Doctoral Dissertations
Date of Award
12-1989
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Major
Microbiology
Major Professor
David A. Brian
Committee Members
Jerry P. Weir, Beth Mullin, W. Stuart Riggsby
Abstract
Bovine coronavirus is one of six coronaviruses that contain the hemagglutinin glycoprotein, a fourth and final major structural protein to be demonstrated for the Coronaviridae. On the virion particle, each hemagglutinin molecule exists as a disulfide-linked homodimeric structure. What biological advantage the hemagglutinin may confer upon BCV is not known. The experiments described in this dissertation examine these issues on a molecular level by cloning and expressing the hemagglutinin gene in both in vitro and in vivo expression systems.
The first 8,955 bases of the 3' end of the BCV genome were cDNA cloned using random or specific oligonucleotide primers. The hemagglutinin gene was found to map on the 5' side of the peplomer gene between bases 8,678 and 7,408 relative to the 3' end of the BCV genome. The hemagglutinin gene was sequenced and found to be 1,272 bases in length. A deduced amino acid sequence shows the hemagglutinin to be 424 amino acids long with a molecular weight of 47,700.
The hemagglutinin gene was subcloned into a pGEM plasmid vector and expressed using wheat germ extract. Partial translocation across the microsomal membranes, signal cleavage, and glycosylation of the hemagglutinin were demonstrated in the presence of microsomes. At no iv time, however, was a dimer form of the in vitro expressed hemagglutinin observed. These results suggest that early stages in the biosynthesis of the hemagglutinin in vivo may be independent of other viral proteins.
The hemagglutinin was found to be anchored in microsomal membranes by its carboxy terminus, indicating that it is a type I membrane glycoprotein. The hemagglutinin gene was transiently expressed in cell culture. Analysis of the hemagglutinin expressed in vivo by immunofluorescence demonstrated that it was transported to the cell surface. These results suggest that the hemagglutinin contains all the information necessary for transport to and insertion into the plasma membrane.
Recommended Citation
Kienzle, Thomas E., "Cloning, sequencing, and expression analysis of the hemagglutinin gene of the bovine enteric coronavirus. " PhD diss., University of Tennessee, 1989.
https://trace.tennessee.edu/utk_graddiss/11705