{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T02:22:47Z","timestamp":1760235767487,"version":"build-2065373602"},"reference-count":97,"publisher":"MDPI AG","issue":"10","license":[{"start":{"date-parts":[[2021,9,22]],"date-time":"2021-09-22T00:00:00Z","timestamp":1632268800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Influenza virus is a highly contagious virus that causes significant human mortality and morbidity annually. The most effective drugs for treating influenza are the neuraminidase inhibitors, but resistance to these inhibitors has emerged, and additional drug discovery research on neuraminidase and other targets is needed. Traditional methods of neuraminidase production from embryonated eggs are cumbersome, while insect cell derived protein is less reflective of neuraminidase produced during human infection. Herein we describe a method for producing neuraminidase from a human cell line, HEK293-6E, and demonstrate the method by producing the neuraminidase from the 1918 H1N1 pandemic influenza strain. This method produced high levels of soluble neuraminidase expression (&gt;3000 EU\/mL), was enhanced by including a secretion signal from a viral chemokine binding protein, and does not require co-expression of additional proteins. The neuraminidase produced was of sufficient quantity and purity to support high resolution crystal structure determination. The structure solved using this protein conformed to the previously reported structure. Notably the glycosylation at three asparagine residues was superior in quality to that from insect cell derived neuraminidase. This method of production of neuraminidase should prove useful in further studies, such as the characterisation of inhibitor binding.<\/jats:p>","DOI":"10.3390\/v13101893","type":"journal-article","created":{"date-parts":[[2021,9,22]],"date-time":"2021-09-22T22:50:48Z","timestamp":1632351048000},"page":"1893","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Optimisation of Neuraminidase Expression for Use in Drug Discovery by Using HEK293-6E Cells"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0178-4017","authenticated-orcid":false,"given":"Ashley C.","family":"Campbell","sequence":"first","affiliation":[{"name":"Department of Biochemistry, University of Otago, 710 Cumberland St., Dunedin 9016, New Zealand"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8314-113X","authenticated-orcid":false,"given":"John J.","family":"Tanner","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA"},{"name":"Department of Chemistry, University of Missouri, Columbia, MO 65211, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3525-1112","authenticated-orcid":false,"given":"Kurt L.","family":"Krause","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, University of Otago, 710 Cumberland St., Dunedin 9016, New Zealand"},{"name":"Maurice Wilkins Centre for Molecular Biodiscovery, Auckland 1010, New Zealand"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,9,22]]},"reference":[{"key":"ref_1","first-page":"1","article-title":"Overview of influenza viruses","volume":"370","author":"Pleschka","year":"2013","journal-title":"Curr. Top. Microbiol. Immunol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"593","DOI":"10.1177\/135965350701200S07.1","article-title":"Neuraminidase inhibitors and their role in avian and pandemic influenza","volume":"12","author":"Crusat","year":"2007","journal-title":"Antivir. Ther."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1016\/j.ejim.2020.12.010","article-title":"Neuraminidase inhibitors are effective and safe in reducing influenza complications: Meta-analysis of randomized controlled trials","volume":"86","author":"Tejada","year":"2021","journal-title":"Eur. J. Intern. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1336","DOI":"10.1056\/NEJM199910283411802","article-title":"Use of the selective oral neuraminidase inhibitor oseltamivir to prevent influenza","volume":"341","author":"Hayden","year":"1999","journal-title":"N. Engl. J. Med."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"104718","DOI":"10.1016\/j.antiviral.2020.104718","article-title":"Global update on the susceptibilities of human influenza viruses to neuraminidase inhibitors and the cap-dependent endonuclease inhibitor baloxavir, 2017\u20132018","volume":"175","author":"Takashita","year":"2020","journal-title":"Antivir. Res."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1111\/irv.12085","article-title":"Antivirals for influenza: A summary of a systematic review and meta-analysis of observational studies","volume":"7","author":"Santesso","year":"2013","journal-title":"Influenza Other Respir. Viruses"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"520","DOI":"10.1097\/QCO.0000000000000498","article-title":"Neuraminidase inhibitor resistance in influenza: A clinical perspective","volume":"31","author":"Lee","year":"2018","journal-title":"Curr. Opin. Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1354","DOI":"10.3201\/eid1309.070164","article-title":"Reduced sensitivity of influenza A (H5N1) to oseltamivir","volume":"13","author":"Selleck","year":"2007","journal-title":"Emerg. Infect. Dis."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"25","DOI":"10.1111\/irv.12047","article-title":"Influenza neuraminidase inhibitors: Antiviral action and mechanisms of resistance","volume":"7","year":"2013","journal-title":"Influenza Other Respir. Viruses"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1038\/303041a0","article-title":"Structure of the catalytic and antigenic sites in influenza virus neuraminidase","volume":"303","author":"Colman","year":"1983","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"111","DOI":"10.1002\/prot.340020205","article-title":"Three-dimensional structure of neuraminidase of subtype N9 from an avian influenza virus","volume":"2","author":"Baker","year":"1987","journal-title":"Proteins"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"418","DOI":"10.1038\/363418a0","article-title":"Rational design of potent sialidase-based inhibitors of influenza virus replication","volume":"363","author":"Wu","year":"1993","journal-title":"Nature"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1687","DOI":"10.1002\/pro.5560031007","article-title":"Influenza virus neuraminidase: Structure, antibodies, and inhibitors","volume":"3","author":"Colman","year":"1994","journal-title":"Protein Sci. A Publ. Protein Soc."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2456","DOI":"10.1128\/JVI.72.3.2456-2462.1998","article-title":"Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2en-derived inhibitors","volume":"72","author":"Sahasrabudhe","year":"1998","journal-title":"J. Virol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"S55","DOI":"10.1016\/S0264-410X(02)00132-9","article-title":"Neuraminidase inhibitors as antivirals","volume":"20","author":"Colman","year":"2002","journal-title":"Vaccine"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2207","DOI":"10.1021\/jm010528u","article-title":"Structural studies of the resistance of influenza virus neuramindase to inhibitors","volume":"45","author":"Smith","year":"2002","journal-title":"J. Med. Chem."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Von Itzstein, M., and Thomson, R. (2009). Anti-influenza drugs: The development of sialidase inhibitors. Handb. Exp. Pharmacol., 111\u2013154.","DOI":"10.1007\/978-3-540-79086-0_5"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/0166-0934(91)90192-3","article-title":"A new method for the purification of the influenza A virus neuraminidase","volume":"32","author":"Caldwell","year":"1991","journal-title":"J. Virol. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Schmidt, P.M., Attwood, R.M., Mohr, P.G., Barrett, S.A., and McKimm-Breschkin, J.L. (2011). A generic system for the expression and purification of soluble and stable influenza neuraminidase. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0016284"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"241","DOI":"10.1006\/viro.2000.0495","article-title":"The 1918 influenza virus: A killer comes into view","volume":"274","author":"Taubenberger","year":"2000","journal-title":"Virology"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"857","DOI":"10.1007\/s11033-011-0809-z","article-title":"Expression, purification and characterization of low-glycosylation influenza neuraminidase in alpha-1,6-mannosyltransferase defective Pichia pastoris","volume":"39","author":"Yang","year":"2012","journal-title":"Mol. Biol. Rep."