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 noncytopathic bovine viral diarrhea virus (BVDV) strain 2724 was used for reverse transcription of viral specific sequences encoding the putative signal 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 strains NADL and 72, and ligated into a plasmid vector. 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 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 glycosylated in the presence of canine microsomal membranes to give a 46 kDa 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 270. Based on signal peptidase cleavage of gp 48 and lack of a membrane anchor, we proposed that gp48 is a glycosylated protein lacking a transmembrane domain, and is analogous to the glycosylated secreted portion of the pre-M protein of flaviviruses.
Silva-Krott
I U
IU
Department of Environmental Practice, University of Tennessee, College of Veterinary Medicine, Knoxville 37901.
Kennedy
M A
MA
Potgieter
L N
LN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry; D001908:Diarrhea Viruses, Bovine Viral / Q000737:chemistry / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D006031:Glycosylation; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000737:chemistry / Q000378:metabolism; D012367:RNA, Viral / Q000737:chemistry; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D004279:DNA, Viral; D021382:Protein Sorting Signals; D012367:RNA, Viral; D014759:Viral Envelope Proteins; C078531:glycoprotein 48, bovine viral diarrhea virus