Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase

Virology Journal

Pascal Gagneux³  Robert T Schooley²  Nissi M Varki³  Hui-Wen Chen¹  Hooman P Senaati³  Xing-Quan Zhang²  Miriam Cohen³ 

[1] Present address: School of Veterinary Medicine, National Taiwan University, 1 Sec. 4 Roosevelt Rd, Taipei 10617, Taiwan;Division of Infectious Disease, University of California San Diego, 9500 Gilman Dr, La Jolla 92093, California, USA;Department of Cellular and Molecular Medicine, University of California San Diego, 9500 Gilman Dr, La Jolla 92093, California, USA

关键词: Saliva;    Infection;    Neuraminidase;    Mucus;    Sialic acids;    Influenza A;   

Others  :  820167
DOI  :  10.1186/1743-422X-10-321

 received in 2013-09-17, accepted in 2013-10-14,  发布年份 2013

【 摘 要 】

Background

Influenza A virus (IAV) neuraminidase (NA) cleaves sialic acids (Sias) from glycans. Inhibiting NA with oseltamivir suppresses both viral infection, and viral release from cultured human airway epithelial cells. The role of NA in viral exit is well established: it releases budding virions by cleaving Sias from glycoconjugates on infected cells and progeny virions. The role of NA in viral entry remains unclear. Host respiratory epithelia secrete a mucus layer rich in heavily sialylated glycoproteins; these could inhibit viral entry by mimicking sialylated receptors on the cell surface. It has been suggested that NA allows influenza to penetrate the mucus by cleaving these sialylated decoys, but the exact mechanism is not yet established.

Methods

We tested IAV interaction with secreted mucus using frozen human trachea/bronchus tissue sections, and bead-bound purified human salivary mucins (HSM) and purified porcine submaxillary mucins (PSM). The protective effect of mucus was analyzed using MDCK cells coated with purified HSM and PSM with known Sia content. Oseltamivir was used to inhibit NA activity, and the fluorescent reporter substrate, 4MU-Neu5Ac, was used to quantify NA activity.

Results

IAV binds to the secreted mucus layer of frozen human trachea/bronchus tissues in a Sia dependent manner. HSM inhibition of IAV infection is Sia dose-dependent, but PSM cannot inhibit infection of underlying cells. HSM competitively inhibits NA cleavage of 4MU-Neu5Ac, reporter substrate. Human IAV effectively cleaves Sias from HSM but not from PSM, and binds to HSM but not to PSM.

Conclusion

IAV interacts with human mucus on frozen tissue sections and mucus-coated beads. Inhibition of IAV infection by sialylated human mucus is dose-dependent, and enhanced when NA is inhibited with oseltamivir. Thus NA cleaves sialylated decoys during initial stages of infection. Understanding IAV interactions with host mucins is a promising new avenue for drug development.

