Doctoral Dissertations

Date of Award

5-1992

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Leon N. D. Potgieter

Committee Members

M. Breider, D. Brian, R. Carroll

Abstract

Total cellular and viral RNA isolated from cells infected with noncytopathic Bovine Viral Diarrhea Virus strain 2724 was used for reverse transcription of viral specific sequences encoding gp48 and its putative protein signal sequence. The cDNA template was amplified twice by the polymerase chain reaction with primers designed from nucleotide sequences of cytopathic NADL and 72 BVD viruses. The amplified product was incorporated into plasmid vector pCRlO00 and subcloned into expression vector pGem-4z. Nucleotide sequence analysis of the cloned cDNA indicated it was 921 base pairs long, encoded 307 amino acid residues, had high sequence homology to other pestiviruses, and had no significant sequence homology to members of the Flaviviridae. In vitro expression of the cDNA yielded a 30kDa protein that was precipitated by BVDV polyclonal antiserum. The protein was glycosylated in the presence of canine microsomal membranes to give a 46kDa product and was secreted into the lumen of the microsomal vesicles. The characteristics of the putative signal peptide were consistent with signal sequences for protein translocation found in eukaryotes. A putative signal peptidase cleavage site was identified at a glycine residue at amino acid position 79. Based on signal peptidase cleavage of gp48 and lack of a protein anchor, I propose that gp48 is a glycosylated secreted protein analog of the glycosylated secreted portion of the pre-M protein of Flaviviruses.

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