This study involves the coordinated application of cryo-electron microscopy (cryoEM), three-dimensional image reconstruction, and X-ray diffraction methods to address fundamental questions about the overall morphology, composition and function of plant viruses. These studies will focus on structural analyses of three plant viruses: cowpea mosaic virus (CPMV), cowpea chlorotic mottle virus (CCMV), and barley yellow dwarf virus (BYDV). Complexes of CPMV with two separate monoclonal Fab fragments will be studied to investigate the detailed interactions between viruses and antibodies as well as the organization of the single-stranded RNA (ssRNA) in CCMV (and also brome mosaic virus). Work with BYDV aims to localize the factor(s) responsible for aphid transmissibility. In addition, studies will be made to coordinate cryoEM and X-ray data to learn how to correct for the effects of the microscope contrast transfer function. This should allow for reconstructed density maps that give a more accurate definition of protein and nucleic acid distributions and also provide the potential to attain higher resolution. %%% These studies provide a powerful way to examine protein-protein and protein-nucleic acid interactions which are involved in establishing virus stability and dictating viral assembly and disassembly (uncoating). The cryoEM techniques are ideally suited for studying large macromolecules and complexes that have proved difficult or "impossible" to crystallize and examine by diffraction methods. Long term goals are to examine and understand virus-antibody interactions, examine protein and nucleic acid organization, and develop enhanced methodology for studying virus structures with complementary biophysical techniques. The plant viruses are excellent model systems for these studies because i) they are readily and inexpensively obtained in large, highly-purified quantities, ii) they are not human pathogens, and iii) their molecular biology and genetics are well characterized.
