Doctoral Dissertations

Date of Award

3-1986

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

David A. Brian

Committee Members

Richard Courtney, Arthur Brown, John Koontz

Abstract

The structural proteins of two hemagglutinating mammalian coronaviruses were characterized at the molecular level. Emphasis was placed on the synthesis and processing of the hemagglutinin protein.

The human respiratory coronavirus 0CA3 (HCV 0CA3) was shown to consist of four major structural proteins. Each of these was shown to have an homologous counterpart in the bovine enteric coronavirus (BCV). The four proteins are:

1. A 190 kDa peplomeric glycoprotein which was cleavable by trypsin into subunits of 110 and 90 kDa. Each subunit represents a different amino acid sequence on the basis of peptide mapping.

2. A 130 kDa glycoprotein, which behaved as a disulfide-linked dimer of 65 kDa molecules. This protein appears to be the virion hemagglutinin on the basis of protease digestion studies.

3. A 55 kDa nucleocapsid phosphoprotein.

4. A 26 kDa glycoprotein, which is apparently a matrix protein. The 190, 130, 55, and 26 kDa species are present at the rates of 12A, 31, 1023, and 1023 molecules per virion, respectively.

Monospecific polyclonal antiserum against each of the major structural proteins of BCV was used to identify the homologous proteins between HCV 0CA3 and BCV. These experiments were extended to the antigenically related, but nonhemagglutinating, mouse hepatitis coronavirus A59, where it was demonstrated for the first time that the mouse hepatitis virus A59 has no molecular counterpart to the hemagglutinin proteins of HCV 0CA3 or BCV. The mouse hepatitis virus A59 is apparently either missing the gene for this protein or the gene, if present, is not expressed.

Further characterization of the BCV hemagglutinin demonstrated that the mature virion protein is a 140 kDa glycoprotein composed of what appears to be two identical glycosylated subunits of 65 kDa. Only one unglycosylated species with a molecular mass of 42.5 kDa was identified in the presence of tunicamycin. The subunits appear to be cotranslationally disulfide-linked and glycosylated with 9-11 N-linked high mannose chains per subunit. Both subunits appear to be identically processed with only 3-4 of these chains being processed to the complex type as the virion passes through the Golgi. A biosynthetic pathway for gpl40 is proposed.

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