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. 1997 May;71(5):3385–3390. doi: 10.1128/jvi.71.5.3385-3390.1997

Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en.

L V Gubareva 1, M J Robinson 1, R C Bethell 1, R G Webster 1
PMCID: PMC191482  PMID: 9094607

Abstract

Here we report the isolation of influenza virus A/turkey/Minnesota/833/80 (H4N2) with a mutation at the catalytic residue of the neuraminidase (NA) active site, rendering it resistant to the novel NA inhibitor 4-guanidino-Neu5Ac2en (GG167). The resistance of the mutant stems from replacement of one of three invariant arginines (Arg 292-->Lys) that are conserved among all viral and bacterial NAs and participate in the conformational change of sialic acid moiety necessary for substrate catalysis. The Lys292 mutant was selected in vitro after 15 passages at increasing concentrations of GG167 (from 0.1 to 1,000 microM), conditions that earlier gave rise to GG167-resistant mutants with a substitution at the framework residue Glu119. Both types of mutants showed similar degrees of resistance in plaque reduction assays, but the Lys292 mutant was more sensitive to the inhibitor in NA inhibition tests than were mutants bearing a substitution at framework residue 119 (Asp, Ala, or Gly). Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en and Neu5Ac2en) varied among mutants resistant to GG167, being lowest for Lys292 and highest for Asp119. All GG167-resistant mutants demonstrated markedly reduced NA activity, only 3 to 50% of the parental level, depending on the particular amino acid substitution. The catalytic mutant (Lys292) showed a significant change in pH optimum of NA activity, from 5.9 to 5.3. All of the mutant NAs were less stable than the parental enzyme at low pH. Despite their impaired NA activity, the GG167-resistant mutants grew as well as parental virus in Madin-Darby canine kidney cells or in embryonated chicken eggs. However, the infectivity in mice was 500-fold lower for Lys292 than for the parental virus. These findings demonstrate that amino acid substitution in the NA active site at the catalytic or framework residues, followed by multiple passages in vitro, in the presence of increasing concentrations of the NA inhibitor GG167, generates GG167-resistant viruses with reduced NA activity and decreased infectivity in animals.

