D型肝炎抗原磷酸化對病毒反向基因體複製機轉 之功能性探討

Abstract

D型肝炎是最簡單的RNA病毒，它可以透過相當獨特的方式進行複製。當病毒進入細胞，病毒本身會先合成小型抗原，並利用細胞內的RNA複製酶進行基因體增殖。過去研究已發現，小型抗原的後轉譯修飾（例如磷酸化、乙醯化與甲基化）可以引導病毒成功完成各階段的生活史，其中小型抗原Ser 177的磷酸化對於病毒反向基因體的複製是相當重要的。目前普遍認為執行D型肝炎病毒反向基因體的RNA複製酶為RNA polymerase II，而且Ser 177在小型抗原與RNA polymerase II之結合能力上可能扮演著舉足輕重的角色，本論文便針對此論點加以進行探討。結果發現，小型抗原在Ser 177進行去磷酸化後與低磷酸化之IIA型RNA polymerase II有較佳的結合力，反觀磷酸化的小型抗原卻與高磷酸化之IIO型RNA polymerase II有較佳的結合力。RNA polymerase II在細胞中合成RNA時，通常會利用磷酸化與否來調控轉錄的各個時期，一般在initiation時期RNA polymerase II會以IIA型態結合在DNA模板的起始點，到了elongation時期則會被細胞中的kinase大量磷酸化而呈現IIO型態，尤其是在其C端Ser 2和Ser 5的位置上。更進一步探討小型抗原磷酸化在病毒反向基因體複製上可能扮演的角色，結果發現Ser 177磷酸化之小型抗原通常會和Ser 2和Ser 5同時被磷酸化的IIO型RNA polymerase II結合，而且kinase抑制劑（DRB）不但會阻斷磷酸化之小型抗原與IIO型RNA polymerase II結合，更會抑制病毒反向基因體之複製。由此結果推斷，Ser 177之磷酸化可能會調控小型抗原與不同型態的RNA polymerase II結合，使RNA polymerase II在進行病毒反向基因體複製時，得以由initiation時期進入elongation時期。然而，除了病毒反向基因體複製酶RNA polymerase II之外，小型抗原是否還可以透過磷酸化來調控與其他細胞內分子的結合力，進而影響病毒反向基因體之複製，則是未來仍需繼續探究的方向。

Hepatitis delta virus (HDV) is the simplest RNA virus that employs a unique strategy for viral replication. Once the virus enters the cells, it uses cellular RNA polymerases and one viral protein, small hepatitis delta antigen (SHDAg), for viral RNA replication. Recent studies have revealed that posttranslational modifications (e.g., phosphorylation, acetylation, and methylation) of SHDAg are conducting the essential functions at successive stages of the HDV life cycle. Phosphorylation of SHDAg at Ser177 is required for HDV replication from antigenomic to genomic RNA, and this residue is crucial for interaction with RNA polymerase II (RNAP II), the enzyme assumed to be responsible for antigenomic RNA replication. This study demonstrated that SHDAg dephosphorylated at Ser177 interacted preferentially with hypophosphorylated RNAP II (RNAP IIA), which generally binds at the transcription initiation sites. In contrast, the Ser177-phosphorylated counterpart (pSer177-SHDAg) exhibited preferential binding to hyperphosphorylated RNAP II (RNAP IIO). In addition, RNAP IIO associated with pSer177-SHDAg was hyperphosphorylated at both the Ser2 and Ser5 residues of its carboxyl-terminal domain (CTD), which is a hallmark of the transcription elongation isoform. Moreover, the RNAP II CTD kinase inhibitor 5,6-dichloro-1-β-D-ribofuranosyl- benzimidazole (DRB) not only blocked the interaction between pSer177-SHDAg and RNAP IIO, but also inhibited HDV antigenomic replication. Our results suggest that the phosphorylation of SHDAg at Ser177 shifted its affinity toward the RNAP IIO isoform and thus may be a switch for HDV antigenome replication, from the initiation to the elongation stage. Except for RNAP II, the study of whether SHDAg interacts with some unknown factors essential for viral replication through a phosphorylation-dependent manner is ongoing.