Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.
Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication.
Xu
J
J
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
Mendez
E
E
Caron
P R
PR
Lin
C
C
Murcko
M A
MA
Collett
M S
MS
Rice
C M
CM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D008969:Molecular Sequence Data; D009154:Mutation; D010447:Peptide Hydrolases; D010576:Pestivirus / Q000502:physiology*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D020365:RNA Helicases; D017386:Sequence Homology, Amino Acid; D012694:Serine; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D010447:Peptide Hydrolases; D011506:Proteins; D020365:RNA Helicases; D012694:Serine; D017361:Viral Nonstructural Proteins; C086730:p80 protein, bovine viral diarrhea virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases