Identification of host cell factors involved in hepatitis C virus replication

Hepatitis C virus (HCV) infections are a major cause of chronic liver disease. Around 71 million people are persistently infected with HCV and around 400,000 people die each year from HCV-related cirrhosis and hepatocellular carcinoma. HCV is a positive strand RNA virus belonging to the genus Hepacivirus. It replicates in the cytoplasm of hepatocytes by utilizing host cell factors and pathways in a still poorly defined manner. Like all positive strand RNA viruses, HCV induces membrane rearrangements with membranes serving as scaffold to assemble the viral replication machinery and to coordinate the different steps of the viral replication cycle. Thus, membranes form a central compartment of the HCV replication factory that consists of cytoplasmic accumulations of double membrane vesicles (DMVs). Their formation is induced and regulated by several viral proteins, including nonstructural protein 4B (NS4B), an integral membrane protein that is thought to serve as a scaffold for DMV formation. Here we sought to identify host cell factors involved in HCV replication and DMV formation. By using unbiased proteome and lipidome analyses of cells containing a stably replicating HCV replicon, we isolated NS4B-associated membranes by affinity purification and determined their protein and lipid composition. A calnexin-associated ER membrane fraction as well as untagged NS4B was used as biological and technical control, respectively. Obtained hits were studied by bioinformatics analyses and are currently validated by using RNA interference-based knock-down approaches and HCV-specific phenotypic assays. Our preliminary results suggest that HCV utilizes distinct host factors to modify the cellular lipid content in a way that renders the cell more permissive for viral replication.