2014-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643165摘要：C 型肝炎病毒(Hepatitis C Virus, HCV)的非結構性蛋白nonstructural protein 5A (NS5A)在其生命週期中扮演極重要的角色，包括調整寄主的抵抗力、改變寄主訊息傳遞的路徑、與參與病毒活性等功能。過去的研究也發現 NS5A的高度磷酸化(hyper-phosphorylation)與病毒的複製能力與組裝有關。但是，我們對NS5A的磷酸化位點和相關的磷酸化酶(kinase)及去磷酸化酶(phosphatase)尚未有清楚的了解。本實驗室利用液相層析搭配質譜技術(liquid chromatography coupled with tandem mass spectrometry, LC-MS/MS)，在受HCV感染的人類肝癌細胞株裏鑑定了3個NS5A的磷酸化位點(phosphorylation site)，包括一個已知的絲氨酸磷酸化位點(S2194)，以及另兩個新的磷酸化位點：絲氨酸(S2202)和蘇胺酸(T2216)。本實驗室進一步發現當S2202被突變成不能被磷酸化的丙氨酸(alanine)後會使病毒停止複製，當T2216突變成丙氨酸則會增加病毒組裝能力。我們並製出S2202磷酸化的專一抗體。我們發現S2202磷酸化隨感染時間增加而增加，S2202磷酸化位於病毒複製的胞內膜狀結構上，顯示S2202磷酸化參與病毒複製。本計畫旨在探討以下課題一、 S2202磷酸化參與病毒複製的機制二、 T2216磷酸化參與病毒組裝的機制三、 S2194、S2202與T2216磷酸化在病毒生命週期的功能與交互關係近10 年來，肝癌死亡率位居台灣癌症死因之首，其中最主要的致病原之一就是C 型肝炎病毒。目前C型肝炎以干擾素與抗病毒藥物Ribavirin之合併療法為主流，但其副作用強，適應症也因人而異。多年來，醫學界一直致力於發展C型肝炎的標靶治療法。本研究計畫已找出與病毒生命週期有關的NS5A的磷酸化位點，將進一步找出其磷酸化酶或去磷酸化酶。我們的成果將有助於發展出治療C型肝炎的新療法，也可助增進我們對HCV病毒的知識。<br> Abstract: The hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays critical roles in HCV life cycle. It modulates host antiviral responses and signaling network. It is indispensable in viral activity. NS5A is a phosphorylated protein whose phosphorylation states are associated viral replication and assembly; however the phosphorylation sites and the kinases/phosphatases involved have not been completely identified. We have identified 3 phosphorylation sites on NS5A using LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) in HCV JFH/J6 strain infected human hepatocarcinoma cells huh7.5.1. One of them (serine 21194, S2194) is previously reported and two are not previously reported: serine 2202 (S2202) and threonine 2216 (T2216). Our preliminary data show that alanine mutation of S2202, which ablates S2202 phosphorylation, abolishes HCV viral replication. In contrast, alanine mutation of T2216 increases HCV viral assembly. We have generated a phospho-specific antibody against NS5A S2202 phosphorylation. Our data show that NS5A S2202 phosphorylation increases with time upon viral infection. NS5A S2202 phosphorylation is localized to intracellular membranous structures where viral replication is thought to take place, consistent with its role in HCV replication. Altogether, we have identified two novel NS5A phosphorylation sites with distinct functions in HCV life cycle. Our Specific Aims are to investigate the molecular mechanisms by which the phosphorylation sites mediate HCV life cycle. 1.The roles of NS5A S2202 phosphorylation in HCV replication2.The roles of NS5A T2216 phosphorylation in HCV assembly3.The relationship among S2194, S2202, and T2216 phosphorylation sites in HCV life cycleHCV is one of the major causes of chronic liver diseases, often progressing to cirrhosis and hepatocellular carcinoma. There is no vaccine available for HCV. Current treatment using interferon  combined with ribavirin is inadequate and non-specific to HCV infection. Our study has identified critical NS5A phosphorylation sites involved in HCV life cycle. We will identify the kinases/phosphatases responsible for their phosphorylation. These kinases/phosphatases may be new targets for HCV-specific treatment. Our results will also enhance our knowledge of HCV.C 型肝炎病毒非結構性蛋白病毒生活史脂肪肝磷酸化蛋白質譜術複製肝癌肝硬化HCVNS5Acancerlivercirrhosisfattyphosphorylationphosphoproteomics