The purpose of this project is to understand the molecular mechanisms involved in the replication of picornaviruses in susceptible target cells. This virus family includes numerous human pathogens (poliovirus, coxsackievirus, echovirus, rhinoviruses, hepatitis A virus). Infection of cells with these viruses leads to drastic changes in the host cells structure and metabolic activity. Cellular protein and RNA synthesis are inhibited; the intracellular membrane network becomes rearranged into a network of vesicles that surround and provide a scaffold for viral RNA replication complexes; cellular proteins are subverted into facilitating viral protein and RNA synthesis. The unique combination of viral and cellular proteins together accomplishes a highly efficient production of viral RNA, proteins, and particles. [unreadable] [unreadable] We are studying the activities of individual viral gene products, expressed alone or in combination, in cultured human cells, to determine their specific roles in the replication process. We have recently identified two viral proteins that bind and recruit different cellular proteins that regulate membrane trafficking and thereby induce the formation of vesicles that comprise the replication complex. These cellular proteins represent a new class of host factors required for virus replication. To help us localize and follow specific viral proteins within the host cell, we developed a method to tag individual proteins with a fluorescent or chemically reactive marker. We also study the biochemical activities and biophysical properties of key viral proteins involved in replicating the viral RNA genome. To accomplish this, we have generated many mutations that produced altered proteins that affect specific properties and activities of the virus, and we construct chimeric proteins composed of partial sequences or domains from different viruses. Understanding the precise biochemical activities of viral proteins in the replication process will allow the development of new strategies for vaccine development and the design of antiviral drugs.