Yonsei Med J.  2012 Sep;53(5):886-893.

N-Linked Glycosylation in the Hemagglutinin of Influenza A Viruses

• Kim JI
• Park MS

Abstract

Since the 1918 influenza A virus (IAV) pandemic, H1N1 viruses have circulated in human populations. The hemagglutinin (HA) of IAV determines viral antigenicity and often undergoes N-linked glycosylation (NLG) at several sites. Interestingly, structural analysis of the 1918 and 2009 H1N1 pandemic viruses revealed antigenic similarities attributable to the conserved epitopes and the NLG statuses of their HA proteins. NLG of the globular head of HA is known to modulate the antigenicity, fusion activity, virulence, receptor-binding specificity, and immune evasion of IAV. In addition, the HA of IAV often retains additional mutations. These supplemental mutations compensate for the attenuation of viral properties resulting from the introduced NLG. In human H1N1 viruses, the number and location of NLG sites has been regulated in accordance with the antigenic variability of the NLG-targeted antibody-binding site. The relationship between the NLG and the antigenic variance in HA appears to be stably controlled in the viral context.

Keyword

Glycosylation; hemagglutinin; influenza virus; pandemic

MeSH Terms

Antigenic Variation
Epitopes
Glycosylation*
Head
Hemagglutinins*
Humans
Immune Evasion
Influenza A virus*
Influenza A Virus, H1N1 Subtype
Influenza, Human*
Orthomyxoviridae
Pandemics
Sensitivity and Specificity
Virulence
Epitopes
Hemagglutinins

Figure

• 

  Fig. 1 NLG of the globular head of H1 HA during each time period. Using the HA crystal structure of the A/California/04/2009 virus (PDB ID: 3LZG),27 the possible N-linked glycans on the head of H1 HA from Table 1 were evaluated. Potential glycosites (red) near the RBS (yellow) are indicated by the H3 numbering. NLG, N-linked glycosylation; HA, hemagglutinin; RBS, receptor-binding site.

• 

  Fig. 2 The prediction of potential glycosites on the head of H1 HA. The 3-D crystal structure of the HA trimer of the A/California/04/2009 virus (PDB ID: 3LZG)27 was modified by the PyMol program to locate potential glycosites (yellow) at the membrane-distal region with top (A) and tilt (B) views. Among the five glycosites, four of them were identified in the SA antigenic site, and glycosite 131 (H3 numbering) was located at the vicinity of the receptor-binding site (RBS in magenta). HA, hemagglutinin; SA, sialic acid.

• 

  Fig. 3 The antigenic variability and the number of N-linked glycans on the head (Sa antigenic site) of H1 HA. The variance in the amino acids (indicated at the left) in the Sa antigenic site of H1 HA was modified from the work of Sun, et al.33 published in 2012. For comparison, the numbers of N-linked glycans (indicated at the right) in the membrane-distal region of H1 HA were collected from the works of Wei, et al.19 and Sun, et al.20 and were supplemented with the HA sequences of the hH1N1 viruses available from the National Center for Biotechnology Information of the United States. NLG, N-linked glycosylation; HA, hemagglutinin.

Reference

1. Taubenberger JK, Kash JC. Influenza virus evolution, host adaptation, and pandemic formation. Cell Host Microbe. 2010. 7:440–451.

2. Salomon R, Webster RG. The influenza virus enigma. Cell. 2009. 136:402–410.

3. Tong S, Li Y, Rivailler P, Conrardy C, Castillo DA, Chen LM, et al. A distinct lineage of influenza A virus from bats. Proc Natl Acad Sci U S A. 2012. 109:4269–4274.

4. Bouvier NM, Palese P. The biology of influenza viruses. Vaccine. 2008. 26:Suppl 4. D49–D53.

5. WHO. Global influenza programme (GIP). Accessed in May 31, 2012. Available from: www.who.int/influenza/en/index.html.

6. Barr IG, McCauley J, Cox N, Daniels R, Engelhardt OG, Fukuda K, et al. Epidemiological, antigenic and genetic characteristics of seasonal influenza A(H1N1), A(H3N2) and B influenza viruses: basis for the WHO recommendation on the composition of influenza vaccines for use in the 2009-2010 Northern Hemisphere season. Vaccine. 2010. 28:1156–1167.

7. de Jong JC, Beyer WE, Palache AM, Rimmelzwaan GF, Osterhaus AD. Mismatch between the 1997/1998 influenza vaccine and the major epidemic A(H3N2) virus strain as the cause of an inadequate vaccine-induced antibody response to this strain in the elderly. J Med Virol. 2000. 61:94–99.

8. Carrat F, Flahault A. Influenza vaccine: the challenge of antigenic drift. Vaccine. 2007. 25:6852–6862.

9. Plotkin JB, Dushoff J, Levin SA. Hemagglutinin sequence clusters and the antigenic evolution of influenza A virus. Proc Natl Acad Sci U S A. 2002. 99:6263–6268.

