Neuraminidase is the main target for current influenza drugs. Reduced susceptibility to oseltamivir, the most widely prescribed neuraminidase inhibitor, has been repeatedly reported. The resistance substitutions I223V and S247N, alone or in combination with the major oseltamivir-resistance mutation H275Y, have been observed in 2009 pandemic H1N1 viruses. We overexpressed and purified the ectodomain of wild-type neuraminidase from the A/California/07/2009 (H1N1) influenza virus, as well as variants containing H275Y, I223V, and S247N single mutations and H275Y/I223V and H275Y/S247N double mutations. We performed enzymological and thermodynamic analyses and structurally examined the resistance mechanism. Our results reveal that the I223V or S247N substitution alone confers only a moderate reduction in oseltamivir affinity. In contrast, the major oseltamivir resistance mutation H275Y causes a significant decrease in the enzyme’s ability to bind this drug. Combination of H275Y with an I223V or S247N mutation results in extreme impairment of oseltamivir’s inhibition potency. Our structural analyses revealed that the H275Y substitution has a major effect on the oseltamivir binding pose within the active site while the influence of other studied mutations is much less prominent. Our crystal structures also helped explain the augmenting effect on resistance of combining H275Y with both substitutions.

Department of Biochemistry Faculty of Science Charles University Hlavova 8 128 00 Prague 2 Czech Republic

Department of Organic Chemistry Faculty of Science Charles University Hlavova 8 128 00 Prague 2 Czech Republic

Faculty of Food and Biochemical Technology University of Chemistry and Technology Technická 5 166 28 Prague 6 Czech Republic

The Czech Academy of Sciences Institute of Molecular Genetics Vídeňská 1083 140 00 Prague 4 Czech Republic

The Czech Academy of Sciences Institute of Organic Chemistry and Biochemistry Flemingovo n 2 166 10 Prague 6 Czech Republic

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Klimov A.I., Garten R., Russell C., Barr I.G., Besselaar T.G., Daniels R., Engelhardt O.G., Grohmann G., Itamura S., Kelso A., et al. WHO recommendations for the viruses to be used in the 2012 southern hemisphere influenza vaccine: Epidemiology, antigenic and genetic characteristics of influenza A(H1N1)pdm09, A(H3N2) and B influenza viruses collected from February to September 2011. Vaccine. 2012;30:6461–6471. doi: 10.1016/j.vaccine.2012.07.089. PubMed DOI PMC

WHO. [(accessed on 10 November 2017)]; Available online: http://www.Who.Int/mediacentre/factsheets/fs211/en/

Murray C.J., Lopez A.D., Chin B., Feehan D., Hill K.H. Estimation of potential global pandemic influenza mortality on the basis of vital registry data from the 1918–1920 pandemic: A quantitative analysis. Lancet. 2006;368:2211–2218. doi: 10.1016/S0140-6736(06)69895-4. PubMed DOI

Taubenberger J.K., Kash J.C. Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe. 2010;7:440–451. doi: 10.1016/j.chom.2010.05.009. PubMed DOI PMC

Garten R.J., Davis C.T., Russell C.A., Shu B., Lindstrom S., Balish A., Sessions W.M., Xu X., Skepner E., Deyde V., et al. Antigenic and genetic characteristics of the early isolates of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science. 2009;325:197–201. doi: 10.1126/science.1176225. PubMed DOI PMC

Pebody R., Warburton F., Ellis J., Andrews N., Potts A., Cottrell S., Johnston J., Reynolds A., Gunson R., Thompson C., et al. Effectiveness of seasonal influenza vaccine for adults and children in preventing laboratory-confirmed influenza in primary care in the United Kingdom: 2015/16 end-of-season results. Euro Surveill. 2016;21 doi: 10.2807/1560-7917.ES.2016.21.38.30348. PubMed DOI PMC

Osterholm M.T., Kelley N.S., Sommer A., Belongia E.A. Efficacy and effectiveness of influenza vaccines: A systematic review and meta-analysis. Lancet Infect. Dis. 2012;12:36–44. doi: 10.1016/S1473-3099(11)70295-X. PubMed DOI

