Randhawa, J. S. (Jaspal Singh) (1993) Molecular characterisation of the pneumonia virus of mice glycoprotein genes. PhD thesis, University of Warwick.

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Abstract

The molecular characterisation of the major glycoproteins of pneumonia virus of mice (PVM) was undertaken to identify the molecular basis for different pathogenicities for two strains of PVM. One is highly pathogenic and passaged entirely in mice (strain J3666) whilst the other is non-pathogenic and has been passaged entirely in tissue culture (strain 15).

Characterisation of the fusion proteins of these two strains revealed little amino acid changes that may account for their pathogenicities. Of the 537 amino acid long fusion protein, only four amino acid changes were observed between the two strains. None of the changes were located within the region encoding the cleavage site of the F₀ polypeptide and the altered amino acids were distributed throughout the fusion protein. The amino acids changes were considered to be fairly conservative and may represent adaptation of the two strains to their different modes of passaging. However, the importance of these changes with regards to cleavage cannot be ruled out since one or more of these changes may be located within the 3-dimensional structure of the F₀ cleavage site.

Nucleotide sequence analysis of the attachment (G) glycoproteins of these two strains of PVM showed only two amino acids changes within the extracellular domain of the protein. However, the G proteins of the two strain differed from each other in that the G protein of strain J3666 possessed a N-terminal extension compared to that of strain 15. This amino acid extension forms the cytoplasmic domain of the G protein in strain J3666 but appears to be lacking in that of strain 15. Furthermore, the G proteins of both strains of PVM were synthesised from the second, but not the same, initiation codon. In PVM strain J3666, the first available initiation codon coded for a short polypeptide of 12 amino acids whereas that of strain 15 coded for a short polypeptide of 33 amino acids.

Expression of the G genes in vitro produced polypeptides which were approximately 3K larger than their predicted molecular weights. Synthesis of polypeptides due to internal initiation during translation were observed for the G gene of strain J3666. Expression of the G genes using recombinant vaccinia virus expressing the T7 RNA polymerase showed that the G proteins of both strains of PVM were expressed on the surface of cells transfected in vitro. Thus, the signals required for the correct processing and transport of the G proteins of PVM are not contained within the cytoplasmic domain of the proteins.

The first steps of an investigation of possible ribosomal frameshifting event occurring within the G gene of PVM strain 15 was undertaken. The initiation codon of the major and short ORF's of the G gene of PVM strain 15 were mutated in a variety of combinations with a view to fusing these mutated fragments to the 5' end of the E. coli β-galactosidase gene that lacks its own initiation codon.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QP Physiology Q Science > QR Microbiology R Medicine > RC Internal medicine S Agriculture > SF Animal culture
Library of Congress Subject Headings (LCSH):	Pneumonia -- Molecular aspects, Viral pneumonia, Virus diseases, Mice as laboratory animals, Mice -- Genetics, Glycoproteins
Official Date:	September 1993
Dates:	Date Event September 1993 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Easton, A. J. (Andrew J.)
Sponsors:	Medical Research Council (Great Britain)
Extent:	xiv, 219 leaves : illustrations
Language:	eng

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