- 學位論文
探討B型肝炎病毒核殼蛋白C端之Serine 170位點磷酸化修飾在HBV生活史之影響
Phosphorylation of C-terminal Domain Serine-170 of Hepatitis B Virus Core Protein in Regulating the Viral Life Cycle
摘要
B型肝炎病毒(Hepatitis B virus；HBV) 所產生的核殼蛋白(HBV core protein；HBc)是由183個胺基酸組成，其中1-149個胺基酸(N-terminal domain；NTD) 為assemble domain，負責病毒核殼(nucleocapsid)的組裝；而150-183個胺基酸(C-terminal domain；CTD) 為nucleic acid-binding domain，負責病毒pgRNA之包裹(encapsidation)。HBc為一磷酸化蛋白，磷酸化主要發生在CTD中的絲氨酸(Serine)位點。先前研究通過絲氨酸突變為丙氨酸(Alanine)，模擬去磷酸化形式HBc，發現Ser155，Ser162，Ser170的磷酸化修飾會參與HBV RNA encapsidation以及DNA synthesis等步驟之調控。然而CTD的磷酸化在wild type HBV複製子中的作用以及潛在的機制仍有待釐清。 在本研究中，我們使用C-S170抗體以檢查不同複製時期的HBc-S170磷酸化，C-S170抗體可專一性識別含有去磷酸化Ser170的HBc。通過引入特定的基因突變在不同階段停止病毒複製，且不影響HBc蛋白密碼子。結果顯示HBc-S170在複製週期中磷酸化呈動態改變，其在pgRNA encapsidation之前為高度磷酸化；而大多數含有核酸的核殼(genome-containing nucleocapsids)為去磷酸化。此外根據在HBc-S170位點不能去磷酸化的HBV突變體中，我們發現pgRNA encapsidation顯著降低，這顯示HBc-S170的去磷酸化對於encapsidation可能是重要的。而通過磷酸酶抑制劑處理下，nucleocapsid所包裹的pgRNA明顯減少，進一步證實了這種可能性。由於在S170A突變體複製子中鑑定出其會降低pgRNA encapsidation，由此可知S170A的磷酸化對於RNA衣殼化之前的某些特定事件也是重要的，目前正在研究中。
並列摘要
Hepatitis B virus (HBV) core protein consists of 183 amino acids, in which 1-149 amino acids (N-terminal domain; NTD) is responsible for the assembly of nucleocapsids and 150-183 amino acids (C-terminal domain; CTD) is responsible for the encapsidation of the viral pregenomic RNA (pgRNA). HBc is a phosphorylated protein, with the phosphorylation sites at Serine residues in CTD. Approached by mutation of Serine to Alanine, mimicking the dephosphorylated form, phosphorylation of Ser155, Ser162, Ser170 has been suggested to participate in the regulation of viral RNA encapsidation and DNA synthesis process. However, the effects in wild-type replicon and also the underlying mechanism are still remained to be addressed. In this study, we used the C-S170 antibody, which specifically recognizes the HBc epitope containing dephosphorylated Ser170, to examine the HBc-S170 phosphorylation pattern of different replication intermediates. The viral replication is stopped at different stages by introducing specific genetic mutations, which however did not affect the HBc protein codon. The results showed a dynamic phosphorylation of HBc-S170 through the replication cycle, which was highly phosphorylated before pgRNA encapsidation and most of the genome-containing nucleocapsids were dephosphorylated. With the evidence from an HBV mutant which could not be dephosphorylated at HBc-S170, we found that pgRNA encapsidation is significantly decreased. It suggested that dephosphorylating of HBc-S170 could be important for encapsidation. This possibility has been further validated by a decrease of pgRNA encapsidated under the treatment of phosphatase inhibitor. However in the previous study, a decreased RNA encapsidation was identified in the S170A mutant replicon, phosphorylation of S170A could be also important for the certain specific event before RNA encapsidation, which is currently under investigation.