Nipsnap1: A Novel Negative Regulator of Hepatitis C Virus Replication

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)慢性感染3%的世界人口[3]。在个体中持续的病毒复制会导致肝细胞癌、肝硬变和慢性肝病[4]。尽管最近在治疗方面取得了相当大的进展，但仍需要做大量工作才能有效地对抗病毒[5-6]。开发新的抗病毒药物的主要障碍之一是丙型肝炎病毒生命周期许多方面的不确定性。尤其是病毒NS5A 蛋白与许多宿主细胞蛋白相互作用，这些蛋白对生产性感染很重要[1]丙型肝炎病毒NS5A蛋白是多功能的，在RNA复制、病毒粒子组装和宿主细胞调节中发挥作用。我们假设，识别与NS5A相关的宿主因素可能有助于揭示病毒操纵的宿主功能，以进行生产性感染。辨别丙型肝炎病毒如何操纵宿主可能有助于更好地理解致癌机制。对感染细胞中NS5A相关蛋白的蛋白质组学分析表明，NipSnap1是一种新的宿主因子。沉默NipSnap1导致RNA复制增加，而过度表达则降低RNA复制。沉默NipSnap1会导致病毒感染性的产生减少。这些观察结果使我们假设NipSnap1是参与调控NS5A在复制和组装中作用的细胞机制的一部分。为了验证这一假说，我们提出了以下具体目标：具体目标1：表征NipSnap1-NS5A的相互作用和作用机制。目前尚不清楚观察到的相互作用需要NS5A和NipSnap1的哪些区域。我们将通过两种方法来评估这一点：(1)通过创建NipSnap1结构域的删除突变体并评估它们与NS5A相互作用的能力；(2)利用先前产生的NS5A结构域和亚结构域删除突变体来评估哪些结构域是与NipSnap1相互作用所必需的。此外，还将创建磷酸酶死亡的NipSnap1突变体来分析NipSnap1的这种活性是否可能改变NS5A的磷酸化状态。具体目标2：研究NipSnap1感染过程中的环境特征。目前尚不清楚NipSnap1在肝细胞中的位置，也不知道该蛋白在感染过程中的定位是否有任何变化。目前还不清楚NipSnap1是否与NS5A以外的其他病毒蛋白相互作用，也不清楚两种蛋白在感染过程中的动力学。我们将使用免疫荧光技术来评估这些问题，这种细胞因子的表达水平将通过siRNA技术和过表达质粒来操纵。具体目标3：确定丙型肝炎病毒调控膜运动的宿主细胞因子。通过免疫沉淀联用质谱学，我们希望产生大量的数据来设计一个模型，在该模型中，丙型肝炎病毒蛋白利用不同的宿主因素来操纵膜进行复制和组装。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) chronically infects 3 percent of the population of the world [3]. Unrelenting viral replication in individuals results in hepatocellular carcinoma, cirrhosis, and chronic liver disease [4]. Although considerable advancement has recently been made in therapeutics, considerable work is needed to effectively combat the virus [5-6]. One of the principal obstacles in development of new anti-virals is the indefinite nature of many aspects of the HCV lifecycle. In particular, the viral NS5A protein interacts with many host cellular proteins that are important for productive infections [1] The HCV NS5A protein is multi-functional, with roles in RNA replication, virion assembly, and host cell modulation. We hypothesized that identification of host factors associated with NS5A might shed light on host functions manipulated by the virus for productive infections. Discerning how HCV manipulates the host might lead to a better understanding of mechanisms of carcinogenesis. A proteomic analysis of NS5A associated proteins from infected cells identified Nipsnap1 as a novel host factor. Silencing Nipsnap1 resulted in increased RNA replication, whereas overexpression decreased it. Silencing Nipsnap1 lead to a decrease in production of virus infectivity. These observations have led us to hypothesize that Nipsnap1 is a part of the cellular machinery involved in regulating NS5A's role in both replication and assembly. To test this hypothesis, we propose the following specific aims: Specific Aim 1: Characterize Nipsnap1-NS5A Interaction and Mechanism of Action. It is unknown what regions of NS5A and Nipsnap1 are required for the observed interaction. We will assess this through two approaches: (1) by creating deletion mutants of the Nipsnap1 domains and evaluating their ability to interact with NS5A; (2) utilize previously generated NS5A domain and sub-domain deletion mutants to evaluate which domains are necessary for interaction with Nipsnap1. Also, phosphatase-dead Nipsnap1 mutants will be created to analyze if this activity of Nipsnap1 might be altering the phosphorylation state of NS5A. Specific Aim 2: Characterize Environment of Nipsnap1 During Infection. It is unknown where Nipsnap1 is in hepatocytes, as well as if there are any changes in localization of this protein during infections. It is also unknown if Nipsnap1 is interacting with ny other viral proteins besides NS5A, or the kinetics of both proteins during infections. We will assess these issues using immunofluorescence techniques, and the expression level of this cellular factor will be manipulated through siRNA technologies and overexpression plasmids. Specific Aim 3: Characterize Host Cellular Factors Manipulated by HCV for Modulation of Membranous Movements. Through immunoprecipitation coupled mass spectrometry, we hope to generate a body of data to devise a model by which hepatitis C viral proteins utilize different host factors to manipulate membranes for replication and assembly.