Molecular cloning and sequence analysis of the 3' end of the bovine enteric coronavirus genome

The structure of the bovine enteric coronavirus (BCV) genome was examined using two experimental approaches. The first approach compared the degree of nucleotide sequences homology between the antigenically related BCV and human respiratory coronavirus 0C43 (HCV 0C43) by two-dimensional T₁ oligonucleotide fingerprinting. From an analysis of comigrating oligonucleotides in a mixing experiment, it was determined that BCV and HCV 0C43 share a sequence homology of approximately 96%. This degree of homology supports the notion that the two viruses are, in fact, not identical, even though they share a very similar protein profile, and are closely related antigenically. The second approach examined the primary structure of the 3' end of the BCV genome. The 3' end of the 20 Kb BCV genome was cDNA cloned and DNA sequenced, and the properties of two genes and a potential third gene were deduced and analyzed from an examination of open reading frames. One clone of 2.16 Kb was sequenced in its entirety and other clones of 4.1, 2.8, and 1.08 were sequenced in part, using the chemical method of Maxam and Gilbert. The largest gene (N) begins at base 1634 from the 3' end of the genome and encodes a 448 amino acid protein of 49,379 molecular weight. It is the nucleocapsid protein gene on the basis of size, chemical properties, and extensive amino acid sequence homology with the murine coronavirus nucleocapsid protein. A hypothetical gene (lORF) identified from a second open reading frame internal to the N gene, encodes a 207 amino acid protein of 23,057 molecular weight beginning at base 1573. The third gene (M) begins at base 2336 from the 3' end of the genome and encodes a 230 amino acid protein of 26,376 molecular weight. It is the matrix protein gene on the basis of its size, hydrophobicity pattern, and extensive amino acid sequence homology with the matrix protein of the murine coronavirus. Both nucleotide and amino acid sequence data show BCV to be closely related to the murine hepatitis coronavirus and very distantly related to the porcine transmissible gastroenteritis coronavirus and the avian infectious bronchitis coronavirus.