Imaging Hepatitis C Virus Morphogenesis

丙型肝炎病毒形态发生成像

• 批准号: 399732171
• 负责人: Professor Dr. Michael Schindler
• 金额: --
• 依托单位: Institut für Medizinische Virologie und Epidemiologie der Viruskrankheiten
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/399732171?language=en
• 关键词: Imaging; Hepatitis; Virus; Morphogenesis

Viruses hijack and dysregulate cellular processes in order to efficiently replicate and produce viral progeny. Therefore, investigating viral replication and production could lead to the discovery of novel cell biological pathways.Mechanisms of hepatitis c virus (HCV) morphogenesis in infected hepatocytes, including viral assembly, budding and release are incompletely understood. In this context, we recently undertook a complementary imaging, biochemical and inhibitor-based approach to characterize the pathway of HCV egress. Our results demonstrate that HCV is released via a non-canonical secretory route. Moreover, our data indicate that assembled HCV particles might be secreted without Golgi passage. In continuation of our previous results we follow up on the overarching hypothesis that HCV could be released via a not-yet elucidated cellular pathway bypassing the Golgi. We will identify host cell factors and characterize this pathway by using HCV as a tool and by employing state-of-the art imaging as well as genetic techniques. We developed and comprehensively characterized fluorescently labelled HCV carrying mCherry or a Nano-luciferase (Nluc) within the structural envelope glycoprotein E1. Cells expressing HCV E1-mCherry/Nluc release particles with virological properties comparable to those of untagged HCV. We will use HCV E1-mCherry as an innovative tool to image the morphogenesis of HCV particles in living hepatoma cells. In more detail, we will visualize virus intracellular protein and particle trafficking with high-resolution live cell imaging and fluorescence recovery after photobleaching (FRAP). We will combine this approach with cotransfection of constructs expressing markers for cellular transport pathways and organelles as fusion proteins with GFP, thus allowing to conclude which intracellular trafficking routes are involved in HCV production. These experiments will be functionally complemented by siRNA-mediated knockdown, treatment of cells with inhibitors of cellular transport pathways and viral mutants which are specifically arrested at certain stages of viral assembly and release. Finally, we will confirm new potential cellular factors involved in HCV release with untagged fully-infectious HCV.Altogether, we propose an innovative and highly complementary approach to identify cellular components involved in the HCV release route.

病毒劫持和失调细胞过程，以便有效复制和产生病毒后代。因此，研究病毒复制和生产可能会发现新的细胞生物学途径。丙型肝炎病毒（HCV）在受感染肝细胞中的形态发生机制，包括病毒组装、出芽和释放，尚不完全清楚。在这种背景下，我们最近采用了一种补充成像、生化和基于抑制剂的方法来表征 HCV 流出途径。我们的结果表明，HCV 通过非典型分泌途径释放。此外，我们的数据表明，组装的 HCV 颗粒可能在没有高尔基体通过的情况下被分泌。延续我们之前的结果，我们跟进了一个总体假设，即丙型肝炎病毒可以通过一条尚未阐明的绕过高尔基体的细胞途径释放。我们将使用 HCV 作为工具并采用最先进的成像和遗传技术来识别宿主细胞因子并表征该途径。我们开发并全面表征了在结构包膜糖蛋白 E1 内携带 mCherry 或纳米荧光素酶 (Nluc) 的荧光标记 HCV。表达 HCV E1-mCherry/Nluc 的细胞释放的病毒学特性与未标记的 HCV 相当。我们将使用 HCV E1-mCherry 作为一种创新工具，对活体肝癌细胞中 HCV 颗粒的形态发生进行成像。更详细地说，我们将通过高分辨率活细胞成像和光漂白后荧光恢复（FRAP）来可视化病毒细胞内蛋白质和颗粒运输。我们将这种方法与表达细胞转运途径标记物和细胞器作为融合蛋白与 GFP 的构建体相结合，从而可以得出哪些细胞内转运途径参与 HCV 生产。这些实验将通过 siRNA 介导的敲低、用细胞转运途径抑制剂和病毒突变体处理细胞来补充，这些突变体在病毒组装和释放的某些阶段被特异性抑制。最后，我们将通过未标记的完全感染性 HCV 来确认参与 HCV 释放的新的潜在细胞因子。总之，我们提出了一种创新且高度互补的方法来识别参与 HCV 释放途径的细胞成分。