Kumar D., Michaels M.G., Morris M.I., Green M., Avery R.K., Liu C., Danziger-Isakov L., Stosor V., Estabrook M., Gantt S., et al. Outcomes from pandemic influenza A H1N1 infection in recipients of solid-organ transplants: A multicentre cohort study. Lancet Infect. Dis. 2010;10:521–526. doi: 10.1016/S1473-3099(10)70133-X. PubMed DOI PMC

Burmeister W.P., Ruigrok R.W., Cusack S. The 2.2 A resolution crystal structure of influenza B neuraminidase and its complex with sialic acid. EMBO J. 1992;11:49–56. PubMed PMC

Varghese J.N., Mckimmbreschkin 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;14:327–332. doi: 10.1002/prot.340140302. PubMed DOI

World Health Organization A revision of the system of nomenclature for influenza viruses: A WHO memorandum. Bull. World Health Organ. 1980;58:585–591. PubMed PMC

Prachanronarong K.L., Ozen A., Thayer K.M., Yilmaz L.S., Zeldovich K.B., Bolon D.N., Kowalik T.F., Jensen J.D., Finberg R.W., Wang J.P., et al. Molecular basis for differential patterns of drug resistance in influenza N1 and N2 neuraminidase. J. Chem. Theory Comput. 2016;12:6098–6108. doi: 10.1021/acs.jctc.6b00703. PubMed DOI

Russell R.J., Haire L.F., Stevens D.J., Collins P.J., Lin Y.P., Blackburn G.M., Hay A.J., Gamblin S.J., Skehel J.J. The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design. Nature. 2006;443:45–49. doi: 10.1038/nature05114. PubMed DOI

Oxford J.S. Chemoprophylaxis and Virus Infections of the Respiratory Tract. Volume 1. CRC Press; Cleveland, OH, USA: 1977. pp. 140–187.

Colman P.M. Neuraminidase inhibitors as antivirals. Vaccine. 2002;20:S55–S58. doi: 10.1016/S0264-410X(02)00132-9. PubMed DOI

Yang X.Y., Steukers L., Forier K., Xiong R.H., Braeckmans K., Van Reeth K., Nauwynck H. A beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus. PLoS ONE. 2014;9:e110026. doi: 10.1371/journal.pone.0110026. PubMed DOI PMC

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., et al. Rational design of potent sialidase-based inhibitors of influenza virus replication. Nature. 1993;363:418–423. doi: 10.1038/363418a0. PubMed DOI

Kim C.U., Lew W., Williams M.A., Liu H., Zhang L., Swaminathan S., Bischofberger N., Chen M.S., Mendel D.B., Tai C.Y., et al. Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: Design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity. J. Am. Chem. Soc. 1997;119:681–690. doi: 10.1021/ja963036t. PubMed DOI

Babu Y.S., Chand P., Bantia S., Kotian P., Dehghani A., El-Kattan Y., Lin T.H., Hutchison T.L., Elliott A.J., Parker C.D., et al. Bcx-1812 (rwj-270201): Discovery of a novel, highly potent, orally active, and selective influenza neuraminidase inhibitor through structure-based drug design. J. Med. Chem. 2000;43:3482–3486. doi: 10.1021/jm0002679. PubMed DOI

Watanabe A., Chang S.C., Kim M.J., Chu D.W., Ohashi Y. Long-acting neuraminidase inhibitor laninamivir octanoate versus oseltamivir for treatment of influenza: A double-blind, randomized, noninferiority clinical trial. Clin. Infect. Dis. 2010;51:1167–1175. doi: 10.1086/656802. PubMed DOI

Varghese J.N., Laver W.G., Colman P.M. Structure of the influenza-virus glycoprotein antigen neuraminidase at 2.9 Å resolution. Nature. 1983;303:35–40. doi: 10.1038/303035a0. PubMed DOI

Colman P.M., Varghese J.N., Laver W.G. Structure of the catalytic and antigenic sites in influenza-virus neuraminidase. Nature. 1983;303:41–44. doi: 10.1038/303041a0. PubMed DOI

Varghese J.N., Smith P.W., Sollis S.L., Blick T.J., Sahasrabudhe A., McKimm-Breschkin J.L., Colman P.M. Drug design against a shifting target: A structural basis for resistance to inhibitors in a variant of influenza virus neuraminidase. Structure. 1998;6:735–746. doi: 10.1016/S0969-2126(98)00075-6. PubMed DOI

Sanjuan R., Nebot M.R., Chirico N., Mansky L.M., Belshaw R. Viral mutation rates. J. Virol. 2010;84:9733–9748. doi: 10.1128/JVI.00694-10. PubMed DOI PMC

Marshall N., Priyamvada L., Ende Z., Steel J., Lowen A.C. Influenza virus reassortment occurs with high frequency in the absence of segment mismatch. PLoS Pathog. 2013;9:e1003421. doi: 10.1371/journal.ppat.1003421. PubMed DOI PMC

Ives J.A.L., Carr J.A., Mendel D.B., Tai C.Y., Lambkin R., Kelly L., Oxford J.S., Hayden F.G., Roberts N.A. The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo. Antivir. Res. 2002;55:307–317. doi: 10.1016/S0166-3542(02)00053-0. PubMed DOI

Meijer A., Lackenby A., Hungnes O., Lina B., van-der-Werf S., Schweiger B., Opp M., Paget J., van-de-Kassteele J., Hay A., et al. Oseltamivir-resistant influenza virus A (H1N1), Europe, 2007–08 season. Emerg. Infect. Dis. 2009;15:552–560. doi: 10.3201/eid1504.181280. PubMed DOI PMC

Bloom J.D., Gong L.I., Baltimore D. Permissive secondary mutations enable the evolution of influenza oseltamivir resistance. Science. 2010;328:1272–1275. doi: 10.1126/science.1187816. PubMed DOI PMC

Abed Y., Baz M., Boivin G. Impact of neuraminidase mutations conferring influenza resistance to neuraminidase inhibitors in the N1 and N2 genetic backgrounds. Antivir. Ther. 2006;11:971–976. PubMed

Collins P.J., Haire L.F., Lin Y.P., Liu J., Russell R.J., Walker P.A., Skehel J.J., Martin S.R., Hay A.J., Gamblin S.J. Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants. Nature. 2008;453:1258–1261. doi: 10.1038/nature06956. PubMed DOI

Vergara-Jaque A., Poblete H., Lee E.H., Schulten K., Gonzalez-Nilo F., Chipot C. Molecular basis of drug resistance in A/H1N1 virus. J. Chem. Inf. Model. 2012;52:2650–2656. doi: 10.1021/ci300343w. PubMed DOI

Woods C.J., Malaisree M., Long B., McIntosh-Smith S., Mulholland A.J. Analysis and assay of oseltamivir-resistant mutants of influenza neuraminidase via direct observation of drug unbinding and rebinding in simulation. Biochemistry. 2013;52:8150–8164. doi: 10.1021/bi400754t. PubMed DOI

Pizzorno A., Abed Y., Bouhy X., Beaulieu E., Mallett C., Russell R., Boivin G. Impact of mutations at residue I223 of the neuraminidase protein on the resistance profile, replication level, and virulence of the 2009 pandemic influenza virus. Antimicrob. Agents Chemother. 2012;56:1208–1214. doi: 10.1128/AAC.05994-11. PubMed DOI PMC

Nguyen H.T., Fry A.M., Gubareva L.V. Neuraminidase inhibitor resistance in influenza viruses and laboratory testing methods. Antivir. Ther. 2012;17:159–173. doi: 10.3851/IMP2067. PubMed DOI

Hurt A.C., Lee R.T., Leang S.K., Cui L., Deng Y.M., Phuah S.P., Caldwell N., Freeman K., Komadina N., Smith D., et al. Increased detection in Australia and Singapore of a novel influenza A(H1N1) 2009 variant with reduced oseltamivir and zanamivir sensitivity due to a S247N neuraminidase mutation. Euro Surveill. 2011;16:2–7. PubMed

Seibert C.W., Rahmat S., Krammer F., Palese P., Bouvier N.M. Efficient transmission of pandemic H1N1 influenza viruses with high-level oseltamivir resistance. J. Virol. 2012;86:5386–5389. doi: 10.1128/JVI.00151-12. PubMed DOI PMC

Paradis E.G., Pinilla L.T., Holder B.P., Abed Y., Boivin G., Beauchemin C.A.A. Impact of the H275Y and I223V mutations in the neuraminidase of the 2009 pandemic influenza virus in vitro and evaluating experimental reproducibility. PLoS ONE. 2015;10:e0126115. doi: 10.1371/journal.pone.0126115. PubMed DOI PMC

