Hepatitis C virus (HCV) infection is a global health concern affecting an estimated 3% of the world's population. Recently, cell culture systems have been established, allowing recapitulation of the complete virus life cycle for the first time. Since the HCV proteins p7 and NS2 are not predicted to be major components of the virion, nor are they required for RNA replication, we investigated whether they might have other roles in the viral life cycle. Here we utilize the recently described infectious J6/JFH chimera to establish that the p7 and NS2 proteins are essential for HCV infectivity. Furthermore, unprocessed forms of p7 and NS2 were not required for this activity. Mutation of two conserved basic residues, previously shown to be important for the ion channel activity of p7 in vitro, drastically impaired infectious virus production. The protease domain of NS2 was required for infectivity, whereas its catalytic active site was dispensable. We conclude that p7 and NS2 function at an early stage of virion morphogenesis, prior to the assembly of infectious virus.Hepatitis C virus (HCV) is a major causative agent of severe liver disease, with an estimated 170 million people infected worldwide (36). HCV is the sole member of the genus Hepacivirus, which, together with the Flavivirus and Pestivirus genera, comprise the family Flaviviridae (reviewed in reference 21). The HCV genome is approximately 9,600 nucleotides in length and is translated from an internal ribosome entry site (IRES) to generate a polyprotein in the order NH 2 -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. Co-and posttranslational processing of the polyprotein by viral and cellular proteases yields the individual viral proteins. Core (C) and envelope proteins (E1 and E2) are the major structural proteins, which, together with a host derived lipid bilayer and the viral RNA, comprise the virion. The nonstructural (NS) proteins, NS3 to NS5B, are essential components of the viral replicase. NS3 possesses helicase and NTPase activities and, along with its cofactor NS4A, comprises the major viral protease. NS4B and NS5A play essential, but as yet undefined roles in RNA replication. NS5B is the RNA-dependent RNA polymerase (RdRp). p7 and NS2 are dispensable for RNA replication, since subgenomic replicons that lack the entire C to NS2 coding region replicate autonomously (2, 22). p7 is a small (63 amino acids) hydrophobic protein that is predicted to span the membrane twice. Both the N and C termini of p7 reside within the endoplasmic reticulum (ER) lumen, with a short, basic intervening loop exposed to the cytoplasm (4). The N and C termini of p7 are released from the polyprotein by host signal peptidase. Incomplete and delayed processing leads to the accumulation of E2-p7 and p7-NS2 precursors, respectively. (10,19,25). While sequence determinants within both p7 and NS2 have been shown to modulate E2-p7 and p7-NS2 cleavage efficiencies (3), the functional importance of these precursors is not known. In chimpanzees, p7 was found to be essential for HCV i...