10. Palese P. Making better influenza virus vaccines? Emerg Infect Dis. 2006. 12:61–65.

11. Kreijtz JH, Fouchier RA, Rimmelzwaan GF. Immune responses to influenza virus infection. Virus Res. 2011. 162:19–30.

12. Corti D, Voss J, Gamblin SJ, Codoni G, Macagno A, Jarrossay D, et al. A neutralizing antibody selected from plasma cells that binds to group 1 and group 2 influenza A hemagglutinins. Science. 2011. 333:850–856.

13. Fleishman SJ, Whitehead TA, Ekiert DC, Dreyfus C, Corn JE, Strauch EM, et al. Computational design of proteins targeting the conserved stem region of influenza hemagglutinin. Science. 2011. 332:816–821.

14. Krause JC, Tsibane T, Tumpey TM, Huffman CJ, Basler CF, Crowe JE Jr. A broadly neutralizing human monoclonal antibody that recognizes a conserved, novel epitope on the globular head of the influenza H1N1 virus hemagglutinin. J Virol. 2011. 85:10905–10908.

15. Whittle JR, Zhang R, Khurana S, King LR, Manischewitz J, Golding H, et al. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin. Proc Natl Acad Sci U S A. 2011. 108:14216–14221.

16. Kilbourne ED, Couch RB, Kasel JA, Keitel WA, Cate TR, Quarles JH, et al. Purified influenza A virus N2 neuraminidase vaccine is immunogenic and non-toxic in humans. Vaccine. 1995. 13:1799–1803.

17. Neirynck S, Deroo T, Saelens X, Vanlandschoot P, Jou WM, Fiers W. A universal influenza A vaccine based on the extracellular domain of the M2 protein. Nat Med. 1999. 5:1157–1163.

18. Nayak B, Kumar S, DiNapoli JM, Paldurai A, Perez DR, Collins PL, et al. Contributions of the avian influenza virus HA, NA, and M2 surface proteins to the induction of neutralizing antibodies and protective immunity. J Virol. 2010. 84:2408–2420.

19. Wei CJ, Boyington JC, Dai K, Houser KV, Pearce MB, Kong WP, et al. Cross-neutralization of 1918 and 2009 influenza viruses: role of glycans in viral evolution and vaccine design. Sci Transl Med. 2010. 2:24ra21.

20. Sun S, Wang Q, Zhao F, Chen W, Li Z. Glycosylation site alteration in the evolution of influenza A (H1N1) viruses. PLoS One. 2011. 6:e22844.

21. Schulze IT. Effects of glycosylation on the properties and functions of influenza virus hemagglutinin. J Infect Dis. 1997. 176:Suppl 1. S24–S28.

22. Aytay S, Schulze IT. Single amino acid substitutions in the hemagglutinin can alter the host range and receptor binding properties of H1 strains of influenza A virus. J Virol. 1991. 65:3022–3028.

23. Gambaryan AS, Marinina VP, Tuzikov AB, Bovin NV, Rudneva IA, Sinitsyn BV, et al. Effects of host-dependent glycosylation of hemagglutinin on receptor-binding properties on H1N1 human influenza A virus grown in MDCK cells and in embryonated eggs. Virology. 1998. 247:170–177.

24. Deshpande KL, Fried VA, Ando M, Webster RG. Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proc Natl Acad Sci U S A. 1987. 84:36–40.

25. Vigerust DJ, Shepherd VL. Virus glycosylation: role in virulence and immune interactions. Trends Microbiol. 2007. 15:211–218.

26. Skehel JJ, Stevens DJ, Daniels RS, Douglas AR, Knossow M, Wilson IA, et al. A carbohydrate side chain on hemagglutinins of Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc Natl Acad Sci U S A. 1984. 81:1779–1783.

27. Xu R, Ekiert DC, Krause JC, Hai R, Crowe JE Jr, Wilson IA. Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science. 2010. 328:357–360.

28. Fukuyama S, Kawaoka Y. The pathogenesis of influenza virus infections: the contributions of virus and host factors. Curr Opin Immunol. 2011. 23:481–486.

29. Luo M. Influenza virus entry. Adv Exp Med Biol. 2012. 726:201–221.

30. Gerhard W. The role of the antibody response in influenza virus infection. Curr Top Microbiol Immunol. 2001. 260:171–190.

31. Okuno Y, Isegawa Y, Sasao F, Ueda S. A common neutralizing epitope conserved between the hemagglutinins of influenza A virus H1 and H2 strains. J Virol. 1993. 67:2552–2558.

32. Corti D, Suguitan AL Jr, Pinna D, Silacci C, Fernandez-Rodriguez BM, Vanzetta F, et al. Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine. J Clin Invest. 2010. 120:1663–1673.

33. Sun S, Wang Q, Zhao F, Chen W, Li Z. Prediction of biological functions on glycosylation site migrations in human influenza H1N1 viruses. PLoS One. 2012. 7:e32119.

34. Das SR, Puigbò P, Hensley SE, Hurt DE, Bennink JR, Yewdell JW. Glycosylation focuses sequence variation in the influenza A virus H1 hemagglutinin globular domain. PLoS Pathog. 2010. 6:e1001211.