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Selected References

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  1. Air G. M., Laver W. G. The neuraminidase of influenza virus. Proteins. 1989;6(4):341–356. doi: 10.1002/prot.340060402. [DOI] [PubMed] [Google Scholar]
  2. Aymard-Henry M., Coleman M. T., Dowdle W. R., Laver W. G., Schild G. C., Webster R. G. Influenzavirus neuraminidase and neuraminidase-inhibition test procedures. Bull World Health Organ. 1973;48(2):199–202. [PMC free article] [PubMed] [Google Scholar]
  3. Baker A. T., Varghese J. N., Laver W. G., Air G. M., Colman P. M. Three-dimensional structure of neuraminidase of subtype N9 from an avian influenza virus. Proteins. 1987;2(2):111–117. doi: 10.1002/prot.340020205. [DOI] [PubMed] [Google Scholar]
  4. Bean W. J., Threlkeld S. C., Webster R. G. Biologic potential of amantadine-resistant influenza A virus in an avian model. J Infect Dis. 1989 Jun;159(6):1050–1056. doi: 10.1093/infdis/159.6.1050. [DOI] [PubMed] [Google Scholar]
  5. Blick T. J., Tiong T., Sahasrabudhe A., Varghese J. N., Colman P. M., Hart G. J., Bethell R. C., McKimm-Breschkin J. L. Generation and characterization of an influenza virus neuraminidase variant with decreased sensitivity to the neuraminidase-specific inhibitor 4-guanidino-Neu5Ac2en. Virology. 1995 Dec 20;214(2):475–484. doi: 10.1006/viro.1995.0058. [DOI] [PubMed] [Google Scholar]
  6. Bossart-Whitaker P., Carson M., Babu Y. S., Smith C. D., Laver W. G., Air G. M. Three-dimensional structure of influenza A N9 neuraminidase and its complex with the inhibitor 2-deoxy 2,3-dehydro-N-acetyl neuraminic acid. J Mol Biol. 1993 Aug 20;232(4):1069–1083. doi: 10.1006/jmbi.1993.1461. [DOI] [PubMed] [Google Scholar]
  7. Burmeister W. P., Henrissat B., Bosso C., Cusack S., Ruigrok R. W. Influenza B virus neuraminidase can synthesize its own inhibitor. Structure. 1993 Sep 15;1(1):19–26. doi: 10.1016/0969-2126(93)90005-2. [DOI] [PubMed] [Google Scholar]
  8. Colman P. M., Hoyne P. A., Lawrence M. C. Sequence and structure alignment of paramyxovirus hemagglutinin-neuraminidase with influenza virus neuraminidase. J Virol. 1993 Jun;67(6):2972–2980. doi: 10.1128/jvi.67.6.2972-2980.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Colman P. M. Influenza virus neuraminidase: structure, antibodies, and inhibitors. Protein Sci. 1994 Oct;3(10):1687–1696. doi: 10.1002/pro.5560031007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crennell S. J., Garman E. F., Laver W. G., Vimr E. R., Taylor G. L. Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza virus neuraminidase. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9852–9856. doi: 10.1073/pnas.90.21.9852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gubareva L. V., Bethell R., Hart G. J., Murti K. G., Penn C. R., Webster R. G. Characterization of mutants of influenza A virus selected with the neuraminidase inhibitor 4-guanidino-Neu5Ac2en. J Virol. 1996 Mar;70(3):1818–1827. doi: 10.1128/jvi.70.3.1818-1827.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gubareva L. V., Penn C. R., Webster R. G. Inhibition of replication of avian influenza viruses by the neuraminidase inhibitor 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid. Virology. 1995 Oct 1;212(2):323–330. doi: 10.1006/viro.1995.1489. [DOI] [PubMed] [Google Scholar]
  13. Hayden F. G., Cote K. M., Douglas R. G., Jr Plaque inhibition assay for drug susceptibility testing of influenza viruses. Antimicrob Agents Chemother. 1980 May;17(5):865–870. doi: 10.1128/aac.17.5.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hayden F. G., Treanor J. J., Betts R. F., Lobo M., Esinhart J. D., Hussey E. K. Safety and efficacy of the neuraminidase inhibitor GG167 in experimental human influenza. JAMA. 1996 Jan 24;275(4):295–299. [PubMed] [Google Scholar]
  15. Lentz M. R., Webster R. G., Air G. M. Site-directed mutation of the active site of influenza neuraminidase and implications for the catalytic mechanism. Biochemistry. 1987 Aug 25;26(17):5351–5358. doi: 10.1021/bi00391a020. [DOI] [PubMed] [Google Scholar]
  16. Liu C., Eichelberger M. C., Compans R. W., Air G. M. Influenza type A virus neuraminidase does not play a role in viral entry, replication, assembly, or budding. J Virol. 1995 Feb;69(2):1099–1106. doi: 10.1128/jvi.69.2.1099-1106.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McKimm-Breschkin J. L., McDonald M., Blick T. J., Colman P. M. Mutation in the influenza virus neuraminidase gene resulting in decreased sensitivity to the neuraminidase inhibitor 4-guanidino-Neu5Ac2en leads to instability of the enzyme. Virology. 1996 Nov 1;225(1):240–242. doi: 10.1006/viro.1996.0595. [DOI] [PubMed] [Google Scholar]
