Imaging Hepatitis C Virus Morphogenesis

病毒劫持和失调细胞过程，以有效地复制和产生病毒后代。因此，研究丙型肝炎病毒（HCV）的复制和产生可能有助于发现新的细胞生物学途径。在这种情况下，我们最近进行了一个互补的成像，生物化学和通道为基础的方法来表征HCV的出口途径。我们的研究结果表明，HCV是通过非典型的分泌途径释放。此外，我们的数据表明，组装的HCV颗粒可能分泌没有高尔基体通过。在我们以前的结果的延续，我们跟进的总体假设，HCV可以通过一个尚未阐明的细胞途径绕过高尔基体释放。我们将确定宿主细胞因子，并通过使用HCV作为工具，并通过采用最先进的成像以及遗传技术来表征这一途径。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）的荧光标记的HCV。表达HCV E1-mCherry/Nluc的细胞释放具有与未标记HCV相当的病毒学特性的颗粒。我们将使用HCV E1-mCherry作为一种创新工具来成像活肝癌细胞中HCV颗粒的形态发生。更详细地说，我们将可视化病毒细胞内的蛋白质和颗粒贩运与高分辨率活细胞成像和荧光恢复后光漂白（FRAP）。我们将结合联合收割机这种方法与共转染的结构表达标记的细胞转运途径和细胞器作为融合蛋白与绿色荧光蛋白，从而允许得出结论的细胞内运输路线参与HCV的生产。这些实验将通过siRNA介导的敲除、用细胞转运途径抑制剂处理细胞以及在病毒组装和释放的某些阶段特异性停滞的病毒突变体在功能上进行补充。最后，我们将确认新的潜在的细胞因子参与HCV释放与未标记的完全感染性HCV。总之，我们提出了一种创新的和高度互补的方法来确定参与HCV释放途径的细胞成分。

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.