Doctoral Dissertations

Date of Award

12-1989

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Microbiology

Major Professor

David A. Brian

Committee Members

Stuart Riggsby, Jerry Weir, Bath Mullin

Abstract

Bovine coronavirus possesses four structural proteins, the spike, hemagglutinin-esterase, matrix and nucleocapsid proteins. The genes for the structural proteins all map within the 3' proximal 9 kilobases of a 20 kilobase, single stranded RNA genome. The genes for the hemagglutininesterase, matrix and nucleocapsid proteins have been cDNA cloned, physically mapped on the genome and sequenced. In this study, five kilobases of the genome mapping between the hemagglutinin-esterase and matrix protein genes and encoding genes for the spike and four potential nonstructural proteins was characterized at the molecular level.

A partial cDNA genomic library was made and the spike protein gene was identified and sequenced. The spike protein gene is 4,089 bases, lies on the 3' side of the hemagglutinin-esterase protein gene, and predicts a protein of 150,825 daltons. An internal lys-arg-arg-ser-arg-arg sequence predicts a protease cleavage site between amino acid 768 and 769 and amino terminal amino acid sequencing of the virion derived 100 kilodalton subunit confirmed the location of this site. The virion 120 and 100 kilodalton spike subunits were therefore established to be the amino-and carboxy-terminal subunits. RNA hybridization analyses using a sequence specific probe demonstrated that the spike protein is encoded on mRNA 3.

Between the spike and matrix protein genes were found four open reading frames that encode in order 5' to 3', potential nonstructural proteins of 4.9, 4.8, 12.7 and 9.5 kilodaltons. A consensus sequence CYAAAC that is thought to play a role in coronavirus transcription precedes the open reading frames for the 4.9, 12.7 and 9.5 kilodalton proteins and predicts that transcripts from each of these sites would be made. RNA hybridization analyses using sequence specific probes confirmed this prediction and established the three transcripts to be mRNAs 3a, 4, and 5 respectively. Translation of in vitro synthesized transcripts of the bovine coronavirus 9.5 kilodalton protein gene was done in an attempt to clarify differences that were observed between the bovine coronavirus and the antigenically related mouse hepatitis coronavirus with respect to the expression of this protein.

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