RNAP I與突變型2 (MT2)病毒基因股RNA的結合情形不受影響。CTCF之cis-element及宿主RNAP I如何參與病毒反基因股RNA複製，仍需進一步探討。

Hepatitis delta virus (HDV), the smallest RNA virus known to infect human, has a rod-like structure RNA genome due to up to 70% intramolecular base-pairing. Because HDV doesn’t encode its own RNA-dependent RNA polymerase (RdRp), HDV is believed to redirect host DNA-dependent RNA polymerase to participate in HDV RNA synthesis. During HDV RNA synthesis, host RNA polymerase II synthesizes the viral genomic RNA and mRNA from antigenomic RNA and genomic RNA, respectively. However, which host RNAP is responsible for HDV antigenomic RNA synthesis still remains controversial. Previous studies from our laboratory indicated that host RNAP I may be involved in HDV life cycle. In the present study, the role of host RNAP I involved in the HDV antigenomic RNA synthesis was investigated. Using transfection system to allow HDV RNA replication occured in cells, the largest subunit of host RNAP I, RPA194, was shown to interact with HDV genomic RNA and small delta antigen required for HDV RNA replication. To mimic HDV infection in vivo, immortalized human hepatocytes were infected with HDV. Results showed that HDV antigenomic RNA synthesis was decreased in RPA194 knockdown cells. These results suggested that host RNAP I may play important roles in HDV antigenomic RNA synthesis. In addition, by using various softwares to analyze whether HDV genomic RNA contains the regulatory elements of host rRNA synthesis, a putative CTCF cis-element at nt 317~321 was identified. Mutations at the CTCF cis-element reduced both HDV antigenomic and genomic RNA levels. Surprisingly, the interaction between host RNAP I and the mutated HDV genomic RNA 1 (MT1) was enhanced, while interaction between host RNAP I and MT2 had no significant change as compared to the wildtype. How the putative CTCF cis-element and host RNAP I are involved in HDV antigenomic RNA synthesis needs to be further investigated.