探討B型肝炎病毒核殼相關蛋白質對病毒複製過程之影響

Abstract

乙型肝炎病毒 (Hepatitis B virus，HBV) 的pregenomic RNA (pgRNA) 除了會轉譯出聚合酶和核殼蛋白 (HBV core protein; HBc) 之外，也會經由剪接(splicing) 所產生的spliced RNAs (spRNAs) 製造出數種病毒蛋白質。在眾多spRNAs中，SP1 spRNA為表現量最多之一員，會轉譯出相較HBc183缺少最後一個胺基酸Cys183之HBc-Cys蛋白質。HBc結構主要可分為兩個domain: 第1-149胺基酸 (N-terminal domain; NTD)負責nucleocapsid的組裝，第150-183胺基酸 (C-terminal domain; CTD)則負責病毒核酸的包裹。由於HBc183與HBc-Cys除了CTD尾端之Cys外，NTD和CTD胺基酸序列完全相同，因此我們提出一個假說是HBc183與HBc-Cys可以一起組裝形成capsid，但在HBV的生活史中各司其職具備不同的功能。 藉由來自濱松大學 (Hamamatsu University) Suzuki教授所提供的HBc-Cys 專一性抗體，我們發現HBc-Cys存在於sucrose gradient 之capsid fraction中，顯示了HBc-Cys會與HBc183一同組裝形成病毒capsid。此外，我們也利用只表現HBc183或HBc-Cys的replicon來研究這兩個蛋白質在capsid組裝的過程中是否有所差異，結果發現HBc183之Cys183參與形成由雙硫鍵鍵結的核殼蛋白dimer結構。然而在CMV-WT和缺少SP1的CMV-A487C replicon所轉染的細胞中，我們觀察到兩者之間無論是在capsid組裝過程、DNA複製和病毒顆粒的釋放皆沒有顯著差異。而同樣的實驗也在貼近正常HBV病毒複製過程的AAV-HBV replicon系統中進行，以進一步探究HBc183或HBc-Cys在病毒複製過程，尤其是capsid的包膜 (envelopment) 和病毒顆粒釋放是否存在差異。本論文研究結果將有機會解開HBc183與HBc-Cys各自在HBV生活史中所扮演的功能性角色。

In addition to encode the polymerase and core (HBc) protein, the pregenomic RNA (pgRNA) of hepatitis B virus (HBV) also produces several viral proteins encoded by the pgRNA derived spliced RNAs (spRNAs). Among them, HBc-Cys encoded by the most abundant SP1 spRNA consists of same protein sequence with HBc but lacks only one cysteine at the very C terminal. As documented, HBc protein contains two functional domains: the N terminal domain (NTD, 1-149 a.a., responsible for nucleocapsid assembly) and the C-termianl domain (CTD, 150-183 a.a., responsible for packaging viral RNA). As HBc and HBc-Cys share the same NTD and CTD, we proposed a hypothesis that both HBc and HBc-Cys can be packaged into the viral capsids, but with different functions in viral replication cycle. Aided by the HBc-Cys-specific antibody kindly provided by Prof. Suzuki (Hamamatsu University), we first found the presence of HBc-Cys protein in the sucrose gradient fractions enriched of viral capsids. It thus suggested the assembly of HBc-Cys together with HBc into the capsids. HBV replicon constructs, expressing either HBc183 or HBc-Cys alone, were then used to examine any difference between HBc and HBc-Cys in capsid assembly process. We found the Cys183 in HBc is involed in the formation of disulfide-bond mediated dimers, the basic subunit of viral capsids. The putative function of HBc183 or HBc-Cys in viral replication cycle has been examined in CMV-WT and SP1 deficient HBV replicons, which however did not show any significant difference for capsid assembly, DNA synthesis and virion release. The same experiment has been applied to the AAV-HBV replicons, which can help examine the envelopment and virion secretion process. We expect the results can help unravel the distinct functions of HBc183 and HBc-Cys in viral replication cycle.