鑑定調控B型肝炎病毒核殼蛋白C端之Serine 170位點磷酸化修飾之激酶

Identification of the Putative Kinase for Serine-170 in C-terminal Domain of HBV Core Protein

劉柔辰(Rou-Chen Liu)

指導教授 ： 葉秀慧

國立臺灣大學/醫學院/微生物學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601997

摘要

B型肝炎病毒(Hepatitis B virus；HBV)所產生之核殼蛋白(HBV core protein；HBc)由183個胺基酸組成，其中1-149胺基酸負責病毒nucleocapsid之組裝；而150-183胺基酸(C-terminal domain；CTD)則負責病毒pregenomic RNA (pgRNA)之包裹(encapsidation)。HBc為一磷酸化蛋白，磷酸化主要發生在CTD中之多個Serine位點，先前研究利用點突變方式發現這些位點的磷酸化可能參與HBV RNA encapsidation以及DNA synthesis等步驟之調控，然這些結果仍有待在wild type HBV replicon中進行確認。為此必須找出負責調控HBc-CTD特定位點磷酸化的激酶 (kinase)，利用影響kinase之活性以探討特定位點磷酸化對病毒複製之影響。雖然目前已有數個kinase被報導可能參與HBc-CTD的磷酸化，然而這些kinase的相關研究主要是利用in vitro kinase assay，無法直接證明這些kinase在細胞內(in vivo) 確實參與HBc蛋白的磷酸化，因此負責HBc-CTD磷酸化之kinase仍有待釐清。
本研究提出利用本實驗室所發展之一可區分HBc蛋白Serine-170位點有無磷酸化的專一性抗體，搭配kinase inhibitor處理細胞之策略，來找尋細胞中負責調控HBc蛋白磷酸化的候選kinase(s)，結果發現Akt可能參與HBc蛋白的磷酸化修飾。進一步利用phosphatase作用檢測HBc蛋白的磷酸化之程度，發現只有部分的HBc蛋白磷酸化是受到Akt inhibitor影響。有鑒於細胞內活化態的Akt主要是分佈於membrane compartment，實驗利用subcellular fractionation分析亦發現主要是分佈在membrane compartment中的HBc蛋白之磷酸化會受到Akt調控。本論文目前的研究結果初步發現Akt可能參與membrane中調控HBc蛋白serine-170位點的磷酸化修飾，未來將進行探討此Akt調控HBc蛋白磷酸化修飾對HBV複製生活史的影響。

關鍵字

B型肝炎病毒 ； 核殼蛋白 ； 磷酸化 ； 激酶

並列摘要

Hepatitis B virus (HBV) core protein (HBc) is a 183-amino acid protein, in which the 1-149 a.a. N-terminal domain is sufficient for nucleocapsid assembly and the 149-183 a.a. C-terminal domain (CTD) is critical for viral RNA encapsidation. The HBc-CTD contains several serine residues, including S155, S162, S170, as the major phosphorylation sites. As revealed by site-directed mutagenesis, the phosphorylation of HBc-CTD might regulate HBV RNA encapsidation and DNA synthesis, which however still awaits to be confirmed in wild-type HBV replicon. Identification of the putative cellular kinase(s) that phosphorylates specific serine residues at HBc-CTD will help address this issue. Though several candidate kinases were identified mainly by in vitro kinase assay, but none of them were proven to occur in cells undergoing active viral replication. The putative kinases are thus still remained to be identified.
The current study proposed to identify the kinase for Serine-170 of HBc-CTD by using the C-S170 antibody, which can specifically recognize the HBc with dephosphorylated S170. The HBc expressing cells were treated with a panel of kinase inhibitors and then processed for the Western blotting by probing with C-S170 antibody. The result pointed out Akt as one candidate in regulating HBc-S170 phosphorylayion. The phosphatase reaction helps illustrate that ~30% of HBc protein in cells is affected by the Akt inhibitor treatment. Regarding to that the Akt is activated at the membrane compartment, the fractionation analysis further demonstrated that the core protein at the membrane compartment is the target HBc to be regulated by Akt. In conclusion, the current study identified that Akt might contribute to the phosphorylation of HBc-S170 at the membrane compartment. This phoshsphorylation in regualating the viral replication is worthy to be further investigated.

並列關鍵字

Hepatitis B virus (HBV) ； core protein ； phosphorylation ； kinase

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