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. 1993 Oct;67(10):6152–6158. doi: 10.1128/jvi.67.10.6152-6158.1993

Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the related pestivirus and flavivirus enzymes.

J A Suzich 1, J K Tamura 1, F Palmer-Hill 1, P Warrener 1, A Grakoui 1, C M Rice 1, S M Feinstone 1, M S Collett 1
PMCID: PMC238037  PMID: 8396675

Abstract

Sequence motifs within the nonstructural protein NS3 of members of the Flaviviridae family suggest that this protein possesses nucleoside triphosphatase (NTPase) and RNA helicase activity. The RNA-stimulated NTPase activity of this protein from prototypic members of the Pestivirus and Flavivirus genera has recently been established and enzymologically characterized. Here, we experimentally demonstrate that the NS3 protein from a member of the third genus of Flaviviridae, human hepatitis C virus (HCV), also possesses a polynucleotide-stimulated NTPase activity. Characterization of the purified HCV NTPase activity showed that it exhibited reaction condition optima with respect to pH, MgCl2, and salt identical to those of the representative pestivirus and flavivirus enzymes. However, each NTPase also possessed several unique properties when compared with one another. Notably, the profile of polynucleotide stimulation of the NTPase activity was distinct for the three enzymes. The HCV NTPase was the only one whose activity was significantly enhanced by a deoxyribopolynucleotide. Additional distinguishing features among the three enzymes relating to the kinetic properties of their NTPase activities are discussed. These studies provide a foundation for investigation of the putative RNA helicase activity of these proteins and for further study of the role of the NS3 proteins of members of the Flaviviridae in the replication cycle of these viruses.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker J. C. Bovine viral diarrhea virus: a review. J Am Vet Med Assoc. 1987 Jun 1;190(11):1449–1458. [PubMed] [Google Scholar]
  2. Bazan J. F., Fletterick R. J. Detection of a trypsin-like serine protease domain in flaviviruses and pestiviruses. Virology. 1989 Aug;171(2):637–639. doi: 10.1016/0042-6822(89)90639-9. [DOI] [PubMed] [Google Scholar]
  3. Chambers T. J., Hahn C. S., Galler R., Rice C. M. Flavivirus genome organization, expression, and replication. Annu Rev Microbiol. 1990;44:649–688. doi: 10.1146/annurev.mi.44.100190.003245. [DOI] [PubMed] [Google Scholar]
  4. Choo Q. L., Kuo G., Weiner A. J., Overby L. R., Bradley D. W., Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science. 1989 Apr 21;244(4902):359–362. doi: 10.1126/science.2523562. [DOI] [PubMed] [Google Scholar]
  5. Collett M. S., Anderson D. K., Retzel E. Comparisons of the pestivirus bovine viral diarrhoea virus with members of the flaviviridae. J Gen Virol. 1988 Oct;69(Pt 10):2637–2643. doi: 10.1099/0022-1317-69-10-2637. [DOI] [PubMed] [Google Scholar]
  6. Collett M. S. Molecular genetics of pestiviruses. Comp Immunol Microbiol Infect Dis. 1992 Jul;15(3):145–154. doi: 10.1016/0147-9571(92)90087-8. [DOI] [PubMed] [Google Scholar]
  7. Dahle J., Liess B. A review on classical swine fever infections in pigs: epizootiology, clinical disease and pathology. Comp Immunol Microbiol Infect Dis. 1992 Jul;15(3):203–211. doi: 10.1016/0147-9571(92)90093-7. [DOI] [PubMed] [Google Scholar]
  8. Feinstone S. M., Alter H. J., Dienes H. P., Shimizu Y., Popper H., Blackmore D., Sly D., London W. T., Purcell R. H. Non-A, non-B hepatitis in chimpanzees and marmosets. J Infect Dis. 1981 Dec;144(6):588–598. doi: 10.1093/infdis/144.6.588. [DOI] [PubMed] [Google Scholar]
  9. Gorbalenya A. E., Donchenko A. P., Koonin E. V., Blinov V. M. N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases. Nucleic Acids Res. 1989 May 25;17(10):3889–3897. doi: 10.1093/nar/17.10.3889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. A conserved NTP-motif in putative helicases. Nature. 1988 May 5;333(6168):22–22. doi: 10.1038/333022a0. [DOI] [PubMed] [Google Scholar]
