Identification of host factors involved in hepatitis C virus assembly and characterization of their potential role in vivo

Hepatitis C virus (HCV) is an important human pathogen that infects the liver and establishes chronic infection in the majority of cases, leading to cirrhosis and hepatocellular carcinoma over the course of many years. Despite recent progress, details of the HCV life cycle are still missing, with the HCV assembly process being particularly poorly understood. Lipid droplets (LDs) appear to play a central role in HCV assembly since all the viral proteins involved in morphogenesis as well as the viral genome accumulate in close proximity of this organelle. Moreover, the viral non-structural (NS) proteins NS2, p7 and NS5A have emerged as central players in the virion assembly process. However, the contribution of host cell factors to HCV morphogenesis remains poorly understood. The objective of this collaborative project is to characterize the contribution of the cell factory to HCV assembly and to determine the pathophysiological consequences of these virus-host interactions. To achieve this goal, we will use a dedicated phenotypic assay relying on the monitoring of NS5A-mediated recruitment to LDs by high content confocal imaging and apply it to the screening of a genome wide small synthetic interfering RNA (siRNA) library and a small lipidic compounds library. We will also use a proteomic strategy using NS2, p7 and NS5A as baits in pull-down experiments followed by interactors identification. The integration of the data from the genetic, chemical and proteomic screens will allow for the identification of key cellular pathways involved in HCV morphogenesis. Subsequently, in depth elucidation of the interactions of some of the host gene products from these pathways with viral proteins that are implicated in virus assembly will be conducted. Next we will determine whether the interactions between these cellular partners and the various viral proteins from HCV are conserved across HCV genotypes and moreover, for other viruses of the Flaviviridae family. Finally, we will determine the potential in vivo relevance of these virus-host interactions on the progression of chronic hepatitis C by exploring the expression of host factors in infected patients. In conclusion, this project will shed light on a poorly understood step of the HCV life cycle and it will help to understand how this virus exploits cellular pathways for its propagation within the liver. Importantly, the variety of advanced techniques and the different approaches encourage us to anticipate the identification of cellular factors involved in lipid metabolism. Furthermore, the participation of clinical partners will also help us to determine the consequences of these virus-host interactions on the progression of the disease and the potential identification of biomarker molecules. Finally, another major outcome of our study will be the validation of new host factors as drug targets for HCV and thus will lead to the establishment of novel target-based cellular assays immediately applicable to the screen of large compounds libraries that will be a benefit to the pharmaceutical efforts for the continuous search of first class novel candidate drugs.

Duration: 36 month