The RNA Helicase and Nucleotide Triphosphatase Activities of the Bovine Viral Diarrhea Virus NS3 Protein Are Essential for Viral Replication

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Abstract
Helicase/nucleoside triphosphatase (NTPase) motifs have been identified in many RNA virus genomes. Similarly, all the members of the Flaviviridae family contain conserved helicase/NTPase motifs in their homologous NS3 proteins. Although this suggests that this activity plays a critical role in the viral life cycle, the precise role of the helicase/NTPase in virus replication or whether it is essential for virus replication is still unknown. To determine the role of the NS3 helicase/NTPase in the viral life cycle, deletion and point mutations in the helicase/NTPase motifs of the bovine viral diarrhea virus (BVDV) (NADL strain) NS3 protein designed to abolish either helicase activity alone (motif II, DEY H to DEY A ) or both NTPase and helicase activity (motif I, G K T to G A 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 expressed in bacteria, purified, and assayed for RNA helicase and ATPase activity. These mutations behaved as predicted with respect to RNA helicase and NTPase activities in vitro. When engineered back into an infectious cDNA for BVDV (NADL strain), point mutations in either the GKT or DEYH motif or deletion of motif VI yielded RNA transcripts that no longer produced infectious virus upon transfection of EBTr cells. Further analysis indicated that these mutants did not synthesize minus-strand RNA. These findings represent the first report unequivocably demonstrating that helicase activity is essential for minus-strand synthesis.