Imaging Hepatitis C Virus Morphogenesis

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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.