CLONING, SEQUENCING, AND IN-VITRO EXPRESSION OF GLYCOPROTEIN GP48 OF A NONCYTOPATHOGENIC STRAIN OF BOVINE VIRAL DIARRHEA VIRUS

Total cellular and viral RNA isolated from cells infected with noncyto pathic bovine viral diarrhea virus (BVDV) strain 2724 was used for rev erse transcription of viral specific sequences encoding the putative s ignal sequence and protein-encoding region of gp48. The cDNA template was amplified twice by the polymerase chain reaction with a nested set of primers designed from nucleotide sequences of cytopathic BVDV stra ins NADL and 72, and ligated into a plasmid vector. Nucleotide sequenc e analysis of the cloned cDNA indicated it was 921 base pairs long, en coded 307 amino acid residues, had high sequence homology to other pes tiviruses, and had no significant sequence homology to members of the Flaviviridae. In vitro expression of the cDNA yielded a 30 kDa protein that was precipitated by BVDV polyclonal antiserum. The protein was g lycosylated in the presence of canine microsomal membranes to give a 4 6 kDa product and was secreted into the lumen of the microsomal vesicl es. 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 270. Based on signal peptidase cleavage of gp48 and lack of a membrane anchor, we proposed that gp48 is a gly cosylated protein lacking a transmembrane domain, and is analogous to the glycosylated secreted portion of the pre-M protein of flaviviruses .