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Ma, Y., Lee, C.J., and Park, J.S. (2020). Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds. Antibiotics, 9.","DOI":"10.3390\/antibiotics9090541"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1111\/j.1432-1033.1997.00332.x","article-title":"Protection of mice against a lethal influenza challenge by immunization with yeast-derived recombinant influenza neuraminidase","volume":"247","author":"Martinet","year":"1997","journal-title":"Eur. J. Biochem."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"484","DOI":"10.1016\/j.fob.2013.10.007","article-title":"Construction of a convenient system for easily screening inhibitors of mutated influenza virus neuraminidases","volume":"3","author":"Shigemori","year":"2013","journal-title":"FEBS Open Bio"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"62","DOI":"10.11594\/jtls.02.03.02","article-title":"Expression of Insoluble Influenza Neuraminidase Type 1 (NA1) Protein in Tobacco","volume":"2","author":"Pua","year":"2012","journal-title":"J. Trop. Life Sci."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"10493","DOI":"10.1128\/JVI.00959-08","article-title":"Structural characterization of the 1918 influenza virus H1N1 neuraminidase","volume":"82","author":"Xu","year":"2008","journal-title":"J. Virol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1107\/S2053230X18017892","article-title":"Structure of an Influenza A virus N9 neuraminidase with a tetrabrachion-domain stalk","volume":"75","author":"Streltsov","year":"2019","journal-title":"Acta Cryst. F Struct. Biol. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2216","DOI":"10.1038\/s41564-019-0522-6","article-title":"The neuraminidase of A(H3N2) influenza viruses circulating since 2016 is antigenically distinct from the A\/Hong Kong\/4801\/2014 vaccine strain","volume":"4","author":"Wan","year":"2019","journal-title":"Nat. Microbiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"5770","DOI":"10.1128\/JVI.00180-16","article-title":"Molecular Characterizations of Surface Proteins Hemagglutinin and Neuraminidase from Recent H5Nx Avian Influenza Viruses","volume":"90","author":"Yang","year":"2016","journal-title":"J. Virol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1016\/j.chom.2019.10.002","article-title":"Structural Basis of Protection against H7N9 Influenza Virus by Human Anti-N9 Neuraminidase Antibodies","volume":"26","author":"Zhu","year":"2019","journal-title":"Cell Host Microbe"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1016\/j.immuni.2020.08.015","article-title":"Human Antibodies Targeting Influenza B Virus Neuraminidase Active Site Are Broadly Protective","volume":"53","author":"Madsen","year":"2020","journal-title":"Immunity"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1016\/j.jviromet.2011.08.010","article-title":"Enhanced expression of secretable influenza virus neuraminidase in suspension mammalian cells by influenza virus nonstructural protein 1","volume":"178","author":"Nivitchanyong","year":"2011","journal-title":"J. Virol. Methods"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Ecker, J.W., Kirchenbaum, G.A., Pierce, S.R., Skarlupka, A.L., Abreu, R.B., Cooper, R.E., Taylor-Mulneix, D., Ross, T.M., and Sautto, G.A. (2020). High-Yield Expression and Purification of Recombinant Influenza Virus Proteins from Stably-Transfected Mammalian Cell Lines. Vaccines, 8.","DOI":"10.3390\/vaccines8030462"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1975","DOI":"10.1002\/pro.3918","article-title":"Drivers of recombinant soluble influenza A virus hemagglutinin and neuraminidase expression in mammalian cells","volume":"29","author":"Turner","year":"2020","journal-title":"Protein Sci. A Publ. Protein Soc."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1016\/j.vascn.2004.08.014","article-title":"HEK293 cell line: A vehicle for the expression of recombinant proteins","volume":"51","author":"Thomas","year":"2005","journal-title":"J. Pharmacol. Toxicol. Methods"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"755","DOI":"10.1177\/1087057111409221","article-title":"The quenching effect of flavonoids on 4-methylumbelliferone, a potential pitfall in fluorimetric neuraminidase inhibition assays","volume":"16","author":"Kongkamnerd","year":"2011","journal-title":"J. Biomol. Screen."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"E9","DOI":"10.1093\/nar\/30.2.e9","article-title":"High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells","volume":"30","author":"Durocher","year":"2002","journal-title":"Nucleic Acids Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1107\/S1744309110018166","article-title":"Crystallization and preliminary X-ray analysis of the chemokine-binding protein from orf virus (Poxviridae)","volume":"66","author":"Fleming","year":"2010","journal-title":"Acta Cryst. Sect. F Struct. Biol. Cryst. Commun."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"242","DOI":"10.1038\/nprot.2010.181","article-title":"Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries","volume":"6","author":"Quan","year":"2011","journal-title":"Nat. Protoc."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"17027","DOI":"10.1073\/pnas.0403069101","article-title":"The VASP tetramerization domain is a right-handed coiled coil based on a 15-residue repeat","volume":"101","author":"Kuhnel","year":"2004","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","article-title":"A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding","volume":"72","author":"Bradford","year":"1976","journal-title":"Anal. Biochem."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Leang, S.-K., and Hurt, A.C. (2017). Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals. J. Vis. Exp., 55570.","DOI":"10.3791\/55570"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1016\/0003-2697(79)90362-2","article-title":"Fluorometric assay of neuraminidase with a sodium (4-methylumbelliferyl-alpha-d-N-acetylneuraminate) substrate","volume":"94","author":"Potier","year":"1979","journal-title":"Anal. Biochem."},{"key":"ref_44","unstructured":"Gupta, R., and Brunak, S. (2002). Prediction of glycosylation across the human proteome and the correlation to protein function. Pac. Symp. Biocomput., 310\u2013322."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1007\/978-1-4939-7015-5_6","article-title":"Predicting Secretory Proteins with SignalP","volume":"1611","author":"Nielsen","year":"2017","journal-title":"Methods Mol. Biol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1107\/S0909049502015170","article-title":"Blu-Ice and the Distributed Control System: Software for data acquisition and instrument control at macromolecular crystallography beamlines","volume":"9","author":"McPhillips","year":"2002","journal-title":"J. Synchrotron Radiat."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"885","DOI":"10.1107\/S1600577518003120","article-title":"MX2: A high-flux undulator microfocus beamline serving both the chemical and macromolecular crystallography communities at the Australian Synchrotron","volume":"25","author":"Aishima","year":"2018","journal-title":"J. Synchrotron Radiat."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1107\/S0907444909047374","article-title":"Integration, scaling, space-group assignment and post-refinement","volume":"66","author":"Kabsch","year":"2010","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1204","DOI":"10.1107\/S0907444913000061","article-title":"How good are my data and what is the resolution?","volume":"69","author":"Evans","year":"2013","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"22","DOI":"10.1107\/S0907444909042589","article-title":"Molecular replacement with MOLREP","volume":"66","author":"Vagin","year":"2010","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"491","DOI":"10.1016\/0022-2836(68)90205-2","article-title":"Solvent content of protein crystals","volume":"33","author":"Matthews","year":"1968","journal-title":"J. Mol. Biol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"213","DOI":"10.1107\/S0907444909052925","article-title":"PHENIX: A comprehensive Python-based system for macromolecular structure solution","volume":"66","author":"Adams","year":"2010","journal-title":"Acta Crystallogr. Sect. D"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"658","DOI":"10.1107\/S0021889807021206","article-title":"Phaser crystallographic software","volume":"40","author":"McCoy","year":"2007","journal-title":"J. Appl. Cryst."