NS3 serine protease of bovine viral diarrhea virus: Characterization of active site residues, NS4A cofactor domain, and protease-cofactor interactions

The gene expression of bovine viral diarrhea virus (BVDV), a pestivirus, oc curs via translation of a hypothetical polyprotein that is processed cotran slationally and posttranslationally by viral and cellular enzymes. A protea se located in the N-terminal region of nonstructural (NS) protein NS3 catal yzes the cleavages, leading to the release of NS4A, NS4B, NS5A, and NS5B. O ur study provides experimental evidence that histidine at position 1658 and aspartic acid at position 1686 constitute together with the previously ide ntified serine at position 1752 (S1752) the catalytic triad of the pestivir al NS3 serine protease. Interestingly, a mutant protease encompassing an ex change of the active site S1752 to threonine still showed residual activity . This finding links the NS3 protease of pestiviruses to the capsid proteas e of Sindbis virus. Furthermore, we observed that the minimal protease doma in of NS3 encompasses about 209 amino acids. The NS3 protease was found to be sensitive to N-terminal truncation because a deletion of 6 amino acids s ignificantly reduced the cleavage efficiency at the NS4A/4B site. Larger N- terminal deletions also impaired the activity of the enzyme with respect to the other cleavage sites but to a different degree at each site. The NS3 p rotease of BVDV has previously been shown to depend on NS4A as cofactor. We demonstrate here that the central region of NS4A represents the cofactor d omain. Furthermore, coprecipitation studies strongly suggest an interaction between NS4A and the N-terminal region of NS3. Besides the remarkable simi larities observed between the pestiviral NS3 protease and the corresponding enzyme of hepatitis C virus (HCV), our results suggest a common ancestry b etween these enzymes and the capsid protease of Sindbis virus. (C) 2000 Aca demic Press.