Purification and Characterization of West Nile Virus Nucleoside Triphosphatase (NTPase)/Helicase: Evidence for Dissociation of the NTPase and Helicase Activities of the Enzyme

Abstract

West Nile virus (WN virus), a member of the family of Flaviviridae, is a small enveloped single-stranded RNA positivestrand virus. The viral genome encodes a monocistronic polyprotein of 3,430 amino acids that is processed into three structural proteins, protein M, capsid protein C, and glycoprotein E, and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (10, 11, 52). The processing of the polyprotein is carried out by the host signal peptidase associated with the endoplasmic reticulum and viral proteases. The polyprotein of WN virus and its processing are similar to those of the pestivirus-and hepatitis C virus (HCV)-related viruses (36,44,55). Sequence analysis of the nonstructural region of WN virus polyprotein revealed numerous conserved motifs specific for serine proteases, RNA helicase with intrinsic RNA-stimulated nucleoside triphosphatase (NTPase) localized in the NS3 protein, and RNA-directed RNA polymerase associated with the NS5 protein (3, 16, 17). These predictions were partially confirmed by verifying the enzymatic properties of a COOH-terminal segment of NS3 released from a membrane fraction of infected cells by subtilisin (54). Further information about the interactions and functions of the viral proteins was obtained by using synthesized recombined proteins of Flaviviridae or HCV-related viruses (19,23,47,49,50).Due to multiple enzymatic and biological activities associated with NS3, this protein appears to be the most promising target for antiviral agents. The protease activity of NS3 is the subject of numerous studies and has been well characterized previously (24, 31). However, despite the importance of enzymes modulating RNA structures in diverse metabolic processes and their critical role in the life cycles of viruses whose genomes are composed of RNA, only limited information on the viral helicases or helicase-like enzymes is available.Helicases are capable of enzymatically unwinding duplex DNA or RNA structures by disrupting the hydrogen bonds that keep the two strands together (18,21). The unwinding reaction is accomplished by the hydrolysis of ␥-phosphate of nucleotide triphosphate (NTP). Based on sequence comparisons, the viral helicases have been divided into three superfamilies. The WN virus helicase is a member of superfamily II (SFII), which includes helicases from bymovirus, potyvirus, pestivirus, herpesvirus, poxvirus, HCV, and other Flaviviridae (22). All of the helicases contain seven highly conserved amino acid sequences (motifs I to VII) that are located on the surfaces of domains 1 and 2 of the three-domain enzymes. The involvement of the motifs in NTP binding, NTP hydrolysis, and the binding of polynucleotide(s) was well explained by resolving the crystal structures of several enzymes (25,57). However, these structures did not elucidate the mechanisms coupling ATP hydrolysis to the unwinding reaction. Although numerous studies about the quantification of the interaction of SFII helicases with NTP and polynucleotides were performed, uniform resul...

Understanding Helicases as a Means of Virus Control

“…Many related viruses including DFV [70,71], WNV [72], and JEV [73] have each been shown to unwind RNA in a 3′ to 5′ direction. Although nucleic acid stimulates ATP hydrolysis catalyzed by flavivirus, pestivirus, and hepacivirus helicases, different nucleic acids stimulate the enzymes differently.…”

NS4A regulates the ATPase activity of the NS3 helicase: a novel cofactor role of the non-structural protein NS4A from West Nile virus

“…The NS proteins are expressed only in the infected host cell and are not packaged into mature particles. Similar to other flaviviruses, the fulllength NS3 peptide sequence of West Nile virus (WNV) represents a multifunctional protein in which the Nterminal 184 residues encode serine proteinase (NS3pro) and the C-terminal 440 residues code for an RNA triphosphatase, an NTPase and an RNA helicase (NS3hel) (Benarroch et al, 2004;Borowski et al, 2001). NS3hel is a member of the DEAH/D-box family within the helicase superfamily 2 (Gorbalenya et al, 1989;Li et al, 1999;Luking et al, 1998;Luo et al, 2008).…”

Synthesis and Evaluation of ATP-Binding Site Directed Potential Inhibitors of Nucleoside Triphosphatases/ Helicases and Polymerases of Hepatitis C and other Selected Flaviviridae Viruses

“…The WNV NTPase/helicase was purified from the medium harvested from cultures of WNV-infected Vero E6 cells as previously described (Borowski et al, 2001).…”

“…Recently tested 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide-5′-triphosphate (ribavirin 5′-triphosphate) appeared a moderate HCV and WNV NTPase/helicase inhibitor (Borowski et al, 2000). In our previous studies we demonstrated that FSBA inhibited efficiently both the NTPase and helicase of the WNV NTPase/helicase, and only slightly the activities of the HCV enzyme (Borowski et al, 2001). It is noteworthy that this inhibition occurred after preincubation of FSBA with the enzymes.…”

“…The released strand (26-mer) was 5′-end labelled with [γ-32 P]ATP using T4 polynucleotide kinase, according to the procedure described previously (Borowski et al, 2001). …”