Nonstructural 4B Protein and Hepatitus C Virus Production

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是一种正链RNA病毒，可在患者中持续感染并导致癌症。我研究的长期目标是阐明非结构4B(NS4B)蛋白促进病毒产生和致病的机制。越来越多的证据表明，NS4B促进了病毒粒子的产生。然而，NS4B从复制模式切换到颗粒形成模式目前尚不清楚。此外，我们对导致NS4B调控病毒粒子产生的病毒与宿主的相互作用知之甚少。因此，这项应用的目的是更好地了解NS4B对病毒产生的贡献和潜在的机制。我们推测：[1]在感染过程中，NS4B与几种病毒蛋白结合并调节它们的活性，在NS5A的情况下，这是通过宿主激酶形成p58来实现的；[2]NS4B蛋白中的特定残基有助于NS5A p58的形成和病毒粒子的产生；[3]NS5A p58形成缺陷的JFH1嵌合体与NS5A相互作用的宿主激酶比Wt JFH1少。通过更好地了解NS4B在病毒粒子生产中的作用，可以发现用于药物开发的新的抗病毒靶点。因此，我们提出了以下具体目标。目的1.定位病毒产生和NS5A p58形成所需的NS4B残基；研究NS5A在JFH1嵌合体中的亚细胞分布；检测适应病毒恢复到Wt NS5A p58水平的可能性和潜在机制。在这个目标中，我们假设NS4B中的特定残基促进了病毒的产生，而其他残基对丙型肝炎病毒的复制是重要的。绘制这些区域/残基是理解NS4B如何在丙型肝炎病毒生命周期中扮演两个重要但不同的角色的关键。因此，我们将：[1]绘制病毒产生和NS5A p58形成所需的NS4B残基图谱，[2]研究嵌合病毒感染期间NS5A p58的亚细胞分布，[3]检查适应病毒恢复到Wt NS5A p58水平的可能性和潜在机制。目的2.确定与NS4B相关的病毒因子或与NS5A p58和NS4B相关的宿主因子；研究它们在病毒粒子产生中的作用。在这里，我们假设NS4B与病毒因子结合，导致从复制到病毒生产的转换。例如，NS4B可能导致NS5A的构象变化，有利于宿主激酶形成p58。相反，NS4B与宿主激酶结合，导致它们过度磷酸化NS5A。因此，我们将：确定[1]NS4B病毒/宿主伙伴，[2]NS5A p58宿主伙伴，[3]使用表面等离子共振来确认结合和特异性以及对丙型肝炎病毒复制和病毒粒子产生的意义。我们将寻找与NS4B或NSS5A p58相关的激酶和其他宿主因子。事实上，激酶调节各种细胞过程，经常是治疗包括癌症在内的许多疾病的靶点。因此，深入了解这些因素在病毒粒子产生中的作用可以导致针对丙型肝炎病毒和由此产生的肝细胞癌的新药的开发。 公共卫生相关性：丙型肝炎病毒(丙型肝炎病毒)是美国肝移植和肝癌的主要原因。NS4B蛋白的表达促进了小鼠丙型肝炎病毒复制复合体的形成、感染性丙型肝炎病毒的产生和肿瘤的形成。了解NS4B蛋白如何与病毒和宿主辅助因子相互作用以促进这些事件，可能会导致开发用于丙型肝炎患者的生物标记物新型抗病毒药物。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is a positive strand RNA virus that establishes both persistent infection in patients and causes cancer. The long-term goal of my research is to elucidate the mechanisms whereby the nonstructural 4B (NS4B) protein facilitates virus production and pathogenesis. There is growing evidence that NS4B facilitates virion production. However, NS4B switch from the replication to the particle formation mode is currently unknown. Further, we know little about the virus and host interactions leading up to NS4B regulation of virion production. Hence, the objective of this application is to gain a better insight into the contribution of NS4B to virus production and the underlying mechanism. We postulate that: [1] During infection, NS4B binds to several virus proteins and regulates their activities, which in the case of NS5A is via p58 formation by host kinases; [2] Specific residues in the NS4B protein contribute to NS5A p58 formation and virion production; [3] JFH1 chimera that are defective in NS5A p58 formation have fewer host kinases interacting with NS5A relative to Wt JFH1. Through a better understanding of NS4B role in virion production, novel antiviral targets for drug development could be uncovered. Thus, we propose the following Specific Aims. Aim 1. Map the NS4B residues required for virus production and NS5A p58 formation; investigate the subcellular distribution of NS5A in the JFH1 chimera; examine the possibility that the adapted viruses have regained the Wt NS5A p58 levels and the underlying mechanism. In this aim, we postulate that specific residues in NS4B are facilitating virus production whereas others are important for HCV replication. Mapping such regions/residues is key to understanding how NS4B can play two important but distinct roles in HCV lifecycle. Hence, we will: [1] Map the NS4B residues required for virus production and NS5A p58 formation, [2] Investigate the subcellular distribution of NS5A p58 during chimeric virus infection and, [3] examine the possibility that the adapted viruses have regained the Wt NS5A p58 levels and the underlying mechanism. Aim 2. Identify virus factors associated NS4B or host factors associated with NS5A p58 and NS4B; examine their roles in virion production. Here, we postulate that NS4B binds to virus factors, leading to a switch from replication to virion production. For instance, NS4B may cause a conformational change in NS5A that favors p58 formation by host kinases. Conversely, NS4B binds to host kinases and causes them to hyperphosphorylate NS5A. Thus, we will: identify [1] NS4B virus/host partners, [2] NS5A p58 host partners and, [3] use surface plasmon resonance to confirm binding and specificity as well as the significance on HCV replication and virion production. We will search for kinases and other host factors associated with NS4B or NSS5A p58. Indeed, kinases regulate various cellular processes and are often targets for treatment of numerous diseases including cancers. Hence, insight into the role of these factors in virion production can lead to the development of novel drugs targeting HCV and the resulting HCC. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is the leading cause of liver transplantation and liver cancer in the United States. Expression of NS4B protein promotes HCV replication complex formation, infectious HCV production and tumor formation in mice. Understanding how NS4B protein interacts with viral and host co-factors to facilitate these events may lead to the development of biomarkers novel antiviral drugs for HCV patients.