Spherical RNA plant viruses are excellent model systems for studying protein-protein and protein-nucleic acid interactions. Tomato Bushy Stunt Virus (TBSV) is the system of choice because x-ray diffraction studies have provided a three-dimensional structure of the icosahedral portion of the virus at 2.9 A resolution. Further refinement of the 2.9 A electron density map is limited by lack of primary sequence information for the TBSV coat protein. We will determine the amino acid sequence of the 40,000 dalton coat protein, and will characterize the 86,000 dalton TBSV minor protein which is present in one copy per virion. RNA-protein crosslinking studies will be used to determine which amino acids of the coat protein contact the viral nucleic acid, and regions of the RNA with a high affinity for coat protein will be isolated and characterized. TBSV also provides an experimental system in which to probe assembly and disassembly of macromolecular complexes, and to identify the structural and chemical basis for control of assembly specificity and disassembly timing. Toward this end, in vitro reconstitution experiments using purified TBSV coat protein, minor protein, and RNA will be performed.
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