The long-term objectives of this project are to elucidate the functions and control mechanisms of proteins encoded by positive-strand RNA viruses. The model virus employed in this project is tobacco etch virus (TEV), a well-characterized virus that belongs to the "picornavirus supergroup". Members of this supergroup, which includes several important human and animal pathogens, employ replication mechanisms that share several common features, including synthesis of large polyproteins that are processed by viral proteinases. The TEV system possesses several unique attributes as a model system to study these viruses, including the ability to manipulate both the virus and the host genomes, and an extremely sensitive and rapid viral replication assay. This project has three specific aims that will provide information concerning the functions, interactions, and partitioning of replication-associated proteins. A. Analysis of nuclear transport of two replication-associated proteins. The role of translocation of two proteins (the VPg/proteinase and RNA polymerase) to the nucleus will be investigated by systematic mutagenesis of localization signals in both proteins. Modified viruses containing these mutations will be tested for replicative abilities using a sensitive assay involving a reporter gene engineered into the TEV genome. B. Analysis of interactions between replication-associated proteins. Genetic and biochemical analyses to investigate protein-protein interactions between VPg/proteinase and RNA polymerase will be conducted. These interactions may be important for formation of replication complexes and for regulating subcellular transport of both proteins. C. Analysis of cis/trans relationships of replication-associated proteins. A novel complementation assay, in which defective viruses are rescued by viral proteins expressed in transgenic cells, will be used to analyze proteolytic processing and polyprotein-linkage requirements of replicative functions.