Coronaviruses cause a wide variety of fatal acute infections in animals, primarily of the respiratory and gastroenteric systems. Life-long persistent infections are associated with chronic disease of the CNS in mice. This study seeks to explain the virally programmed regulation of viral RNA levels that vary dramatically throughout the infectious process. Regulation during acute infection is observed as a progressively faster accumulation of the smaller 3'coterminal subgenomic mRNAs, a process involving a leader-fusion event (we hypothesize involvement of transcriptionally active subgenomic mRNA-length replicative forms), and a coordinated early shut-off of minus-strand synthesis (we hypothesize involvement of a pseudoknot in the 3' UTR). This study focuses on three specific aims, each examining a separate set of structural elements known to function in RNA synthesis: To (1) define the 3' terminal cis-acting signal for RNA replication, (2) define the 5' terminal cis-acting signal directing leader fusion onto the genome during a high frequency leader reversion event, and (3) define the internal cis- acting signal for leader fusion during subgenomic mRNA synthesis. Experimental approaches will involve extensive site-directed mutagenesis o the cis-acting elements and analysis of effects on structure, function, and RNA-protein interaction. The primary molecule to be used for this study is a cloned reporter- containing replicon (a defective-interfering RNA) of the bovine enteric coronavirus (a close relative of the human respiratory coronavirus IC43 and mouse hepatitis virus). The DI RNA genome is a 2.2 kilobase simple chimera of the viral genomic termini that replicates after transfection into helper virus-infected cells, becomes packaged, and has been engineered to produce subgenomic mRNAs.
