Nipsnap1: A Novel Negative Regulator of Hepatitis C Virus Replication

描述（由申请人提供）：丙型肝炎病毒（HCV）慢性感染全球3%的人口。病毒在个体中的持续复制导致肝细胞癌、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，但要有效地对抗这种病毒还需要做大量的工作[5-6]。开发新抗病毒药物的主要障碍之一是丙型肝炎病毒生命周期的许多方面的不确定性。特别是病毒NS5A

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.