McKimm-Breschkin J.L. Influenza neuraminidase inhibitors: Antiviral action and mechanisms of resistance. Influenza Other Respir. 2013;7:25–36. doi: 10.1111/irv.12047. PubMed DOI PMC

Jiang L., Liu P., Bank C., Renzette N., Prachanronarong K., Yilmaz L.S., Caffrey D.R., Zeldovich K.B., Schiffer C.A., Kowalik T.F., et al. A balance between inhibitor binding and substrate processing confers influenza drug resistance. J. Mol. Biol. 2016;428:538–553. doi: 10.1016/j.jmb.2015.11.027. PubMed DOI

Huang L., Cao Y., Zhou J., Qin K., Zhu W., Zhu Y., Yang L., Wang D., Wei H., Shu Y. A conformational restriction in the influenza a virus neuraminidase binding site by R152 results in a combinational effect of I222T and H274Y on oseltamivir resistance. Antimicrob. Agents Chemother. 2014;58:1639–1645. doi: 10.1128/AAC.01848-13. PubMed DOI PMC

Van der Vries E., Veldhuis Kroeze E.J., Stittelaar K.J., Linster M., Van der Linden A., Schrauwen E.J., Leijten L.M., van Amerongen G., Schutten M., Kuiken T., et al. Multidrug resistant 2009 A/H1N1 influenza clinical isolate with a neuraminidase I223R mutation retains its virulence and transmissibility in ferrets. PLoS Pathog. 2011;7:e1002276. doi: 10.1371/journal.ppat.1002276. PubMed DOI PMC

Samson M., Pizzorno A., Abed Y., Boivin G. Influenza virus resistance to neuraminidase inhibitors. Antivir. Res. 2013;98:174–185. doi: 10.1016/j.antiviral.2013.03.014. PubMed DOI

Itzstein M. Influenza Virus Sialidase—A Drug Discovery Target. Springer; Basel, Switzerland: 2012.

Potier M., Mameli L., Belisle M., Dallaire L., Melancon S.B. Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-d-n-acetylneuraminate) substrate. Anal. Biochem. 1979;94:287–296. doi: 10.1016/0003-2697(79)90362-2. PubMed DOI

Barinka C., Rinnova M., Sacha P., Rojas C., Majer P., Slusher B.S., Konvalinka J. Substrate specificity, inhibition and enzymological analysis of recombinant human glutamate carboxypeptidase II. J. Neurochem. 2002;80:477–487. doi: 10.1046/j.0022-3042.2001.00715.x. PubMed DOI

Schmidt T.G., Skerra A. The Strep-tag system for one-step purification and high-affinity detection or capturing of proteins. Nat. Protoc. 2007;2:1528–1535. doi: 10.1038/nprot.2007.209. PubMed DOI

Schmidt T.G.M., Batz L., Bonet L., Carl U., Holzapfel G., Kiem K., Matulewicz K., Niermeier D., Schuchardt I., Stanar K. Development of the Twin-Strep-tag® and its application for purification of recombinant proteins from cell culture supernatants. Protein Expr. Purif. 2013;92:54–61. doi: 10.1016/j.pep.2013.08.021. PubMed DOI

Williams J.W., Morrison J.F. The kinetics of reversible tight-binding inhibition. Methods Enzymol. 1979;63:437–467. PubMed

Dixon M. The determination of enzyme inhibitor constants. Biochem. J. 1953;55:170–171. doi: 10.1042/bj0550170. PubMed DOI PMC

Fukada H., Takahashi K. Enthalpy and heat capacity changes for the proton dissociation of various buffer components in 0.1 M potassium chloride. Proteins. 1998;33:159–166. doi: 10.1002/(SICI)1097-0134(19981101)33:2<159::AID-PROT2>3.0.CO;2-E. PubMed DOI

Mueller U., Darowski N., Fuchs M.R., Forster R., Hellmig M., Paithankar K.S., Puhringer S., Steffien M., Zocher G., Weiss M.S. Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin. J. Synchrotron Radiat. 2012;19:442–449. doi: 10.1107/S0909049512006395. PubMed DOI PMC

Krug M., Weiss M.S., Heinemann U., Mueller U. Xdsapp: A graphical user interface for the convenient processing of diffraction data using XDS. J. Appl. Crystallogr. 2012;45:568–572. doi: 10.1107/S0021889812011715. DOI