  11. Gorbalenya A. E., Koonin E. V., Donchenko A. P., Blinov V. M. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 1989 Jun 26;17(12):4713–4730. doi: 10.1093/nar/17.12.4713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Grakoui A., McCourt D. W., Wychowski C., Feinstone S. M., Rice C. M. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J Virol. 1993 May;67(5):2832–2843. doi: 10.1128/jvi.67.5.2832-2843.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grakoui A., Wychowski C., Lin C., Feinstone S. M., Rice C. M. Expression and identification of hepatitis C virus polyprotein cleavage products. J Virol. 1993 Mar;67(3):1385–1395. doi: 10.1128/jvi.67.3.1385-1395.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hodgman T. C. A new superfamily of replicative proteins. Nature. 1988 May 5;333(6168):22–23. doi: 10.1038/333022b0. [DOI] [PubMed] [Google Scholar]
  15. Keegan K., Collett M. S. Use of bacterial expression cloning to define the amino acid sequences of antigenic determinants on the G2 glycoprotein of Rift Valley fever virus. J Virol. 1986 May;58(2):263–270. doi: 10.1128/jvi.58.2.263-270.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laín S., Martín M. T., Riechmann J. L., García J. A. Novel catalytic activity associated with positive-strand RNA virus infection: nucleic acid-stimulated ATPase activity of the plum pox potyvirus helicaselike protein. J Virol. 1991 Jan;65(1):1–6. doi: 10.1128/jvi.65.1.1-6.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Laín S., Riechmann J. L., García J. A. RNA helicase: a novel activity associated with a protein encoded by a positive strand RNA virus. Nucleic Acids Res. 1990 Dec 11;18(23):7003–7006. doi: 10.1093/nar/18.23.7003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Laín S., Riechmann J. L., Martín M. T., García J. A. Homologous potyvirus and flavivirus proteins belonging to a superfamily of helicase-like proteins. Gene. 1989 Oct 30;82(2):357–362. doi: 10.1016/0378-1119(89)90063-2. [DOI] [PubMed] [Google Scholar]
  19. Linder P., Lasko P. F., Ashburner M., Leroy P., Nielsen P. J., Nishi K., Schnier J., Slonimski P. P. Birth of the D-E-A-D box. Nature. 1989 Jan 12;337(6203):121–122. doi: 10.1038/337121a0. [DOI] [PubMed] [Google Scholar]
  20. Miller R. H., Purcell R. H. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and flaviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2057–2061. doi: 10.1073/pnas.87.6.2057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Rosenfeld S. J., Yoshimoto K., Kajigaya S., Anderson S., Young N. S., Field A., Warrener P., Bansal G., Collett M. S. Unique region of the minor capsid protein of human parvovirus B19 is exposed on the virion surface. J Clin Invest. 1992 Jun;89(6):2023–2029. doi: 10.1172/JCI115812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schmid S. R., Linder P. D-E-A-D protein family of putative RNA helicases. Mol Microbiol. 1992 Feb;6(3):283–291. doi: 10.1111/j.1365-2958.1992.tb01470.x. [DOI] [PubMed] [Google Scholar]
  23. Tamura J. K., Gellert M. Characterization of the ATP binding site on Escherichia coli DNA gyrase. Affinity labeling of Lys-103 and Lys-110 of the B subunit by pyridoxal 5'-diphospho-5'-adenosine. J Biol Chem. 1990 Dec 5;265(34):21342–21349. [PubMed] [Google Scholar]
  24. Tamura J. K., Warrener P., Collett M. S. RNA-stimulated NTPase activity associated with the p80 protein of the pestivirus bovine viral diarrhea virus. Virology. 1993 Mar;193(1):1–10. doi: 10.1006/viro.1993.1097. [DOI] [PubMed] [Google Scholar]
  25. Thompson N. E., Hager D. A., Burgess R. R. Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase. Biochemistry. 1992 Aug 4;31(30):7003–7008. doi: 10.1021/bi00145a019. [DOI] [PubMed] [Google Scholar]
  26. Walker J. E., Saraste M., Runswick M. J., Gay N. J. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1982;1(8):945–951. doi: 10.1002/j.1460-2075.1982.tb01276.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Warrener P., Tamura J. K., Collett M. S. RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria. J Virol. 1993 Feb;67(2):989–996. doi: 10.1128/jvi.67.2.989-996.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wassarman D. A., Steitz J. A. RNA splicing. Alive with DEAD proteins. Nature. 1991 Feb 7;349(6309):463–464. doi: 10.1038/349463a0. [DOI] [PubMed] [Google Scholar]
  29. Wengler G., Wengler G. The carboxy-terminal part of the NS 3 protein of the West Nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an RNA-stimulated NTPase. Virology. 1991 Oct;184(2):707–715. doi: 10.1016/0042-6822(91)90440-m. [DOI] [PubMed] [Google Scholar]
  30. Wiskerchen M., Collett M. S. Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing. Virology. 1991 Sep;184(1):341–350. doi: 10.1016/0042-6822(91)90850-b. [DOI] [PubMed] [Google Scholar]

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