The object of this grant proposal is to further the understanding of coronavirus multiplication strategies, especially viral RNA replication, transcription and RNA packaging, through the use of mouse hepatitis virus (MHV) as a model system. Coronaviruses are enveloped single-stranded, positive-sense RNA viruses which cause the common cold, gastroenteritis and demyelinating disease in man and livestock. I have established a system in which defective-interfering (DI) RNA synthesized from a complete DI cDNA clone replicates extremely efficiently in DI RNA-transfected, and MHV- infected cells. This MHV DI cDNA system will be utilized extensively to study the following four subjects. (1) Identification of the sequences essential for MHV RNA replication. Deletions and linker-scanning mutations will be introduced at both the 5'-and 3'-end non-coding regions of a complete DI cDNA clone. The replication of DI RNAs which are transcribed from mutated DI cDNA clones will be examined in MHV-infected cells to identify sequences essential for MHV RNA replication. (2) Identification of the MHV RNA packaging signal. The primary structure of DI RNA which is packaged efficiently into the virion will be studied. The packaging signal will be identified either by virus uptake of a series of DI RNAs which have internal deletions or by incorporation of DI RNA fragments, expressed via a Sindbis virus vector, into MHV virions. (3) Identification of the sequence requirement for a biologically unique, discontinuous transcription mechanism involving a free leader RNA species. The sequence DI cDNA will be altered to that it will contain an MHV intergenic sequence where free leader RNA species can bind. By site-specific mutagenesis and deletion of this intergenic sequence, the precise sequence requirement for MHV mRNA synthesis will be determined. (4) Analysis of the leader RNA switching event that occurs between DI RNA and its helper virus RNA. Attempts will be made to identify sequences present both in the DI RNA and helper virus which are responsible for leader RNA switching. Together, these four studies will identify the essential recognition sequences involved in the replication, transcription and packaging of MHV RNA.
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