9514271 Gehrke The long-range goals of this project are to understand how specific ribonucleoprotein complexes form; to define their structure, and most importantly, to understand their biological significance. The immediate experimental goals of this work will be accomplished by studying a model virus system that has been developed collaboratively in two laboratories. Although the coat proteins of alfalfa mosaic virus and the ilarviruses are both unrelated at the primary amino acid level and serologically distinct, they are functionally interchangeable in their capacity to bind different viral RNAs and to activate virus replication. These investigations will biochemically identify common nucleotide determinants in alfalfa mosaic virus and ilarvirus RNAs that are bound specifically by the viral coat proteins. The biochemical data will then be extended by evaluating RNA and/or protein sequence mutations in the context of functional virus replication and assembly. Two experimental approaches will be followed to identify the nucleotide sequences and structures required for coat protein binding. First, electrophoretic mobility bandshift analysis, hydroxyl radical footprinting, and chemical modification interference analysis will be used to analyze the binding of citrus variegation virus (CVV) coat protein and citrus leaf rugose virus (CiLRV) coat protein to the 3' untranslated region of AlMV RNA (170 nucleotides) and to a 3'-terminal 39-nucleotide minimal coat protein binding site that has been described previously. These experiments will permit comparison of the binding determinants recognized by the cognate (AlMV) and heterologous (CVV/CiLRV) coat proteins. Second, in vitro selection of RNA ligands from a pool of random or semi-random oligoribonucleotides will be used to identify 1) sequences and/or structures in the RNA determinant that are essential for binding AlMV, CVV, or CiLRV coat proteins, and 2) new RNA sequences and/or structures that display enhanced binding affinity for AlMV and il arvirus coat proteins. The biological significance of the identified RNA-protein interactions will be evaluated using two distinct assay systems: 1) a virus replication assay in tobacco protoplasts, in which the ability of the small selected RNAs to inhibit virus production will be analyzed and 2) an assay to evaluate AIMV RNA 3 replication and assembly in cells or protoplasts that transiently or constitutively express AIMV replicase proteins. The work has significance for greater understanding of RNA-protein interactions and the life cycle of viruses that infect a wide range of plants. ***
