The objective of this research is to systematically study (a) coronavirus molecular structure and (b) the molecular biology of coronavirus replication. We will use a bovine coronavirus and a porcine coronavirus which can be cultured to titers of greater than 10 to the 8th power pfu/ml and therefore used for biochemical studies. Virion genomic RNA will be examined with respect to the following properties: its infectiousness, its ability to function as mRNA in vitro, its 2 degrees structure, its molecular weight as determined by denaturing gel electrophoresis and oligonucleotide fingerprinting, the nature of its methylated 5' end and the nature of the internal methylated nucleosides. Intracellular virus-specific RNA species will be characterized to identify the size, number, and sequence specificity of mRNA species and to identify replicative forms, replicative intermediates, and minus-strand RNA. Gene products of the mRNA's will be identified and the arrangement of genes along the viral genome will be determined. Caps and methylation patterns in mRNA's will be characterized. Virion polypeptides will be characterized with respect to size, number, and stoichiometric arrangement on the virion, location of dissulfide bonds, glycosylation, phosphorylation, hemagglutinating properties, and role in inducing neutralizing antibodies. The size, number, and the kinetics of synthesis of intracellular virus-specific proteins will be characterized using pulse-chase studies, immunoprecipitation and gel electrophoresis. We will initiate experiments to prepare DNA copies of viral genomic RNA by reverse transcription in an initial effort to make probes for tissue location of viral RNA and for use in analyzing gene expression.