Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing.
Bovine viral diarrhea virus (BVDV), the prototypic pestivirus, possesses a positive-strand RNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. We have endeavored to elucidate the mechanisms involved in protein biogenesis by this pestivirus. Here, we present our studies on gene expression from the viral nonstructural protein coding region encompassing the carboxy-terminal 60% of the ORF. Previous sequence and modeling analyses predicted the amino-terminal region of the BVDV nonstructural protein p80 to be a trypsin-like serine proteinase. Using a mammalian cell transient expression system, we show that this region indeed possessed a proteolytic activity and, further, required the serine residue previously predicted to be the putative serine proteinase catalytic site. We found the p80-region proteinase activity was required for proteolytic processing of all viral nonstructural proteins. Cleavage by this activity at the amino and carboxy termini of the p80 protein itself likely occurred intramolecularly (in cis), since we were unable to demonstrate activity in trans at these sites. Cleavages at the three processing sites downstream of the carboxy terminus of p80 were shown to occur in trans. However, p80 proteinase activity alone was not sufficient for cleavage of the last of these sites. Another viral gene product, or specific condition, is implicated as a necessary cofactor for p80 proteinase activity at this site. Pestivirus polyprotein processing can now be compared to similar events by viruses of other groups. Finally, the potential role of p80 proteinase activity in the phenotype of cytopathic biotypes of BVDV is discussed.
Wiskerchen
M
M
Medimmune, Inc., Gaithersburg, Maryland 20878.
Collett
M S
MS
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics*; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005814:Genes, Viral; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016366:Open Reading Frames; D010447:Peptide Hydrolases; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D020365:RNA Helicases; D012367:RNA, Viral / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification; D015678:Viral Structural Proteins / Q000235:genetics*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D010450:Endopeptidases; D015345:Oligonucleotide Probes; D010447:Peptide Hydrolases; D020365:RNA Helicases; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D015678:Viral Structural Proteins; C086730:p80 protein, bovine viral diarrhea virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases