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...

To enhance the inhibitory potential of 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) vs hepatitis C virus (HCV) NTPase/helicase, ribavirin-5'-triphosphate (ribavirin-TP) was synthesized and investigated. Ribavirin-TP was prepared with the use of modified Yoshikawa-Ludwig-Mishra-Broom procedure (cf. Mishra & Broom, 1991, J. Chem. Soc., Chem. Commun, 1276-1277) involving phosphorylation of unprotected nucleoside. Kinetic analysis revealed enhanced inhibitory potential of ribavirin-TP (IC50=40 microM) as compared to ribavirin (IC50 > 500 microM). Analysis of the inhibition type by means of graphical methods showed a competitive type of inhibition with respect to ATP. In view of the relatively low specificity towards nucleoside-5'-triphosphates (NTP) of the viral NTPase/helicases, it could not be ruled out that the investigated enzyme hydrolyzed the ribavirin-TP to less potent products. Investigations on non- hydrolysable analogs of ribavirin-TP or ribavirin-5'-diphosphate (ribavirin-DP) are currently under way.

RNA nucleoside triphosphatases (NTPase)/helicases represent a large family of proteins that are ubiquitously distributed over a wide range of organisms. The enzymes play essential role in cell development and differentiation, and some of them are involved in transcription and replication of viral single-stranded RNA genomes. The enzymatic activities of a NTPase/helicase were also detected in the carboxyl-terminal non-structural protein 3 (NS3) of members of the Flaviviridae family. The crucial role of the enzyme for the virus life cycle was demonstrated in knock out experiments and by using NTPase/helicase specific inhibitors. This makes the enzyme an attractive target for development of Flaviviridae-specific antiviral therapies. This review will summarize our knowledge about the function and structure of the enzyme, update the spectrum of inhibitors of the enzymatic activities of the NTPase/helicase and describe the different mechanisms by which the compounds act. Some of the compounds reviewed herein could show potential utility as antiviral agents against Flaviviridae viruses.

In the presented study the ribavirin-TP--an established inhibitor of the NTPase activity of the superfamily NTPase/helicases II--was investigated as an inhibitor of the unwinding activity of the hepatitis C virus (HCV) NTPase/helicase. The kinetics of the reaction revealed that ribavirin-TP reduces the turnover number of the helicase reaction by a mechanism that does not correspond to that of the inhibition of the NTPase activity. Our results suggest that derivatives of ribavirin-TP with enhanced stability towards hydrolytic attack may be effective inhibitors of the enzyme.

No abstract