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Title: | 非結構性蛋白質5A絲氨酸235磷酸化介導C型 肝炎病毒複製之分子機制探討 Molecular Mechanisms of NS5A S235 Phosphorylation-Mediated Hepatitis C Virus Replication |
Authors: | Kuan-Ying Lee 李冠潁 |
Advisor: | 余明俊 |
Keyword: | C型肝炎病毒,非結構性蛋白質5A,絲胺酸235,人類囊泡相關膜蛋白相關蛋白A的宿主因子, HCV,NS5A,S235,hVAP-A, |
Publication Year : | 2016 |
Degree: | 碩士 |
Abstract: | C型肝炎病毒(Hepatitis C virus, HCV)非結構性蛋白質5A (Non-structural protein 5A, NS5A)第235號絲胺酸(serine 235, S235)的磷酸化對病毒的複製來說是一個非常重要的轉譯後修飾作用。基於病毒學與醫學上的研究興趣,了解哪些激酶負責NS5A S235磷酸化是有其必要的。使用HEK293T細胞作為篩選平臺,我們發現第二型鈣調蛋白依賴激酶(Calmodulin-dependent kinase II, CaMKII)抑制劑及第一型酪蛋白激酶(Casein kinase I α, CKIα)抑制劑兩者皆在無細胞毒性的前提下能降低NS5A S235的磷酸化。然而在感染HCV的Huh7.5.1細胞中只有CKIα的gene knockdown能有效抑制NS5A S235的磷酸化及HCV的核醣核酸的數量,這顯示由CKIα所促使的NS5A S235磷酸化在HCV的複製中扮演關鍵的角色。HCV的複製通常需要許多宿主因子的協助,在病毒感染的細胞中一個稱為hVAP-A(人類囊泡相關膜蛋白相關蛋白A的宿主因子)的宿主因子在細胞質有聚集成簇的現象,然而這樣的現象卻在進行CKIα的gene knockdown後就消失了。上述的結果顯示在感染細胞中CKIα所介導的NS5A S235磷酸化能協助HCV複製複合體形成並幫助病毒的複製。我們最近也正在使用液相層析質譜儀來分析235磷酸化與不磷酸化之間不同的交互作用蛋白質差異。我們的分析結果將可為究竟有哪些蛋白質會參與在235磷酸化的NS5A上提供完整的說明。 Phosphorylation at serine 235 of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a critical post-translational modification required for viral replication. Because of virological and medical interests, it is necessary to identify the kinases responsible for NS5A S235 phosphorylation. Using HEK293T cells as a platform, we found both calmodulin-dependent kinase II (CaMKII) and casein kinase I α (CKIα) inhibitor reduced NS5A S235 phosphorylation without cytotoxicity. However, only shRNA-mediated CKIα knockdown reduced S235 phosphorylation and HCV RNA levels in the HCV-infected Huh7.5.1 cells, indicating a critical role CKIα-mediated NS5A S235 phosphorylation in HCV replication. HCV replication often requires host factors such as the vesicle-associated membrane protein-associated protein A (hVAP-A) that forms clusters in the HCV-infected cells. Upon CKIα knockdown, the hVAP-A clusters dispersed throughout the cytosol. The above data suggest that CKIα-mediated NS5A S235 phosphorylation facilitates HCV replication complex formation in the infected cells. We are currently using LC-MS/MS based protein mass spectrometry to profile the host protein factors that interact with S235D vs. S235A NS5A. Our findings will potentially provide a complete protein repertoire that participates in NS5A S235 phosphorylation-mediated HCV replication. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/50485 |
DOI: | 10.6342/NTU201601420 |
Fulltext Rights: | 有償授權 |
Appears in Collections: | 生物化學暨分子生物學科研究所 |
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ntu-105-1.pdf Restricted Access | 1.26 MB | Adobe PDF |
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