Coronaviruses are a group of enveloped RNA viruses which infect diverse species of animals causing respiratory, gastrointestinal and neurological diseases. Mouse hepatitis virus serves as a model system for the study of viral induced demyelination. The goal of our laboratory is to understand the unique mechanisms of coronavirus replication. A novel mechanism of leader-RNA primed transcription has been proposed to describe the synthesis of coronavirus mRNAs. In this model, leader RNA is synthesized, dissociates from the genomic template RNA and rebinds to the negative-strand RNA template at intergenic regions where there is a complementary sequence between the 3'-end of the leader RNA and the RNA template. Transcription initiates off the leader RNA primer and continues to the end of the template. I have developed an in vitro system to determine what proteins and leader RNA sequences are required for this unique mechanism of transcription. This system utilizes exogenous leader RNA which differs slightly in sequence from the viral leader RNA. The synthetic leader RNA is added to cytoplasmic extracts prepared from coronavirus infected cells. I adapted the protocol of lysolecithin treatment of infected cells to set up the in vitro transcription system and developed a very sensitive PCR method to detect transcription products. Using this system, I was able to demonstrate leader RNA priming of viral mRNAs. This system represents the first direct biochemical demonstration of the role of free leader RNA in coronavirus mRNA synthesis. We propose to use this system to analyze the mechanism of coronavirus transcription. Another unusual feature of coronavirus is its high rate of genome recombination. The high frequency recombination is proposed to be due to the discontinuous nature of coronavirus RNA replication which results in the synthesis of RNA intermediates. Using exogenous RNA in the in vitro transcription system, I have been able to demonstrate copy-choice recombination. We propose to investigate the sequence requirements for coronavirus RNA recombination using the in vitro system. The unique transcription and recombination mechanisms of coronavirus implicate a polymerase with interesting -properties such as a putative endonucleolytic activity which would recognize base mismatches. Our understanding of the functions of the coronavirus polymerase have been hampered because of the large size of the gene (22 kb) and its gene product (> 800 kDa). We have shown that the protein product is a polyprotein which is cleaved into subunits, but little is known about other functional domains. We proposed to investigate the functional domains of the polyprotein by characterizing temperature sensitive mutants which map to this gene. and by generating antibodies to putative functional domains of the polyprotein. The antibodies will be used to identify proteins in virus infected cells and tested in the in vitro transcription system for inhibition of RNA synthesis.
