Hepatitis C continues to be a major global public health problem despite significant advances in interferon- based treatment. A new generation of specific antivirals is entering clinical trials but early results, even after short-term administration, suggest that resistant variants do emerge and that combination therapy will be needed for effective virus control and eradication. Most efforts to date have focused on viral targets involved in genome RNA translation or RNA replication. The advent of efficient cell culture systems mimicking the complete HCV replication cycle, including virion assembly, egress and entry, opens up new opportunities for basic and applied studies. This proposal is focused on defining the cellular molecules required for HCV entry into host cells and the sequence of events required for productive entry. HCV E2 glycoprotein binding cellular cell surface molecules such as the tetraspannin CD81 and scavenger receptor SR-BI participate in HCV entry, but they are neither sufficient for entry nor have their precise roles been defined. We surveyed human CD81+ SR-B1+ cell lines and identified several that were unable to support HCV entry. One of these, 293T cells, was used to screen a novel recyclable retrovirus cDNA library made from HCV-permissive Huh- 7.5 cells. This screen identified a new molecule required for HCV entry, Claudin-1 (CLDN1). CLDN1 is a multiple membrane spanning cell surface protein previously found in tight junctions. CLDN1 expression in 293T cells renders them fully permissive for infection by HCV pseudoparticles (HCVpp) and cell culture produced HCV (HCVcc). CLDN1 dependent HCV entry is observed for diverse HCV envelopes and requires CD81. CLDN1 expression correlates with the ability of HCVpp to enter target cells. We propose to map the functional determinants of CLDN1 required for HCV entry, define additional molecules required for HCV entry into human and murine cells, and dissect the roles of these molecules in HCV entry. These studies will provide a detailed picture of the cellular interactions required for HCV entry with implications for the development of new antiviral approaches and small animal models.