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 polyprote ins, In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for proc essing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4E/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 ana lysis of the NS4A, NS4B, NS5A, and NS5B proteins,. A conserved leucine residue is found at the P1 position of all four cleavage sites, follo wed 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 preferen ce, a structural model of the pestivirus NS3 proteinase predicts a hig hly hydrophobic P1 specificity pocket. trans-Processing experiments im plicate the 64-residue NS4A protein as an NS3 proteinase cofactor requ ired 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.