The RNA helicase and nucleotide triphosphatase activities of the bovine viral diarrhea virus NS3 protein are essential for viral replication

Helicase/nucleoside triphosphatase (NTPase) motifs have been identified in many RNA virus genomes. Similarly, all the members of the Flaviviridae fami ly contain conserved helicase/NTPase motifs in their homologous NS3 protein s. Although this suggests that this activity plays a critical role in the v iral life cycle, the precise role of the helicase/NTFase in virus replicati on or whether it is essential for virus replication is still unknown. To de termine the role of the NS3 helicase/NTPase in the viral life cycle, deleti on and point mutations in the helicase/NTPase motifs of the bovine viral di arrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish either he licase activity alone (motif II, DBY (H) under bar to DEY (A) under bar) or both NTPase and helicase activity (motif I, G (K) under bar T to G (A) und er bar T and deletion of motif VI) were generated. The C-terminal domain of NS3 (BVDV amino acids 1854 to 2362) of these mutants and wild type was exp ressed in bacteria, purified, and assayed for RNA helicase and ATPase activ ity. These mutations behaved as predicted with respect to RNA helicase and NTPase activities in vitro. When engineered back into an infectious cDNA fo r BVDV (NADL strain), point mutations in either the GKT or DEYH motif or de letion of motif VI yielded RNA transcripts that no longer produced infectio us virus upon transfection of EBTr cells. Further analysis indicated that t hese mutants did not synthesize minus-strand RNA. These findings represent the first report unequivocably demonstrating that helicase activity is esse ntial for minus-strand synthesis.