The long-term objectives are to understand the molecular details of coronavirus structure and replication, especially with regard to the function of specific gene products, and mechanisms regulating replication during a cytopathogenic acute infection and those regulating replication during a noncytopathogenic persistent infection. Coronaviruses cause primarily severe to mild acute gastrointestinal and respiratory diseases in animals and humans, and chronic multiorgan diseases in some animals and possible humans.
The specific aims of this proposal are: (1) To complete the sequencing of the genome of the porcine transmissible gastroenteritis coronavirus (TGEV) (11Kb remaining of a 20 Kb genome), a region that encodes the RNA-dependent RNA polymerase. cDNA clones that map in this region will be sequenced by a combination of the chemical and dideoxy methods and more cDNA clones will be generated to close the remaining gaps. A strategy for reconstructing a full-length (genome) cDNA will be developed in order to generate infectious RNA transcripts. (2) To characterize the products of open reading frames in the genome of TGEV and the bovine enteric coronavirus (BCV), regions that have been sequenced, with regard to their structure, localization, and function during virus replication. Open reading frames will be subcloned into expression vectors to generate products in vitro and in vivo. Antibodies will be produced to the individual proteins and the localization and orientation of the protein in cell organelles will be studied. The effects of stably expressed products on virus replication will be determined. (3) To block expression of specific genes and RNA replication by site-directed nucleic acid inhibition in order to analyze specific coronavirus replicational events. Both externally applied oligodeoxynucleotides (and modified oligodeoxynucleotides) and internally expressed """"""""antisense"""""""" sequences will be studied. Inhibition of virus replication will be studied by blocking specific events separately in acute and persistent infections in cell culture.