Brunger A.T. Free R-value—A novel statistical quantity for assessing the accuracy of crystal-structures. Nature. 1992;355:472–475. doi: 10.1038/355472a0. PubMed DOI

Adams P.D., Afonine P.V., Bunkoczi G., Chen V.B., Davis I.W., Echols N., Headd J.J., Hung L.W., Kapral G.J., Grosse-Kunstleve R.W., et al. Phenix: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D. 2010;66:213–221. doi: 10.1107/S0907444909052925. PubMed DOI PMC

Vagin A., Teplyakov A. An approach to multi-copy search in molecular replacement. Acta Crystallogr. D Biol. Crystallogr. 2000;56:1622–1624. doi: 10.1107/S0907444900013780. PubMed DOI

Albinana C.B., Machara A., Rezacova P., Pachl P., Konvalinka J., Kozisek M. Kinetic, thermodynamic and structural analysis of tamiphosphor binding to neuraminidase of H1N1 (2009) pandemic influenza. Eur. J. Med. Chem. 2016;121:100–109. doi: 10.1016/j.ejmech.2016.05.016. PubMed DOI

Murshudov G.N., Vagin A.A., Dodson E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 1997;53:240–255. doi: 10.1107/S0907444996012255. PubMed DOI

Collaborative Computational Project The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 1994;50:760–763. PubMed

Emsley P., Cowtan K. Coot: Model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 2004;60:2126–2132. doi: 10.1107/S0907444904019158. PubMed DOI

Lovell S.C., Davis I.W., Arendall W.B., 3rd, de Bakker P.I., Word J.M., Prisant M.G., Richardson J.S., Richardson D.C. Structure validation by Calpha geometry: Phi, psi and Cbeta deviation. Proteins. 2003;50:437–450. doi: 10.1002/prot.10286. PubMed DOI

Krissinel E., Henrick K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004;60:2256–2268. doi: 10.1107/S0907444904026460. PubMed DOI

Hawthorne M.F., Young D.C., Wegner P.A. Carbametallic boron hydride derivatives. I. Apparent analogs of ferrocene and ferricinium ion. J. Am. Chem. Soc. 1965;87:1818–1819. doi: 10.1021/ja01086a053. DOI

Betts M.J., Sternberg M.J. An analysis of conformational changes on protein-protein association: Implications for predictive docking. Protein Eng. 1999;12:271–283. doi: 10.1093/protein/12.4.271. PubMed DOI

Rezac J., Riley K.E., Hobza P. S66: A well-balanced database of benchmark interaction energies relevant to biomolecular structures. J. Chem. Theory Comput. 2011;7:2427–2438. doi: 10.1021/ct2002946. PubMed DOI PMC

Collins P.J., Haire L.F., Lin Y.P., Liu J., Russell R.J., Walker P.A., Martin S.R., Daniels R.S., Gregory V., Skehel J.J., et al. Structural basis for oseltamivir resistance of influenza viruses. Vaccine. 2009;27:6317–6323. doi: 10.1016/j.vaccine.2009.07.017. PubMed DOI

Oakley A.J., Barrett S., Peat T.S., Newman J., Streltsov V.A., Waddington L., Saito T., Tashiro M., McKimm-Breschkin J.L. Structural and functional basis of resistance to neuraminidase inhibitors of influenza B viruses. J. Med. Chem. 2010;53:6421–6431. doi: 10.1021/jm100621s. PubMed DOI PMC

Li Q., Qi J., Zhang W., Vavricka C.J., Shi Y., Wei J., Feng E., Shen J., Chen J., Liu D., et al. The 2009 pandemic H1N1 neuraminidase N1 lacks the 150-cavity in its active site. Nat. Struct. Mol. Biol. 2010;17:1266–1268. doi: 10.1038/nsmb.1909. PubMed DOI

Le L., Lee E.H., Hardy D.J., Truong T.N., Schulten K. Molecular dynamics simulations suggest that electrostatic funnel directs binding of tamiflu to influenza N1 neuraminidases. PLoS Comput. Biol. 2010;6 doi: 10.1371/journal.pcbi.1000939. PubMed DOI PMC

Structural and Thermodynamic Analysis of the Resistance Development to Pimodivir (VX-787), the Clinical Inhibitor of Cap Binding to PB2 Subunit of Influenza A Polymerase