The structures of several icosahedral RNA viruses are known to atomic resolution, but little is known about the molecular interactions that are involved in their assemble and disassembly during virus infection. Further research in this area will help to elucidate these fundamental steps of viral replication and may allow new strategies for the control of viral infections to be designed. The long-term objective of this research is to define the RNA-protein and protein-protein interactions which are involved in the assemble and disassembly of southern bean mosaic virus (SBMV), the type member of the sobemovirus group. Several features make SBMV an attractive model system for these studies. The structure of the virus is known, the viral genome is compact (4.2 kilobases) and has been sequenced, a coat protein binding site has been identified on the viral RNA, the virus has been reassembled in vitro from purified components, and co-translational disassembly by a cap-independent mechanism has been demonstrated in vitro. In addition, a full-length cDNA clone of the viral genome has been constructed as is being tested for biological activity. Biochemical and genetic approaches will be used to define the RNA-protein interactions involved in assemble and to determine the function of the 5' UTR of the viral RNA in the early events, disassemble and translation, of the viral infection. Three specific questions will be addressed in this research proposal: 1. What are the requirements for ribosome entry on SBMV RNA? A model for SBMV translation will be tested using a genetic approach to determine if the SBMV 5' UTR directs internal ribosome entry in vitro. If it does, then the requirements for viral RNA elements and cellular proteins in this activity will be determined. 2. Is the SBMV coat protein binding site required for selective assembly? A coat protein binding site has been identified on SBMV RNA. The specificity of the interaction between the SBMV coat protein and its binding site will be determined by filter-binding and gel retardation. If the full-length SBMV clone is functional, then mutations will be constructed in the binding site to determine its function in SBMV assembly in vivo. 3. Which amino acids and structural elements of the SBMV coat protein are required for RNA binding? Mutations in the SBMV coat protein gene will be constructed to determine the function of specific amino acids and secondary structure of the coat protein in RNA binding.
