Nipsnap1: A Novel Negative Regulator of Hepatitis C Virus Replication

Nipsnap1：丙型肝炎病毒复制的新型负调节因子

• 批准号: 8526205
• 负责人: Briana M Weiser
• 金额: $ 2.25万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-16 至 2014-04-25
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8526205
• 关键词: Affect; Biology; Carcinogenesis Mechanism; Cells; Cellular Membrane; Cellular biology; Centers for Disease Control and Prevention (U.S.); Chronic; Cirrhosis; Complex; Coupled; Data; Development; Drug Targeting; Environment; Fluorescent Antibody Technique; Genetic; Hepatitis C; Hepatitis C virus; Hepatitis C-Like Viruses; Hepatocyte; Image; Immunoprecipitation; Individual; Infection; Integration Host Factors; Kinetics; Lead; Life; Life Cycle Stages; Light; Link; Liver Cirrhosis; Mass Spectrum Analysis; Membrane; Metalloproteins; Methods; Modeling; Movement; Mutation; Nature; Pathway interactions; Patients; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Plasmids; Play; Population; Primary carcinoma of the liver cells; Process; Production; Proteins; Proteomics; RNA replication; Research; Role; Small Interfering RNA; Staging; Technology; Testing; Therapeutic; Vesicle; Viral; Viral Proteins; Virion; Virus; Virus Replication; Work; Zinc; carcinogenesis; chronic liver disease; combat; mutant; novel; overexpression; protein expression; research study; viral RNA

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.

描述（由申请人提供）：丙型肝炎病毒 (HCV) 长期感染世界上 3% 的人口 [3]。病毒在个体中不断复制会导致肝细胞癌、肝硬化和慢性肝病[4]。尽管最近在治疗方面取得了相当大的进展，但有效对抗病毒还需要做大量工作[5-6]。开发新型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。特别是，病毒 NS5A HCV NS5A 蛋白与许多对生产性感染很重要的宿主细胞蛋白相互作用 [1] HCV 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。我们假设与 NS5A 相关的宿主因素的鉴定可能有助于了解病毒操纵宿主功能以实现生产性感染。了解丙型肝炎病毒如何操纵宿主可能有助于更好地了解致癌机制。对感染细胞中 NS5A 相关蛋白的蛋白质组学分析确定 Nipsnap1 是一种新的宿主因子。沉默 Nipsnap1 会导致 RNA 复制增加，而过度表达则会减少 RNA 复制。沉默 Nipsnap1 会导致病毒感染性的产生降低。这些观察结果使我们推测 Nipsnap1 是参与调节 NS5A 在复制和组装中的作用的细胞机制的一部分。为了检验这一假设，我们提出以下具体目标： 具体目标 1：表征 Nipsnap1-NS5A 相互作用和作用机制。目前尚不清楚 NS5A 和 Nipsnap1 的哪些区域需要观察到的相互作用。我们将通过两种方法对此进行评估：（1）通过创建 Nipsnap1 结构域的缺失突变体并评估它们与 NS5A 相互作用的能力； (2) 利用先前生成的 NS5A 结构域和子结构域缺失突变体来评估哪些结构域对于与 Nipsnap1 相互作用是必需的。此外，还将创建磷酸酶死亡的 Nipsnap1 突变体，以分析 Nipsnap1 的这种活性是否可能改变 NS5A 的磷酸化状态。具体目标 2：表征 Nipsnap1 感染期间的环境。目前尚不清楚 Nipsnap1 在肝细胞中的位置，以及感染期间该蛋白的定位是否发生任何变化。目前还不清楚 Nipsnap1 是否与 NS5A 之外的任何其他病毒蛋白相互作用，或者这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评估这些问题，并通过 siRNA 技术和过表达质粒来操纵该细胞因子的表达水平。具体目标 3：表征 HCV 操纵的宿主细胞因子以调节膜运动。通过免疫沉淀耦合质谱法，我们希望生成大量数据来设计一个模型，通过该模型，丙型肝炎病毒蛋白利用不同的宿主因子来操纵膜进行复制和组装。