Hepatitis C virus (HCV) is a major human bloodborne pathogen. Growth of HCV in cell culture is controversial, and the lack of an """"""""in vitro"""""""" infection system is an obstacle to study cell entry of HCV. Therefore, our approach to study cell entry of HCV consists of developing viral pseudotypes containing the HCV envelope and genomes of RNA viruses capable of replicating in cell culture. These viral pseudotypes will be used as probes to identify HCV cellular receptors. To do so, the extracellular domains of E1 and E2 envelope proteins of HCV were fused with to the transmembrane and cytoplasmic domains of the G protein of VSV. The expression of the E1/E2 proteins at the cell surface of human 293 cells was determined with monoclonal antibodies and a cell surface ELISA. Cell lines that expressed the E1/E2 envelope proteins of HCV will be used to produce pseudotyped viruses. We are also overexpressing the core, E1, E2, and P7 structural proteins of HCV in CHO cells to produce virus-like particles (VLPs). Formation and budding of VLPs will be studied in the CHO system to determine whether VLPs accumulate in the ER, Golgi, or extracellular compartments. We are constructing HVC and BVDV replicons to be encapsidated and pseudotyped into HCV envelops.
