Forms of HCV NS5A in vivo

描述(由申请人提供)：这是一项探索性研究拨款的申请，目的是识别在受感染的人类肝脏组织中产生的新型丙型肝炎病毒(丙型肝炎病毒)非结构蛋白，并开发其形成和功能的模型。丙型肝炎病毒在全球范围内仍然是一个重要的公共卫生问题。尽管多年来的研究成果，但无法研究细胞培养中的多种丙型肝炎病毒仍然是我们理解这种病毒用来建立持久性的机制的障碍，这最终可能导致肝细胞癌(HCC)的发展。丙型肝炎病毒非结构蛋白5A(NS5A)是大多数正链RNA病毒中唯一的非结构蛋白。它的三个结构域中有两个是固有的无序的，它包含大量的丝氨酸/苏氨酸激酶的磷酸化位点，包括cAMP激活的蛋白激酶(PKA)、酪蛋白激酶1家族成员和酪蛋白激酶2。我们一直感兴趣的是，NS5A磷酸化的特定模式产生了该蛋白独特的构象和功能，从而解释了NS5A与如此多的细胞蛋白质和途径相互作用的能力。我们对这种可能性的实验测试导致了对 NS5A被PKA磷酸化的位置和免疫学试剂的发展，以证明在复制1b和2a两种基因型的丙型肝炎病毒RNA的细胞中存在PKA磷酸化的NS5A。此外，对丙型肝炎儿童病例的活检组织和一例终末期丙型肝炎病例的组织进行检查后发现，在感染的肝组织中观察到的NS5A形式与在复制丙型肝炎病毒RNA的人肝癌细胞系中观察到的形式不同。在肝组织中检测到的其他形式的NS5A与激活caspase时在细胞系中观察到的NS5A非常相似，并且PKA磷酸化物种的丰度与肝脏损伤的程度很好地相关。我们的假设是，NS5A的caspase裂解可能为丙型肝炎病毒提供了一种监测肝细胞抗病毒状态的机制；NS5A的磷酸化可能向丙型肝炎病毒发出信号，表明肝细胞中存在适合复制的抗凋亡状态。这些被切割和磷酸化的形式可能会扩大NS5A蛋白质组，产生病毒增殖所必需的蛋白质形式。如果是这样的话，那么大多数丙型肝炎病毒序列在肝癌细胞系中不能复制的部分原因可能与不能产生形式的NS5A或其他非结构蛋白有关，这些蛋白在肝细胞中很容易产生，并且是最佳病毒增殖所必需的。我们的工作模型将通过以下具体目标进行探索：(1)体内感染过程中产生的各种形式的丙型肝炎病毒非结构蛋白的鉴定和表征；(2)体内感染过程中促凋亡和促存活(抗凋亡)反应的评估；以及(3)临床相关性的阐明。这些研究有能力发现感染肝细胞特有的各种形式的丙型肝炎病毒非结构蛋白，并揭示这些新形式的水平与临床结果之间的相关性。 公共卫生相关性：丙型肝炎病毒是世界范围内肝脏疾病的主要原因；只有两种丙型肝炎病毒序列和一种细胞系可用于体外研究丙型肝炎病毒的生命周期。我们发现，在丙型肝炎病毒感染的肝脏中观察到的某些形式的丙型肝炎病毒非结构蛋白在细胞系中不产生，我们认为这些形式在细胞系中的缺失限制了丙型肝炎病毒的体外增殖。因此，这项研究的完成可能会揭示新的方法来研究更多的丙型肝炎病毒，并促进新的抗病毒疗法的开发。

DESCRIPTION (provided by applicant): This is an application for an exploratory research grant to identify new forms of the hepatitis C virus (HCV) non- structural proteins produced in infected human liver tissue and to develop models for their formation and function. HCV continues to be a significant public-health concern of global proportion. In spite of years of effot, the inability to study the numerous genotypes of HCV in cell culture remains an obstacle to our understanding of the mechanisms employed by this virus to establish persistence, which can ultimately lead to the development of hepatocellular carcinoma (HCC). HCV non-structural protein 5A (NS5A) is unique among non-structural proteins of most positive-strand RNA viruses. Two of its three domains are intrinsically disordered, and it contains numerous sites of phosphorylation for myriad serine/threonine kinases, including cAMP-activated protein kinase (PKA), casein kinase 1 family members and casein kinase 2. We have been intrigued by the possibility that specific patterns of NS5A phosphorylation produce unique conformations and functions for this protein, thus explaining the ability of NS5A to interact with so many cellular proteins and pathways. Our experimental tests of this possibility have led to the identification of the site of NS5A phosphorylation by PKA and to the development of immunological reagents to demonstrate the existence of the PKA-phosphorylated form of NS5A in cells replicating HCV RNA of both genotypes 1b and 2a. In addition, examination of biopsies from pediatric cases of hepatitis C and tissue from an end-stage case of hepatitis C, led to the discovery that forms of NS5A observed in infected liver tissue are different than those observed in human hepatoma cell lines replicating HCV RNA. The additional forms of NS5A detected in liver tissue are remarkably similar to those observed in cell lines when caspases are activated and the abundance of the PKA- phosphorylated species correlates well with the amount of liver injury. It is our hypothesis that caspase cleavage of NS5A could provide a mechanism for HCV to monitor the antiviral state of the hepatocyte; phosphorylation of NS5A could signal to HCV that an anti-apoptotic state suitable for replication exists in the hepatocyte. These cleaved and phosphorylated forms may expand the NS5A proteome, creating forms of the protein essential for virus multiplication. If this is the case, then the inability of most HCV sequences to replicat in hepatoma cell lines may be related, in part, to the inability to produce forms of NS5A, and perhaps other non-structural proteins, that are readily produced in hepatocytes and that are required for optimal virus multiplication. Our working model will be explored by pursuing the following specific aims: (1) Identification and characterization of forms of HCV non-structural proteins produced during infection in vivo; (2) Evaluation of pro-apoptotic and pro-survival (anti-apoptotic) responses during HCV infection in vivo; and (3) Elucidation of clinical correlations. These studies have the ability to discover forms of HCV non-structural proteins unique to the infected hepatocyte and to reveal correlations between the level of these new forms and clinical outcomes. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a major cause of liver disease worldwide; only two HCV sequences and one cell line are available to study the HCV lifecycle in vitro. We have discovered that forms of some HCV non-structural proteins observed in HCV-infected liver are not produced in cell lines, and we propose that the absence of these forms in cell lines limits HCV multiplication in vitro. Therefore, completion of this study may reveal new approaches to study more HCV genotypes and facilitate the development of new antiviral therapies.