The purpose of this work is to study the fundamental processes of genome replication, viral protein synthesis and virion assembly and their interrelationship for certain mRNA viruses. We are especially concerned with these processes at the molecular level and thus have selected for study three viruses whose physical and genetic structure is especially well understood, and for which appropriate synthesizing systems and methodologies are available. We will work with a) brome mosaic virus (BMV), a divided-genome, single-stranded RNA plant virus; b) black beetle virus (BBV) a divided-genome, single-stranded RNA insect virus; and c) southern bean mosaic virus (SBMV) a single genome, mRNA plant virus. For BMV and BBV, and as appropriate for SBMV, we will identify, and kinetically and structurally relate, the intermediate and final products of synthesis of the genomic and especially the subgenomic RNAs. We will quantitate and measure the time course of the syntheses by direct measurements of fractionated radioactive products and by probing their presence with radioactive cDNA and cRNA. We will study binding of viral RNA and RNA fragments with protein and protein fragments to analyze the interactions governing RNA replication and virion assembly. We will genetically engineer viral cDNA to give large-scale production of particular viral proteins for enzymatic reconstitution studies, antibody-mediated inhibition studies and RNA binding studies. We will devise systems in which viral cDNAs are infectious and utilize these for functional studies of regulatory and coding sequences, the construction of genetic vectors and the development of synthetic defective interfering particles for the control of viral infections.
