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

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, DEYH to DEYA) or both NTPase and helicase activity (motif I, GKT to GAT 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.The Flaviviridae family is comprised of three genera, Flavivirus (such as Yellow fever virus and Dengue virus types 1 to 4), Hepacivirus (such as Hepatitis C virus [HCV]), and Pestivirus (such as Bovine viral diarrhea virus [BVDV]) (28). BVDV infection represents an economically important disease of cattle, and BVDV has been identified as the causative agent of viral diarrhea-mucosal disease (reviewed in references 1, 12, 25, and 37). Like the other members of the Flaviviridae, BVDV is an enveloped, plus-stranded RNA virus whose genome consists of a nonsegmented single-stranded RNA molecule. BVDV genomic RNA is approximately 12.5 kb and encodes a single open reading frame of approximately 3,900 amino acids (7-9, 24). The polyprotein translated from the open reading frame is subsequently processed by virally encoded and cellular proteases into 12 individual proteins (13,30,31,35,44). These individual proteins function either as structural components of the virion or presumably, at least in part, as components of the viral RNA replicase complex as described for other Flaviviridae family members (2, 6, 17). RNA replicons derived from defective interfering particles have shown that the 5Ј and 3Ј nontranslated regions (NTRs) along with the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B can support RNA replication (4, 45). However, the essentiality of the individual nonstructural proteins has not yet been tested.The NS5B proteins of both BVDV (46) and the related HCV (3, 23) display RNA...

Influence of NS5A protein of classical swine fever virus (CSFV) on CSFV internal ribosome entry site-dependent translation

“…The NS5B protein serves as the viral RNAdependent RNA polymerase (RdRp) (Steffens et al, 1999;Xiao et al, 2002) and contains a conserved GDD motif necessary for catalytic activity (Wang et al, 2007). NS3 is a multifunctional protein possessing serine protease, RNA helicase and nucleoside triphosphatase activities, and is important for virus replication and even for promoting viral and cellular translation (Gu et al, 2000;Piccininni et al, 2002;Sheng et al, 2007;Suzich et al, 1993;Warrener & Collett, 1995;Xiao et al, 2008;Xu et al, 1997). The 39UTR is probably involved in initiation of pestiviral genome replication (Isken et al, 2003(Isken et al, , 2004Pankraz et al, 2005;Xiao et al, 2004;Yu et al, 1999), whilst the 59UTR contains an internal ribosome entry site (IRES), located between nt 40 and 350 at the 59 terminus of the genome, which is able to facilitate translation of the viral genome (Fletcher & Jackson, 2002).…”

Modulation of the Nucleoside Triphosphatase/RNA Helicase and 5′-RNA Triphosphatase Activities of Dengue Virus Type 2 Nonstructural Protein 3 (NS3) by Interaction with NS5, the RNA-dependent RNA Polymerase

“…24 and 25). RNA helicase activities of NS3 of hepatitis C virus, a member of Hepacivirus genus (26 -35), and the p80 of bovine virus diarrhea virus, a member of the Pestivirus genus (36,37), have been well characterized. However, the RNA helicases of mosquito-borne flaviviruses have not been well studied.…”

An Overview of the Immune Evasion Strategies Adopted by Different Viruses with Special Reference to Classical Swine Fever Virus

“…The cleaved NS3 protein is produced essentially during the first few hours post-infection. Besides being a protease the NS3 protein has also helicase [103,104] and NTPase activity [105,106]. The NS4A protein is an essential co-factor of the NS3 protease [107].…”