以質譜儀鑑定C型肝炎病毒高度及低磷酸化態非結構性蛋白質5A的交互作用蛋白質

Abstract

C型肝炎病毒（hepatitis C virus , HCV）非結構性蛋白質5A （Non-structure protein 5A , NS5A）在病毒生活史中扮演重要的角色。非結構性蛋白質5A有兩種磷酸化態，包含：低度（hypo）磷酸化態，以及高度（hyper）磷酸化態。利用免疫墨點法，我們可以偵測到低度磷酸化態的NS5A位在56kDa的位置，而高度磷酸化態的NS5A則是在58kDa的位置。目前，已有許多研究將NS5A上被認為對病毒生活史滿重要的絲氨酸（serine, S）位點進行突變，發現NS5A的高度磷酸化態對於HCV的複製扮演非常重要的角色。然而，對於低度磷酸化態的NS5A是如何參與在HCV的病毒生活史中目前還不是那麼了解。所以我的研究目標是利用質譜儀去分析是否高度磷酸化的NS5A與低度磷酸化的NS5A會跟不同的蛋白進行交互作用進而有不同的功能。實驗室先前就已經製作會辨認高度磷酸化態NS5A的抗體（anti-p58），我們也做了另外的抗體（anti-p56）去辨認低度磷酸化態的NS5A。我們利用這兩支抗體，透過高解析度顯微鏡發現低度磷酸化態的NS5A與高度磷酸化態的NS5A在細胞內的分布不一樣，我們認為這可能跟NS5A不同磷酸化程度可能有不同的功能有關。所以我們接著利用這兩支抗體分別對HCV感染後的人類肝癌細胞Huh7.5.1進行免疫沉澱實驗。並且將會與高度磷酸化態NS5A交互作用的蛋白質與會與低度磷酸化態NS5A交互作用的蛋白質藉由非標記定量（label-free）的蛋白質體學技術進行分析。我們總共做了兩次實驗，一共鑑定到534個蛋白質，其中有104個蛋白質顯示較傾向與高度磷酸化態的NS5A進行交互作用，有14個蛋白質顯示較傾向與低度磷酸化態的NS5A進行交互作用。進一步利用DAVID基因本體(Gene Ontology)分析發現高度磷酸化態的NS5A在細胞遷移（cell migration）與HCV RNA的複製中扮演關鍵的角色。

The non-structural protein 5A (NS5A) is a critical hepatitis C virus (HCV) protein required for the viral life cycle. It is a phosphoprotein with two phosphorylation states: hypo- and hyper-phosphorylated NS5A that appear at 56 (p56) and 58 (p58) kDa on immunoblots. Numerous genetic mutations followed by functional assays have pinpointed several serine residues that are critical for NS5A hyper-phosphorylation and for viral replication ability, indicating a role of hyper-phosphorylated NS5A in HCV replication. The functions of hypo-phosphorylated NS5A are less understood. My thesis work aimed to distinguish the proteins that bound to hypo- vs. hyper-phosphorylated NS5A using co-immunoprecipitation followed by LC-MS/MS analysis. We have previously generated a hyper-phosphorylation specific antibody that specifically detected hyper-phosphorylated NS5A. Here, we generated a hypo-phosphorylation specific antibody that detected only hypo-phosphorylated NS5A. Using stimulated-emission depletion based super resolution microscopy, the hypo- and hyper-phosphorylated NS5A were found in discrete intracellular localization, suggesting that these two phosphorylation forms of NS5A may have discrete functions. Co-immunoprecipitation followed by label-free quantitative proteomics identified proteins that bound to hypo- vs. hyper-phosphorylated NS5A. A total of 534 proteins were quantified in two replicates. Among the quantified proteins, 104 proteins showed favorable interacting with hyper-phosphorylated NS5A and 14 proteins showed favorable interacting with hypo-phosphorylated NS5A. DAVID Gene Ontology term analysis of the proteins bound to hyper-phosphorylated NS5A revealed terms predominantly associated with cell migration and RNA replication.