Forms of HCV NS5A in vivo

描述（由申请人提供）：这是一项探索性研究资助申请，旨在鉴定受感染人肝组织中产生的丙型肝炎病毒（HCV）非结构蛋白的新形式，并开发其形成和功能的模型。丙型肝炎病毒仍然是一个重大的公共卫生问题的全球比例。尽管多年的effot，无法研究细胞培养中的HCV的许多基因型仍然是我们理解这种病毒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。HCV非结构蛋白5A（NS 5A）是大多数正链RNA病毒非结构蛋白中唯一的一种。它的三个结构域中的两个本质上是无序的，并且它包含无数丝氨酸/苏氨酸激酶的许多磷酸化位点，包括cAMP活化蛋白激酶（PKA）、酪蛋白激酶1家族成员和酪蛋白激酶2。我们一直对NS 5A磷酸化的特定模式产生这种蛋白质的独特构象和功能的可能性感兴趣，从而解释了NS 5A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已经导致了 通过PKA的NS 5A磷酸化的位点，并开发免疫试剂以证明在复制基因型1b和2a的HCV RNA的细胞中存在PKA磷酸化形式的NS 5A。此外，对来自儿科丙型肝炎病例的活检和来自终末期丙型肝炎病例的组织的检查导致发现，在感染的肝组织中观察到的NS 5A的形式不同于在复制HCV RNA的人肝癌细胞系中观察到的那些。在肝组织中检测到的其他形式的NS 5A与当半胱天冬酶被激活时在细胞系中观察到的那些非常相似，并且PKA-磷酸化物质的丰度与肝损伤的量很好地相关。我们假设NS 5A的半胱天冬酶裂解可以为HCV提供一种监测肝细胞抗病毒状态的机制; NS 5A的磷酸化可以向HCV发出信号，表明肝细胞中存在适合复制的抗凋亡状态。这些切割和磷酸化的形式可以扩增NS 5A蛋白质组，产生病毒增殖所必需的蛋白质形式。如果是这种情况，那么大多数HCV序列不能在肝癌细胞系中复制可能部分与不能产生NS 5A形式以及可能其他非结构蛋白有关，这些非结构蛋白容易在肝细胞中产生并且是最佳病毒增殖所需的。我们的工作模型将通过追求以下具体目标进行探索：（1）体内感染期间产生的HCV非结构蛋白形式的鉴定和表征;（2）体内HCV感染期间促凋亡和促存活（抗凋亡）反应的评价;和（3）阐明临床相关性。这些研究有能力发现感染肝细胞特有的HCV非结构蛋白的形式，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（HCV）是世界范围内肝脏疾病的主要原因;只有两个HCV序列和一个细胞系可用于体外研究HCV生命周期。我们已经发现，在HCV感染的肝脏中观察到的一些HCV非结构蛋白的形式在细胞系中不产生，并且我们提出，细胞系中这些形式的缺乏限制了HCV在体外的增殖。因此，本研究的完成可能会揭示新的方法来研究更多的HCV基因型，并促进新的抗病毒治疗的发展。

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.