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"2126","DOI":"10.1107\/S0907444904019158","article-title":"Coot: Model-building tools for molecular graphics","volume":"60","author":"Emsley","year":"2004","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1107\/S2059798316020143","article-title":"Ligand fitting with CCP4","volume":"73","author":"Nicholls","year":"2017","journal-title":"Acta Crystallogr. Sect. D"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1107\/S0907444909042073","article-title":"MolProbity: All-atom structure validation for macromolecular crystallography","volume":"66","author":"Chen","year":"2010","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1107\/S0021889892009944","article-title":"PROCHECK: A program to check the stereochemical quality of protein structures","volume":"26","author":"Laskowski","year":"1993","journal-title":"J. Appl. Crystallogr."},{"key":"ref_58","doi-asserted-by":"crossref","unstructured":"L\u00fctteke, T., and von der Lieth, C.W. (2004). pdb-care (PDB carbohydrate residue check): A program to support annotation of complex carbohydrate structures in PDB files. BMC Bioinform., 5.","DOI":"10.1186\/1471-2105-5-69"},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"223","DOI":"10.1107\/S2059798317001061","article-title":"CheckMyMetal: A macromolecular metal-binding validation tool","volume":"73","author":"Zheng","year":"2017","journal-title":"Acta Cryst. D Struct. Biol."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"1916","DOI":"10.1016\/j.str.2017.10.009","article-title":"Validation of Structures in the Protein Data Bank","volume":"25","author":"Gore","year":"2017","journal-title":"Structure"},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"774","DOI":"10.1016\/j.jmb.2007.05.022","article-title":"Inference of macromolecular assemblies from crystalline state","volume":"372","author":"Krissinel","year":"2007","journal-title":"J. Mol. Biol."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"D355","DOI":"10.1093\/nar\/gkn860","article-title":"PDBsum new things","volume":"37","author":"Laskowski","year":"2009","journal-title":"Nucleic Acids Res."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"2240","DOI":"10.1107\/S0907444904013253","article-title":"The Uppsala Electron-Density Server","volume":"60","author":"Kleywegt","year":"2004","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"148","DOI":"10.1107\/S2059798316018210","article-title":"Polder maps: Improving OMIT maps by excluding bulk solvent","volume":"73","author":"Liebschner","year":"2017","journal-title":"Acta Cryst. D Struct. Biol."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"3359","DOI":"10.1093\/bioinformatics\/btv362","article-title":"IBS: An illustrator for the presentation and visualization of biological sequences","volume":"31","author":"Liu","year":"2015","journal-title":"Bioinformatics"},{"key":"ref_66","doi-asserted-by":"crossref","unstructured":"Fang, Q., and Shen, B. (2010). Optimization of polyethylenimine-mediated transient transfection using response surface methodology design. Electron. J. Biotechnol., 13.","DOI":"10.2225\/vol13-issue5-fulltext-6"},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"61","DOI":"10.1016\/j.pep.2011.02.008","article-title":"Design of Experiment in CHO and HEK transient transfection condition optimization","volume":"78","author":"Bollin","year":"2011","journal-title":"Protein Expr. Purif."},{"key":"ref_68","first-page":"493","article-title":"Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring","volume":"66","author":"Beer","year":"2013","journal-title":"Cytotechnology"},{"key":"ref_69","doi-asserted-by":"crossref","unstructured":"Sun, S., Wang, Q., Zhao, F., Chen, W., and Li, Z. (2011). Glycosylation Site Alteration in the Evolution of Influenza A (H1N1) Viruses. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0022844"},{"key":"ref_70","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1002\/prot.340140302","article-title":"The structure of the complex between influenza virus neuraminidase and sialic acid, the viral receptor","volume":"14","author":"Varghese","year":"1992","journal-title":"Proteins"},{"key":"ref_71","doi-asserted-by":"crossref","first-page":"39","DOI":"10.3389\/fmicb.2019.00039","article-title":"Influenza Virus Neuraminidase Structure and Functions","volume":"10","author":"McAuley","year":"2019","journal-title":"Front Microbiol."},{"key":"ref_72","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1107\/S0907444900019168","article-title":"Geometry of metal-ligand interactions in proteins","volume":"57","author":"Harding","year":"2001","journal-title":"Acta Crystallogr. Sect. D Biol. Crystallogr."},{"key":"ref_73","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1038\/nature05114","article-title":"The structure of H5N1 avian influenza neuraminidase suggests new opportunities for drug design","volume":"443","author":"Russell","year":"2006","journal-title":"Nature"},{"key":"ref_74","doi-asserted-by":"crossref","first-page":"7764","DOI":"10.1021\/ja0723535","article-title":"Remarkable Loop Flexibility in Avian Influenza N1 and Its Implications for Antiviral Drug Design","volume":"129","author":"Amaro","year":"2007","journal-title":"J. Am. Chem. Soc."},{"key":"ref_75","doi-asserted-by":"crossref","first-page":"8431","DOI":"10.1128\/JVI.00638-11","article-title":"Influenza A virus N5 neuraminidase has an extended 150-cavity","volume":"85","author":"Wang","year":"2011","journal-title":"J. Virol."},{"key":"ref_76","doi-asserted-by":"crossref","first-page":"689","DOI":"10.1110\/ps.41801","article-title":"Analysis of inhibitor binding in influenza virus neuraminidase","volume":"10","author":"Smith","year":"2001","journal-title":"Protein Sci. A Publ. Protein Soc."},{"key":"ref_77","doi-asserted-by":"crossref","unstructured":"Vavricka, C.J., Li, Q., Wu, Y., Qi, J., Wang, M., Liu, Y., Gao, F., Liu, J., Feng, E., and He, J. (2011). Structural and functional analysis of laninamivir and its octanoate prodrug reveals group specific mechanisms for influenza NA inhibition. PLoS Pathog., 7.","DOI":"10.1371\/journal.ppat.1002249"},{"key":"ref_78","doi-asserted-by":"crossref","first-page":"2871","DOI":"10.1038\/srep02871","article-title":"Structural basis for a class of nanomolar influenza A neuraminidase inhibitors","volume":"3","author":"Kerry","year":"2013","journal-title":"Sci. Rep."},{"key":"ref_79","doi-asserted-by":"crossref","first-page":"1266","DOI":"10.1038\/nsmb.1909","article-title":"The 2009 pandemic H1N1 neuraminidase N1 lacks the 150-cavity in its active site","volume":"17","author":"Li","year":"2010","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_80","doi-asserted-by":"crossref","first-page":"26","DOI":"10.32607\/20758251-2009-1-2-26-32","article-title":"Influenza Virus Neuraminidase: Structure and Function","volume":"1","author":"Shtyrya","year":"2009","journal-title":"Acta Nat."},{"key":"ref_81","doi-asserted-by":"crossref","first-page":"174","DOI":"10.1089\/vim.2017.0141","article-title":"Influenza Virus: Dealing with a Drifting and Shifting Pathogen","volume":"31","author":"Kim","year":"2018","journal-title":"Viral Immunol."},{"key":"ref_82","unstructured":"(2020). Antiviral drugs for influenza for 2020\u20132021. Med. Lett. Drugs Ther., 62, 169\u2013172."},{"key":"ref_83","doi-asserted-by":"crossref","first-page":"913","DOI":"10.1056\/NEJMoa1716197","article-title":"Baloxavir Marboxil for Uncomplicated Influenza in Adults and Adolescents","volume":"379","author":"Hayden","year":"2018","journal-title":"N. Engl. J. Med."},{"key":"ref_84","doi-asserted-by":"crossref","first-page":"105126","DOI":"10.1016\/j.antiviral.2021.105126","article-title":"Baloxavir-oseltamivir combination therapy inhibits the emergence of resistant substitutions in influenza A virus PA gene in a mouse model","volume":"193","author":"Park","year":"2021","journal-title":"Antivir. Res."