Hepatitis C Virus Replication and Intracellular Membrane Rearrangement

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)是世界范围内导致肝癌的主要原因。在感染过程中，丙型肝炎病毒诱导形成重排的膜结构，称为网。在单独表达丙型肝炎病毒NS4B蛋白后，体外也观察到这些膜。各种报道表明，该网络含有丙型肝炎病毒非结构蛋白和病毒基因组，提示这种新的膜可能是丙型肝炎病毒基因组复制发生的地方。这一提议将检验以下假设：(I)网络在丙型肝炎病毒基因组复制中发挥关键作用，(Ii)网络包含几个宿主因子，包括Rab5，它们在丙型肝炎病毒的生命周期中发挥直接或间接的作用。为了检验这些假设，我们提出了以下具体目标： 目的I.检测网络在丙型肝炎病毒基因组复制中是否起关键作用。人们普遍认为，网络为丙型肝炎病毒基因组复制提供了支架。然而，没有直接的实验证据来证明这一假设。我们目前的网络形成方法包括点状NS4B染色和内源性Rab5B蛋白的共同定位。这种化验方法通常通过对纤网的电子显微镜检测而得到证实。为此，我们将(IA)在NS4B中引入突变(S)以取消网络形成，并(IB)确定取消网络是否抑制丙型肝炎病毒基因组复制。我们将(1C)测试NS4B寡聚是否在网络形成和基因组复制中发挥作用。目的II.确定网络中存在Rab5蛋白的意义。以前的报道表明，网络诱导蛋白NS4B主要与内质网(ER)室相关。我们的实验室最近发现，早期的内体标记Rab5和EEA1与网络诱导的NS4B相关。此外，还发现Rab5与NS4B相互作用。在这个目标中，我们将(IIA)研究Rab5是如何融入网络的。我们将(IIB)鉴定与丙型肝炎病毒NS4B相互作用的Rab5相关蛋白。最后，我们将(IIC)确定网络是否改变了细胞内吞作用。目的III.确定网络的蛋白质图谱。网络诱导的NS4B蛋白与ER和EE标记物Rab5的相关性表明，这种新的膜具有复杂的来源和组成。在我们的初步结果中，我们使用lodixano(Optiprep)梯度来分离富含病毒蛋白的膜组分，这些蛋白参与网络形成和基因组复制。在这个目标中，我们将(IIIA)结合Optiprep梯度和免疫沉淀来分离网络富集膜。我们将使用质谱仪(IIIB)来检测免疫隔离膜的蛋白质图谱。 我们希望这些研究将解决丙型肝炎病毒相关的超微结构变化在病毒基因组复制和病毒持久性中的作用。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is a leading cause of liver cancer worldwide. During infection, HCV induces the formation of rearranged membrane structures termed web. These membranes are also observed in vitro following expression of HCV NS4B protein alone. Various reports have shown that the web contains HCV nonstructural proteins as well as the viral genome, suggesting that this novel membrane might be the site where HCV genome replication occurs. This proposal will test the following hypotheses: (i) the web plays a critical role in HCV genome replication, (ii) the web contains several host factors, including Rab5, which play a direct or indirect role in HCV life cycle. To test these hypotheses, we propose the following Specific Aims: Aim I. Test whether the web plays a critical role in HCV genome replication. It is widely believed that the web provides the scaffold for HCV genome replication. However, there is no direct experimental evidence to prove this hypothesis. Our current assay for web formation consists of co-localization of punctate NS4B staining with endogenous Rab5B protein. This assay is generally confirmed by electron microscopy detection of the web. In this Aim, we will (IA) introduce mutation(s) in NS4B to abolish web formation and (IB) determine whether abolishing the web inhibits HCV genome replication. We will (1C) test whether NS4B oligomerization plays a role in web formation and genome replication. Aim II. Determine the significance of the presence of Rab5 protein in the web. Previous reports have shown that the web-inducing protein, NS4B, is associated mostly with the endoplasmic reticulum (ER) compartment. Our laboratory has recently found that early endosome markers, Rab5 and EEA1, are associated with web-inducing NS4B. In addition, Rab5 was found to interact with NS4B. In this Aim, we will (IIA) examine how Rab5 gets incorporated into the web. We will (IIB) identify the Rab5-associated proteins interacting with HCV NS4B. Finally, we will (IIC) determine whether the web alters endocytosis in the cell. Aim III. Determine the protein profile of the web. The association of web-inducing NS4B protein with ER and EE marker, Rab5, suggests that this novel membrane has a complex origin and composition. In our preliminary results, we have used lodixanol (Optiprep) gradient to isolate membrane fractions enriched in viral proteins involved in web formation and genome replication. In this Aim, we will (IIIA) combine Optiprep gradient with immunoprecipitation to isolate web-enriched membranes. We will (IIIB) use mass spectrometry to examine the protein profile of the immuno-isolated membrane. We hope that these studies will address the role of HCV-associated ultrastructural changes in the viral genome replication and viral persistence.