The major object of this proposal is to determine the mechanism of coronavirus nucleocapsid (N) protein assembly with RNA, both to understand the unique processes of coronavirus replication as well as to gain insight into a mode of protein-RNA interaction that may be a paradigm for some cellular processes. Coronaviruses are a family of single-stranded, positive-sense RNA viruses whose nucleocapsids are assembled into helical structures. Through a combination of interstrain sequence comparison, RNA-binding studies and mutant analysis, we have developed a model of the functional domains of the N protein of the coronavirus mouse hepatitis virus (MHV). The RNA-binding domain, which we have localized, will be further characterized by site- directed mutagenesis of critical amino acid residues and by the analysis of isolated N protein mutants and revertants. An attempt will be made to introduce defined N gene mutations back into the RNA genome of MHV. The conditions required for N protein to exhibit sequence-specific binding will be determined, and then specific RNA sequences in the MHV genome to which N binds with high affinity will be examined by in vitro experiments with engineered RNAs. Other viral and cellular interactions of N protein will also be studied, particularly as these affect N-RNA binding. The location and functional significance of the phosphorylation sites in N will be resolved. Protein- protein interactions between the N protein and the carboxy-terminal, cytoplasmic (or virion-internal) domain of the MHV membrane glycoprotein (M) will be examined. The carboxy-terminal tail of M will be expressed in vitro as a separate, soluble polypeptide. This protein fragment will be tested for its ability to bind to isolated MHV virion nucleocapsid as well as to expressed MHV N protein. Interacting regions of the N and M proteins will be identified, and the effect of M protein binding to N protein on the specificity and affinity of N-RNA interactions will be determined. Coronavirus are important respiratory, neurologic and enteric pathogens for humans and domestic animals, and an understanding of their molecular biology and pathogenesis is critical for their control and prophylaxis. The studies proposed will provide insights into the coronavirus life cycle and potential targets for antiviral chemotherapy.
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