},{"key":"ref_85","doi-asserted-by":"crossref","first-page":"670","DOI":"10.1007\/s00018-004-4464-6","article-title":"Hsp70 chaperones: Cellular functions and molecular mechanism","volume":"62","author":"Mayer","year":"2005","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_86","doi-asserted-by":"crossref","unstructured":"\u00d6stbye, H., Gao, J., Martinez, M.R., Wang, H., de Gier, J.W., and Daniels, R. (2020). N-Linked Glycan Sites on the Influenza A Virus Neuraminidase Head Domain Are Required for Efficient Viral Incorporation and Replication. J. Virol., 94.","DOI":"10.1128\/JVI.00874-20"},{"key":"ref_87","doi-asserted-by":"crossref","unstructured":"Bao, D., Xue, R., Zhang, M., Lu, C., Ma, T., Ren, C., Zhang, T., Yang, J., Teng, Q., and Li, X. (2021). N-Linked Glycosylation Plays an Important Role in Budding of Neuraminidase Protein and Virulence of Influenza Viruses. J. Virol., 95.","DOI":"10.1128\/JVI.02042-20"},{"key":"ref_88","doi-asserted-by":"crossref","unstructured":"Kim, P., Jang, Y.H., Kwon, S.B., Lee, C.M., Han, G., and Seong, B.L. (2018). Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs. Viruses, 10.","DOI":"10.3390\/v10040183"},{"key":"ref_89","doi-asserted-by":"crossref","first-page":"524","DOI":"10.1016\/j.bbrc.2016.03.096","article-title":"Core-6 fucose and the oligomerization of the 1918 pandemic influenza viral neuraminidase","volume":"473","author":"Wu","year":"2016","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_90","doi-asserted-by":"crossref","first-page":"749","DOI":"10.1016\/j.bbrc.2008.12.139","article-title":"Active 1918 pandemic flu viral neuraminidase has distinct N-glycan profile and is resistant to trypsin digestion","volume":"379","author":"Wu","year":"2009","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_91","doi-asserted-by":"crossref","first-page":"104","DOI":"10.1080\/07391102.2013.855142","article-title":"Interface dynamics explain assembly dependency of influenza neuraminidase catalytic activity","volume":"33","author":"Wallnoefer","year":"2015","journal-title":"J. Biomol. Struct. Dyn."},{"key":"ref_92","doi-asserted-by":"crossref","first-page":"5011","DOI":"10.1128\/jvi.69.8.5011-5017.1995","article-title":"Steps in maturation of influenza A virus neuraminidase","volume":"69","author":"Saito","year":"1995","journal-title":"J. Virol."},{"key":"ref_93","doi-asserted-by":"crossref","first-page":"9638","DOI":"10.1073\/pnas.1003587107","article-title":"Function of human Rh based on structure of RhCG at 2.1 \u212b","volume":"107","author":"Gruswitz","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_94","doi-asserted-by":"crossref","first-page":"1179","DOI":"10.1016\/j.jmb.2007.03.007","article-title":"Crystal Structure of a Thermally Stable Rhodopsin Mutant","volume":"372","author":"Standfuss","year":"2007","journal-title":"J. Mol. Biol."},{"key":"ref_95","doi-asserted-by":"crossref","first-page":"85","DOI":"10.1038\/nature12533","article-title":"X-ray structure of dopamine transporter elucidates antidepressant mechanism","volume":"503","author":"Penmatsa","year":"2013","journal-title":"Nature"},{"key":"ref_96","doi-asserted-by":"crossref","unstructured":"Li, J., zu Dohna, H., Cardona, C.J., Miller, J., and Carpenter, T.E. (2011). Emergence and Genetic Variation of Neuraminidase Stalk Deletions in Avian Influenza Viruses. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0014722"},{"key":"ref_97","doi-asserted-by":"crossref","unstructured":"Li, Y., Chen, S., Zhang, X., Fu, Q., Zhang, Z., Shi, S., Zhu, Y., Gu, M., Peng, D., and Liu, X. (2014). A 20-amino-acid deletion in the neuraminidase stalk and a five-amino-acid deletion in the NS1 protein both contribute to the pathogenicity of H5N1 avian influenza viruses in mallard ducks. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0095539"}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/10\/1893\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:03:24Z","timestamp":1760166204000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/13\/10\/1893"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,9,22]]},"references-count":97,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2021,10]]}},"alternative-id":["v13101893"],"URL":"https:\/\/doi.org\/10.3390\/v13101893","relation":{},"ISSN":["1999-4915"],"issn-type":[{"type":"electronic","value":"1999-4915"}],"subject":[],"published":{"date-parts":[[2021,9,22]]}}}