Hepatitis C virus (HCV) is the etiologic agent of parenterally transmitted non-A, non-B viral hepatitis. It encodes for a single polyprotein that is cleaved to generate ten distinct viral proteins (core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The development of several new cell culture systems which exhibit robust virus replication and virion production promises to allow for in-depth study of the virus life cycle. NS2, in particular, is one such protein in which complete understanding of its autoproteolytic function and other potential roles requires a live-virus system. Studies of other related pesti- and flaviviruses, including bovine viral diarrhea, kunjin, and yellow fever viruses, indicate the involvement of NS2 protein in virus assembly possibly at the level of RNA encapsidation. Preliminary data utilizing a bicistronic HCV genome, where an encephalomyocarditis virus (EMCV) IRES was introduced between NS2 and NS3 within the J6/JFH chimera, suggests that HCV NS2 may also contribute to virus assembly. We propose to examine the mechanism of NS2 involvement in virus assembly and the requirement of cellular and viral factors in NS2 activity. Our approach is to use comparative genetic analysis of the NS2 protein expressed from a H77 (genotype 1a) and J6 (2a) background, which in the context of the HCV cell culture systems have shown dramatic differences in virus production. Furthermore, we plan to utilize proteomic analysis, including mass spectrometry, to determine NS2 binding partners essential for its activity. By examining the activity of NS2 in the HCV lifecycle, we hope to clarify an essential part of HCV infection. The importance of this research is underscored by the fact that in the US, HCV infection is the leading cause of liver transplants. Furthermore, interferon-based therapies are only 50% effective against the prevailing strain within the US. Therefore, the long-term goal of our research is to develop therapeutic agents targeting NS2 based on its role in the HCV life cycle.