Chronic hepatitis C virus (HCV) infection develops in 70-80% of all exposed individuals and affects 3% of the world's population. While new drugs that directly target the viral NS3-4A serine protease have been approved for clinical use this year, there remains a need for more effective and better-tolerated HCV therapies. Our long- term goals are to define host-HCV molecular interactions and in so doing, to identify novel therapeutic agents for HCV therapy. HCV, like all other positive-sense single-stranded RNA viruses studied to date, induces the formation of specific membranous structures (called membranous webs) within infected cells for its own replication. Multiple RNAi screens have identified a critical role for a host protein phosphatidylinositol 4-kinase (PI 4-kinase) named PI4KA in HCV replication, most likely at the level of membranous web formation. Intriguingly, three other positive-sense single-stranded RNA viruses subvert the related PI 4-kinase PI4KB for their own replication. The objective of this proposal is to understand how the host hepatocyte protein PI4KA and its product PI 4-phosphate (PI(4)P) support HCV replication. Our central hypothesis is that HCV replication requires the local generation of PI(4)P, which in turn is necessary for the recruitment of effector proteins to the nascent membranous web. The specific aims of the project are to (1) Define the functional domains of PI4KA and its regulation; (2) Identify the phosphoinositide-dependent steps in the HCV life cycle; (3) Identify and characterize downstream effectors of PI4KA and PI(4)P in the HCV life cycle. We will use a variety of biochemical, cell-based, and imaging techniques to accomplish the proposed research. The proposed research is innovative as (1) the model of web assembly presented in this proposal is novel; (2) the mechanisms by which PI4KA is regulated in the host cell and in the context of HCV replication have not been studied (Aim 1); (3) The mechanisms by which PI(4)P and other phosphoinositides support HCV replication has not been previously pursued (Aims 2 and 3); (4) The use of inducible and selective depletion of phosphoinositides (Aim 2) has not been previously used to study viral replication. We expect that the proposed research may have direct public health benefits, as a better understanding of the mechanisms by which PI4KA and host phosphoinositides support viral replication may lead to novel approaches to the treatment of chronic HCV infection. A better understanding of the HCV-host relationship is a stated goal of the NIH Action Plan for Liver Disease Research (Goal B2a). Moreover, completion of the proposed aims will also lead to new insights into the physiological functions and regulation of PI4KA, areas that are currently poorly understood.