【 授权许可】

   
2013 Cohen et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Andrewes CH: Nomenclature of viruses. Nature 1954, 173:620-621.
• [2]Burnet FM: Mucoproteins in relation to virus action. Physiol Rev 1951, 31:131-150.
• [3]Fahy JV, Dickey BF: Airway mucus function and dysfunction. N Engl J Med 2010, 363:2233-2247.
• [4]Cone RA: Barrier properties of mucus. Adv Drug Deliv Rev 2009, 61:75-85.
• [5]Duez JM, Sixt N, Pechinot A: Influenza virus infection: don’t forget the role of the mucociliary system! J Antimicrob Chemother 2009, 63:421-422.
• [6]Matrosovich M, Klenk HD: Natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding. Rev Med Virol 2003, 13:85-97.
• [7]Lillehoj ER, Kim KC: Airway mucus: its components and function. Arch Pharm Res 2002, 25:770-780.
• [8]Knowles MR, Boucher RC: Mucus clearance as a primary innate defense mechanism for mammalian airways. J Clin Invest 2002, 109:571-577.
• [9]von Itzstein M, Thomson R: Anti-influenza drugs: the development of sialidase inhibitors. Handb Exp Pharmacol 2009, 189:111-154.
• [10]Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD: Neuraminidase is important for the initiation of influenza virus infection in human airway epithelium. J Virol 2004, 78:12665-12667.
• [11]Huang RT, Rott R, Wahn K, Klenk HD, Kohama T: The function of the neuraminidase in membrane fusion induced by myxoviruses. Virology 1980, 107:313-319.
• [12]Huang RT, Dietsch E, Rott R: Further studies on the role of neuraminidase and the mechanism of low pH dependence in influenza virus-induced membrane fusion. J Gen Virol 1985, 66:295-301.
• [13]Zalewska A, Zwierz K, Zolkowski K, Gindzienski A: Structure and biosynthesis of human salivary mucins. Acta Biochim Pol 2000, 47:1067-1079.
• [14]Klein A, Carnoy C, Wieruszeski JM, Strecker G, Strang AM, van Halbeek H, Roussel P, Lamblin G: The broad diversity of neutral and sialylated oligosaccharides derived from human salivary mucins. Biochemistry 1992, 31:6152-6165.
• [15]Thornton DJ, Rousseau K, McGuckin MA: Structure and function of the polymeric mucins in airways mucus. Annu Rev Physiol 2008, 70:459-486.
• [16]Gagneux P, Cheriyan M, Hurtado-Ziola N, van der Linden EC, Anderson D, McClure H, Varki A, Varki NM: Human-specific regulation of alpha 2-6-linked sialic acids. J Biol Chem 2003, 278:48245-48250.
• [17]Gerken TA, Jentoft N: Structure and dynamics of porcine submaxillary mucin as determined by natural abundance carbon-13 NMR spectroscopy. Biochemistry 1987, 26:4689-4699.
• [18]Varki NM, Strobert E, Dick EJJ, Benirschke K, Varki A: Biomedical differences between human and nonhuman hominids: potential roles for uniquely human aspects of sialic acid biology. Annu Rev Pathol 2011, 6:365-393.
• [19]Van Poucke SG, Nicholls JM, Nauwynck HJ, Van Reeth K: Replication of avian, human and swine influenza viruses in porcine respiratory explants and association with sialic acid distribution. Virol J 2010, 7:38. BioMed Central Full Text
• [20]Liu X, Luo M, Guo C, Yan Z, Wang Y, Engelhardt JF: Comparative biology of rAAV transduction in ferret, pig and human airway epithelia. Gene Ther 2007, 14:1543-1548.
• [21]Matrosovich MN, Matrosovich TY, Gray T, Roberts NA, Klenk HD: Human and avian influenza viruses target different cell types in cultures of human airway epithelium. Proc Natl Acad Sci U S A 2004, 101:4620-4624.
• [22]Ozawa M, Kawaoka Y: Cross talk between animal and human influenza viruses. Annu Rev Anim Biosci 2013, 1:21-42.
• [23]Nicholls JM, Bourne AJ, Chen H, Guan Y, Peiris JS: Sialic acid receptor detection in the human respiratory tract: evidence for widespread distribution of potential binding sites for human and avian influenza viruses. Respir Res 2007, 8:73. BioMed Central Full Text
• [24]Jayaraman A, Chandrasekaran A, Viswanathan K, Raman R, Fox JG, Sasisekharan R: Decoding the distribution of glycan receptors for human-adapted influenza a viruses in ferret respiratory tract. PLoS One 2012, 7:e27517.
• [25]Cohen M, Varki NM, Jankowski MD, Gagneux P: Using unfixed, frozen tissues to study natural mucin distribution. J Vis Exp 2012, 67:e3928.
• [26]Varki A, Kannagi R, Toole BP: Glycosylation Changes in Cancer: Chapter 44. In Essentials of Glycobiology. 2nd edition. Edited by Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 2009:617-632.
• [27]Gahmberg CG, Andersson LC: Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride. J Biol Chem 1977, 252:5888-5894.
• [28]White MR, Helmerhorst EJ, Ligtenberg A, Karpel M, Tecle T, Siqueira WL, Oppenheim FG, Hartshorn KL: Multiple components contribute to ability of saliva to inhibit influenza viruses. Oral Microbiol Immunol 2009, 24:18-24.
• [29]Sung JC, Van Wynsberghe AW, Amaro RE, Li WW, McCammon JA: Role of secondary sialic acid binding sites in influenza N1 neuraminidase. J Am Chem Soc 2010, 132:2883-2885.
• [30]Nayak DP, Reichl U: Neuraminidase activity assays for monitoring MDCK cell culture derived influenza virus. J Virol Methods 2004, 122:9-15.
