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

基本信息

  • 批准号:
    8526205
  • 负责人:
  • 金额:
    $ 2.25万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 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

项目摘要

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)慢性感染世界人口的3%[3]。病毒在个体中持续复制导致肝细胞癌、肝硬化和慢性肝病[4]。虽然最近在治疗方面取得了相当大的进展,但仍需要大量的工作来有效地对抗病毒[5-6]。开发新的抗病毒药物的主要障碍之一是HCV生命周期的许多方面的不确定性。特别是病毒NS 5A HCV NS 5A蛋白与许多对生产性感染重要的宿主细胞蛋白相互作用[1] HCV NS 5A蛋白是多功能的,在RNA复制、病毒体组装和宿主细胞调节中起作用。我们假设,与NS 5A相关的宿主因子的鉴定可能揭示由病毒操纵的生产性感染的宿主功能。辨别HCV如何操纵宿主可能会导致更好地理解致癌机制。对感染细胞中NS 5A相关蛋白的蛋白质组学分析确定Nipsnap 1为一种新的宿主因子。沉默Nipsnap 1导致RNA复制增加,而过表达则降低RNA复制,沉默Nipsnap 1导致病毒感染性降低。这些观察使我们假设Nipsnap 1是参与调节NS 5A在复制和组装中的作用的细胞机制的一部分。为了验证这一假设,我们提出了以下具体目标:具体目标1:表征Nipsnap 1-NS 5A相互作用和作用机制。目前尚不清楚NS 5A和Nipsnap 1的哪些区域是观察到的相互作用所必需的。我们将通过两种方法来评估这一点:(1)通过创建Nipsnap 1结构域的缺失突变体并评估其与NS 5A相互作用的能力;(2)利用先前生成的NS 5A结构域和亚结构域缺失突变体来评估哪些结构域是与Nipsnap 1相互作用所必需的。此外,将创建磷酸酶死亡的Nipsnap 1突变体,以分析Nipsnap 1的这种活性是否可能改变NS 5A的磷酸化状态。具体目标2:表征Nipsnap 1在感染期间的环境。目前尚不清楚Nipsnap 1在肝细胞中的位置,以及在感染期间这种蛋白质的定位是否有任何变化。目前还不清楚Nipsnap 1是否与NS 5A以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,并将通过siRNA技术和过表达质粒操纵这种细胞因子的表达水平。具体目标3:表征由HCV操纵的用于调节膜运动的宿主细胞因子。通过免疫沉淀耦合质谱,我们希望产生一个数据体,设计一个模型,丙型肝炎病毒蛋白利用不同的宿主因子来操纵膜复制和组装。

项目成果

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Briana M Weiser其他文献

Briana M Weiser的其他文献

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{{ truncateString('Briana M Weiser', 18)}}的其他基金

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:
    8394628
  • 财政年份:
    2012
  • 资助金额:
    $ 2.25万
  • 项目类别:

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