  18. Palese P., Compans R. W. Inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-N-trifluoroacetylneuraminic acid (FANA): mechanism of action. J Gen Virol. 1976 Oct;33(1):159–163. doi: 10.1099/0022-1317-33-1-159. [DOI] [PubMed] [Google Scholar]
  19. Paulson J. C., Weinstein J., Dorland L., van Halbeek H., Vliegenthart J. F. Newcastle disease virus contains a linkage-specific glycoprotein sialidase. Application to the localization of sialic acid residues in N-linked oligosaccharides of alpha 1-acid glycoprotein. J Biol Chem. 1982 Nov 10;257(21):12734–12738. [PubMed] [Google Scholar]
  20. Ryan D. M., Ticehurst J., Dempsey M. H. GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is a potent inhibitor of influenza virus in ferrets. Antimicrob Agents Chemother. 1995 Nov;39(11):2583–2584. doi: 10.1128/aac.39.11.2583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ryan D. M., Ticehurst J., Dempsey M. H., Penn C. R. Inhibition of influenza virus replication in mice by GG167 (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is consistent with extracellular activity of viral neuraminidase (sialidase). Antimicrob Agents Chemother. 1994 Oct;38(10):2270–2275. doi: 10.1128/aac.38.10.2270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Staschke K. A., Colacino J. M., Baxter A. J., Air G. M., Bansal A., Hornback W. J., Munroe J. E., Laver W. G. Molecular basis for the resistance of influenza viruses to 4-guanidino-Neu5Ac2en. Virology. 1995 Dec 20;214(2):642–646. doi: 10.1006/viro.1995.0078. [DOI] [PubMed] [Google Scholar]
  23. Taylor N. R., von Itzstein M. Molecular modeling studies on ligand binding to sialidase from influenza virus and the mechanism of catalysis. J Med Chem. 1994 Mar 4;37(5):616–624. doi: 10.1021/jm00031a011. [DOI] [PubMed] [Google Scholar]
  24. Varghese J. N., Colman P. M. Three-dimensional structure of the neuraminidase of influenza virus A/Tokyo/3/67 at 2.2 A resolution. J Mol Biol. 1991 Sep 20;221(2):473–486. doi: 10.1016/0022-2836(91)80068-6. [DOI] [PubMed] [Google Scholar]
  25. Varghese J. N., Epa V. C., Colman P. M. Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase. Protein Sci. 1995 Jun;4(6):1081–1087. doi: 10.1002/pro.5560040606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Varghese J. N., Laver W. G., Colman P. M. Structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 A resolution. Nature. 1983 May 5;303(5912):35–40. doi: 10.1038/303035a0. [DOI] [PubMed] [Google Scholar]
  27. Varghese J. N., McKimm-Breschkin J. L., Caldwell J. B., Kortt A. A., Colman P. M. The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor. Proteins. 1992 Nov;14(3):327–332. doi: 10.1002/prot.340140302. [DOI] [PubMed] [Google Scholar]
  28. Warner T. G., O'Brien J. S. Synthesis of 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and detection of skin fibroblast neuraminidase in normal humans and in sialidosis. Biochemistry. 1979 Jun 26;18(13):2783–2787. doi: 10.1021/bi00580a014. [DOI] [PubMed] [Google Scholar]
  29. Webster R. G. Antigenic hybrids of influenza A viruses with surface antigens to order. Virology. 1970 Nov;42(3):633–642. doi: 10.1016/0042-6822(70)90309-0. [DOI] [PubMed] [Google Scholar]
  30. White C. L., Janakiraman M. N., Laver W. G., Philippon C., Vasella A., Air G. M., Luo M. A sialic acid-derived phosphonate analog inhibits different strains of influenza virus neuraminidase with different efficiencies. J Mol Biol. 1995 Feb 3;245(5):623–634. [PubMed] [Google Scholar]
  31. Woods J. M., Bethell R. C., Coates J. A., Healy N., Hiscox S. A., Pearson B. A., Ryan D. M., Ticehurst J., Tilling J., Walcott S. M. 4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid is a highly effective inhibitor both of the sialidase (neuraminidase) and of growth of a wide range of influenza A and B viruses in vitro. Antimicrob Agents Chemother. 1993 Jul;37(7):1473–1479. doi: 10.1128/aac.37.7.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. von Itzstein M., Wu W. Y., Kok G. B., Pegg M. S., Dyason J. C., Jin B., Van Phan T., Smythe M. L., White H. F., Oliver S. W. Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature. 1993 Jun 3;363(6428):418–423. doi: 10.1038/363418a0. [DOI] [PubMed] [Google Scholar]

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