探討B型肝炎病毒核殼蛋白 Serine 170 位點磷酸化調控病毒複製之影響

Abstract

構成B型肝炎病毒(Hepatitis B virus；HBV)核殼(nucleocapsid)的核殼蛋白(HBV core protein；HBc)為一磷酸化蛋白質，透過其C端上Ser 155、Ser 162、Ser 170三個主要磷酸化位點調控HBV pregenomic RNA (pgRNA) encapsidation和DNA synthesis。近年研究指出HBc C端磷酸化在HBV生活史中呈現動態調控，HBc在進行pgRNA encapsidation前呈現高度磷酸化，而完成pgRNA encapsidation後則呈現低度磷酸化。然而對於HBc-S170如何透過磷酸化修飾調控pgRNA encapsidation及中間磷酸化轉變去磷酸化的分子機制仍不清楚。 本研究針對Ser 170磷酸化位點，探討其功能如何調控pgRNA encapsidation。利用專一辨認Ser 170位點去磷酸化之抗體C-S170，我們發現HBc-S170磷酸化位點在HBV 生活史中同樣呈現動態調控，在pgRNA encapsidation前後分別出現高度和低度磷酸化。透過回補的方式，顯示Ser170位點透過polymerase調控pgRNA encapsidation。除此之外，亦發現HBc和HBc-Cys蛋白上之Ser 170磷酸化位點均扮演調控pgRNA encapsidation進行之重要角色。本論文研究結果因此指出HBc和HBc-Cys蛋白上之Ser 170 為點之磷酸化有可能會藉由改變其蛋白質C端胺基酸序列之結構而影響其與polymerase蛋白之結合而增加pgRNA encapsidation之假說，值得未來進一步探討。

HBV core protein (HBc), the base subunit of Hepatitis B virus (HBV) nucleocapsid, is a phosphorylated protein regulating HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis through three major phosphorylation sites at Ser155, Ser162, Ser170 on its C-terminal domain (CTD). Recent studies have demonstrated that HBc C-terminal undergoes dynamic phosphorylation during HBV life cycle. HBc is hyperphosphorylated before pgRNA encapsidation and hypophosphorylated after pgRNA encapsidation. Nevertheless, the molecular mechanisms for the phosphorylation of HBc-Ser170 in regulating pgRNA encapsidation and the specific events occurring from phosphorylation to dephosphorylation are still unclear. This study focused on HBc-Ser170 (S170) phosphorylation site for investigating its function in regulating the RNA encapsidation process, in the replicon transfected cells. Using the dephosphorylated specific C-S170 Ab, we found a dynamic phosphorylation pattern of HBc-S170 in different replication stages, highly phosphorylated before RNA encapsidation but de-phosphorylated afterwards. By complementation assay, we demonstrated that HBV pgRNA encapsidation could be mediated through the recruited polymerase. Moreover, we found the phosphorylation of S170 is essential for both HBc and HBc-Cys proteins to proceed the RNA encapsidation process. The possibility that phosphorylation of S170 in HBc and HBc-Cys protein could be critical for recruitment of polymerase via inducing a conformational change of their C-terminal domain structure is worthy to be tested in future studies.