The proposed project will initiate a structure-function analysis of the alphavirus nucleocapsid protein. The alphaviruses are a group of arthropod-borne plus-strand RNA viruses, many of which cause encephalitis, arthritis, myositis, and fever in humans. We will utilize the recently solved high resolution three-dimensional structure of the Sindbis nucleocapsid protein, three alphavirus cDNA clones(Sindbis, Semliki Forest, Ross River), in vivo and in vitro biochemical assays, and in vitro mutagenesis schemes to carry out our objectives. Site-directed and random mutagenesis will be used to construct mutations in the nucleocapsid designed to study proteolytic activity, protein-protein and protein-nucleic and interactions, assembly/disassembly, and interactions involving the mature capsid and the cytoplasmic domain of E2 glycoprotein. Mutants will be analyzed by a variety of in vitro and in vivo assay. Proteolytic processing will be studied using an in vitro translation system. RNA binding assays will quantitatively analyze mutants defective in nucleic acid binding. These assays will discriminate between defects in viral RNA binding activity and defects in ribosomal RNA binding activity. An in vitro assembly assay will be utilized to study defects in the assembly pathway that are expected with some mutations. All mutations will be reconstructed into full-length cDNA clones from which infectious RNA can be generated for rescue and analysis of the resulting viruses. These viruses will be studied in vivo using analyses designed to determine the nature of the defect. A cDNA clone of Sindbis virus will be used that will allow expression of genetically altered nucleocapsid protein independent of the other structural proteins thus negating the requirement for proteolytic processing in the replication pathway. E coli expression systems will be used to generate purified nucleocapsid protein containing genetically engineered mutations. In collaboration with Dr. M. Rossmann, the structures of these altered nucleocapsid proteins will then be determined. Alterations in nucleocapsid structure will then be correlated with defects in activity, and a structure-function relationship will be determined. The proposed studies will contribute to the elucidation of the alphavirus nucleocapsid function and further our knowledge of macromolecular interactions and assembly.
