C型肝炎病毒非結構性蛋白5A磷酸化位點之鑑定與功能探討

Abstract

C型肝炎為一慢性的肝發炎疾病，也是引發肝癌的原因之一。其致病原 ─ C型肝炎病毒(Hepatitis C virus, HCV)為單一正股RNA病毒。HCV之基因體可轉譯出一條多蛋白 (Polyprotein)，透過蛋白酶的切割，此多蛋白能產生具有功能的結構性(Structural protein)及非結構性蛋白(Nonstructural protein)。其中，C型肝炎病毒的非結構性蛋白5A (Non-structural protein 5A, NS5A)為一重要的抗病毒藥物標的，但NS5A在HCV生命周期中的詳細功能尚不清楚。過去報導顯示NS5A會受磷酸化調控，而其磷酸化會影響C型肝病毒的複製能力。但NS5A的磷酸化位點尚未清楚。 利用液相層析串聯式質譜術 (Liquid chromatography coupled with tandem mass spectrometry)，我們鑑定出一個全新的NS5A磷酸化位點 (Serine 2207, S2207)，並發現此位點存在於各基因型的C型肝炎病毒中。為了探討此位點功能，我們把帶有報導基因之HCV載體的serine 2207突變為alanine，以抑制該位點的磷酸化。將此載體表現於人類肝腫瘤細胞株(Huh7.5.1)，結果顯示，HCV突變株的報導基因活性無法隨感染時間增加。在進一步的研究中，我們發現NS5A蛋白S2207位點的磷酸化程度隨病毒感染時間增加，亦觀察到S2207磷酸化的NS5A與另一參與病毒複製的蛋白─非結構性蛋白3 (NS3)有共位現象(co-localization)。這些結果確認S2207的磷酸化存在於HCV生命周期中，並暗示S2207的磷酸化在HCV複製之重要性。透過利用casein kinase I (CKI)的抑制劑進行實驗，我們發現S2207的磷酸化隨抑制劑劑量增加而下降，顯示CKI或許參與在S2207的磷酸化過程中。 綜上所述，本研究發現一個全新的NS5A磷酸化位點，並證明此位點之磷酸化在病毒複製中的重要性。這些發現增進我們對NS5A的認識，並將有助於我們探討調控HCV複製的詳細機轉。

Phosphorylation of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is implicated in HCV life cycle. Disrupting NS5A phosphorylation has been reported to affect viral replication and hence may be of clinical relevance; however, specific sites of NS5A phosphorylation have been elusive. In this study, we identified 1,776 unique phosphorylation sites in 1,088 proteins of HCV (J6/JFH1 strain, genotype 2a) infected Huh7.5.1 cells via liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). NS5A protein was found to be phosphorylated at serine 2194, a previously reported site, and serine 2207 (S2207), a new site. Mutation of S2207 to a phosphorylation-ablative residue in an HCV reporter construct abolished HCV replication. Immunoblotting showed a time-dependent increase in S2207 phosphorylated NS5A upon HCV infection. Immunofluorescence staining revealed that S2207 phosphorylated NS5A co-localized with an HCV replication complex component, nonstructural protein 3 (NS3) and localized in cytoplasmic membranous structures, where viral replication takes place. These findings suggest a role of NS5A S2207 phosphorylation in HCV RNA replication. Furthermore, we discovered that a casein kinase I inhibitor, D4476, reduced S2207 phosphorylation in a dose-dependent manner, thus suggesting a role of casein kinase in S2207 phosphorylation. In conclusion, our study identifies a new casein kinase inhibitor sensitive NS5A phosphorylation site (serine 2207) which is involved in HCV replication.