• [31]Nadano D, Iwasaki M, Endo S, Kitajima K, Inoue S, Inoue Y: A naturally occurring deaminated neuraminic acid, 3-deoxy-D-glycero-D-galacto-nonulosonic acid (KDN): Its unique occurrence at the nonreducing ends of oligosialyl chains in polysialoglycoprotein of rainbow trout eggs. J Biol Chem 1986, 261:11550-11557.
• [32]Angata T, Varki A: Chemical diversity in the sialic acids and related alpha-keto acids: an evolutionary perspective. Chem Rev 2002, 102:439-469.
• [33]Koerner I, Matrosovich MN, Haller O, Staeheli P, Kochs G: Altered receptor specificity and fusion activity of the haemagglutinin contribute to high virulence of a mouse-adapted influenza a virus. J Gen Virol 2012, 93:970-979.
• [34]Childs RA, Palma AS, Wharton S, Matrosovich T, Liu Y, Chai W, Campanero-Rhodes MA, Zhang Y, Eickmann M, Kiso M, Hay A, Matrosovich M, Feizi T: Receptor-binding specificity of pandemic influenza A (H1N1) 2009 virus determined by carbohydrate microarray. Nat Biotechnol 2009, 27:797-799.
• [35]Suzuki Y, Matsunaga M, Matsumoto M: N-Acetylneuraminyllactosylceramide, GM3-NeuAc, a new influenza a virus receptor which mediates the adsorption-fusion process of viral infection: binding specificity of influenza virus A/Aichi/2/68 (H3N2) to membrane-associated GM3 with different molecular species of sialic acid. J Biol Chem 1985, 260:1362-1365.
• [36]Matrosovich MN, Gambaryan AS, Tuzikov AB, Byramova NE, Mochalova LV, Golbraikh AA, Shenderovich MD, Finne J, Bovin NV: Probing of the receptor-binding sites of the H1 and H3 influenza A and influenza B virus hemagglutinins by synthetic and natural sialosides. Virology 1993, 196:111-121.
• [37]Tamura S, Kurata T: Defense mechanisms against influenza virus infection in the respiratory tract mucosa. Jpn J Infect Dis 2004, 57:236-247.
• [38]Vareille M, Kieninger E, Edwards MR, Regamey N: The airway epithelium: soldier in the fight against respiratory viruses. Clin Microbiol Rev 2011, 24:210-229.
• [39]Lillehoj EP, Kato K, Lu W, Kim KC: Cellular and molecular biology of airway mucins. Int Rev Cell Mol Biol 2013, 303:139-202.
• [40]Hartshorn KL, White MR, Mogues T, Ligtenberg T, Crouch E, Holmskov U: Lung and salivary scavenger receptor glycoprotein-340 contribute to the host defense against influenza A viruses. Am J Physiol Lung Cell Mol Physiol 2003, 285:L1066-L1076.
• [41]Meindl P, Tuppy H: 2-Deoxy-2,3-dehydrosialic acids. II. Competitive inhibition of Vibrio cholerae neuraminidase by 2-deoxy-2,3-dehydro-N-acylneuraminic acids. Hoppe Seylers Z Physiol Chem 1969, 350:1088-1092.
• [42]Schauer R, Haverkamp J, Wember M, Kamerling JP, Vliegenthart JF: N-acetyl-9-O-L-lactylneuraminic acid, a new acylneuraminic acid from bovine submandibular gland. Eur J Biochem 1976, 62:237-242.
• [43]Stevens J, Blixt O, Paulson JC, Wilson IA: Glycan microarray technologies: tools to survey host specificity of influenza viruses. Nat Rev Microbiol 2006, 4:857-864.
• [44]Monti E, Bonten E, D’Azzo A, Bresciani R, Venerando B, Borsani G, Schauer R, Tettamanti G: Sialidases in vertebrates: a family of enzymes tailored for several cell functions. Adv Carbohydr Chem Biochem 2010, 64:403-479.
• [45]Toovey S, Rayner C, Prinssen E, Chu T, Donner B, Thakrar B, Dutkowski R, Hoffmann G, Breidenbach A, Lindemann L, Carey E, Boak L, Gieschke R, Sacks S, Solsky J, Small I, Reddy D: Assessment of neuropsychiatric adverse events in influenza patients treated with oseltamivir: a comprehensive review. Drug Saf 2008, 31:1097-1114.
• [46]Smith SM, Gums JG: Antivirals for influenza: strategies for use in pediatrics. Paediatr Drugs 2010, 12:285-299.
• [47]Holmes EC, Ghedin E, Halpin RA, Stockwell TB, Zhang XQ, Fleming R, Davey R, Benson CA, Mehta S, Taplitz R, Liu YT, Brouwer KC, Wentworth DE, Lin X, Schooley RT: Extensive geographical mixing of 2009 human H1N1 influenza A virus in a single university community. J Virol 2011, 85:6923-6929.
• [48]Lu Q, Zhang XQ, Pond SL, Reed S, Schooley RT, Liu YT: Detection in 2009 of the swine origin influenza A (H1N1) virus by a subtyping microarray. J Clin Microbiol 2009, 47:3060-3061.
• [49]Banda K, Gregg CJ, Chow R, Varki NM, Varki A: Metabolism of vertebrate amino sugars with N-glycolyl groups: mechanisms underlying gastrointestinal incorporation of the non-human sialic acid xeno-autoantigen N-glycolylneuraminic acid. J Biol Chem 2012, 287:28852-28864.
• [50]Faure M, Moennoz D, Montigon F, Fay LB, Breuille D, Finot PA, Ballevre O, Boza J: Development of a rapid and convenient method to purify mucins and determine their in vivo synthesis rate in rats. Anal Biochem 2002, 307:244-251.
• [51]Hara S, Yamaguchi M, Takemori Y, Furuhata K, Ogura H, Nakamura M: Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography. Anal Biochem 1989, 179:162-166.
• [52]Kimble B, Nieto GR, Perez DR: Characterization of influenza virus sialic acid receptors in minor poultry species. Virol J 2010, 7:365. BioMed Central Full Text