The major object of this proposal is to further develop and apply a genetic system for the study of coronaviruses. Coronoaviruses are a family of single-stranded, positive-sense RNA viruses whoe helical nucleocapsids are packaged within host-derived membrane envelopes containing a small complement of viral membrane glycoproteins. The RNA genomes of coronaviruses are the largest mature RNA molecules yet discovered, making their genetic manipulation unapproachable by the techniques that have been used with other RNA viruses. We have established a system for the site-specific mutagenesis of the prototype coronavirus mouse hepatitis virus (HMV) which takes advantage of the high rate of RNA-RNA recombination in MHV by transducing a site-specific mutation into the viral genome by recombination with a synthetic RNA introduced into infected cells. Single point mutations and extensive substitutions have been engineered into all of the structural protein genes of MHV in this fashion. This procedure will be enhanced further by the advantages offered by a powerful host-range-based selection system and by efforts to obtain an infectious full-length cDNA of the MHV genome. The genetic system will be used to answer basic questions about the structure and function of the components of MHV virions: the spike glycoprotein, the matrix glycoprotein, the small envelope protein, and the nucleocapsid protein. This will provide valuable insights into the roles these proteins play in viral replication and how they interact with both viral and host components. Coronaviruses are important respiratory, neurologic and enteric pathogens, and an understanding of their molecular biology is critical for their control and prophylaxis. The studies proposed will provide fundamental insights into the coronavirus life cycle and assembly, potential targets for antiviral chemotherapy, and a possible means to manipulate these infectious agents for vaccine design.