35. Das SR, Hensley SE, David A, Schmidt L, Gibbs JS, Puigbò P, et al. Fitness costs limit influenza A virus hemagglutinin glycosylation as an immune evasion strategy. Proc Natl Acad Sci U S A. 2011. 108:E1417–E1422.

36. Schwarz F, Aebi M. Mechanisms and principles of N-linked protein glycosylation. Curr Opin Struct Biol. 2011. 21:576–582.

37. Alberts B, Wilson JH, Hunt T. Molecular biology of the cell. 2008. 5th ed. New York: Garland Science.

38. Ohuchi M, Ohuchi R, Feldmann A, Klenk HD. Regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety. J Virol. 1997. 71:8377–8384.

39. Wagner R, Wolff T, Herwig A, Pleschka S, Klenk HD. Interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics. J Virol. 2000. 74:6316–6323.

40. Tsuchiya E, Sugawara K, Hongo S, Matsuzaki Y, Muraki Y, Li ZN, et al. Effect of addition of new oligosaccharide chains to the globular head of influenza A/H2N2 virus haemagglutinin on the intracellular transport and biological activities of the molecule. J Gen Virol. 2002. 83(Pt 5):1137–1146.

41. Vigerust DJ, Ulett KB, Boyd KL, Madsen J, Hawgood S, McCullers JA. N-linked glycosylation attenuates H3N2 influenza viruses. J Virol. 2007. 81:8593–8600.

42. Caton AJ, Brownlee GG, Yewdell JW, Gerhard W. The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (H1 subtype). Cell. 1982. 31(2 Pt 1):417–427.

43. Reading PC, Pickett DL, Tate MD, Whitney PG, Job ER, Brooks AG. Loss of a single N-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice. Respir Res. 2009. 10:117.

44. Tate MD, Job ER, Brooks AG, Reading PC. Glycosylation of the hemagglutinin modulates the sensitivity of H3N2 influenza viruses to innate proteins in airway secretions and virulence in mice. Virology. 2011. 413:84–92.

45. Tate MD, Brooks AG, Reading PC. Specific sites of N-linked glycosylation on the hemagglutinin of H1N1 subtype influenza A virus determine sensitivity to inhibitors of the innate immune system and virulence in mice. J Immunol. 2011. 187:1884–1894.

46. Rudneva IA, Ilyushina NA, Timofeeva TA, Webster RG, Kaverin NV. Restoration of virulence of escape mutants of H5 and H9 influenza viruses by their readaptation to mice. J Gen Virol. 2005. 86(Pt 10):2831–2838.

47. O'Donnell CD, Vogel L, Wright A, Das SR, Wrammert J, Li GM, et al. Antibody Pressure by a Human Monoclonal Antibody Targeting the 2009 Pandemic H1N1 Virus Hemagglutinin Drives the Emergence of a Virus with Increased Virulence in Mice. MBio. 2012. 3:pii: e00120-12.

48. Reading PC, Tate MD, Pickett DL, Brooks AG. Glycosylation as a target for recognition of influenza viruses by the innate immune system. Adv Exp Med Biol. 2007. 598:279–292.

49. Rogers GN, D'Souza BL. Receptor binding properties of human and animal H1 influenza virus isolates. Virology. 1989. 173:317–322.

50. Connor RJ, Kawaoka Y, Webster RG, Paulson JC. Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology. 1994. 205:17–23.

51. Rogers GN, Paulson JC, Daniels RS, Skehel JJ, Wilson IA, Wiley DC. Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature. 1983. 304:76–78.

52. Matrosovich M, Tuzikov A, Bovin N, Gambaryan A, Klimov A, Castrucci MR, et al. Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J Virol. 2000. 74:8502–8512.

53. Nobusawa E, Ishihara H, Morishita T, Sato K, Nakajima K. Change in receptor-binding specificity of recent human influenza A viruses (H3N2): a single amino acid change in hemagglutinin altered its recognition of sialyloligosaccharides. Virology. 2000. 278:587–596.

54. Crecelius DM, Deom CM, Schulze IT. Biological properties of a hemagglutinin mutant of influenza virus selected by host cells. Virology. 1984. 139:164–177.

55. Deom CM, Caton AJ, Schulze IT. Host cell-mediated selection of a mutant influenza A virus that has lost a complex oligosaccharide from the tip of the hemagglutinin. Proc Natl Acad Sci U S A. 1986. 83:3771–3775.

56. Gamblin SJ, Haire LF, Russell RJ, Stevens DJ, Xiao B, Ha Y, et al. The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science. 2004. 303:1838–1842.

57. Wang W, Lu B, Zhou H, Suguitan AL Jr, Cheng X, Subbarao K, et al. Glycosylation at 158N of the hemagglutinin protein and receptor binding specificity synergistically affect the antigenicity and immunogenicity of a live attenuated H5N1 A/Vietnam/1203/2004 vaccine virus in ferrets. J Virol. 2010. 84:6570–6577.

58. Gao Y, Zhang Y, Shinya K, Deng G, Jiang Y, Li Z, et al. Identification of amino acids in HA and PB2 critical for the transmission of H5N1 avian influenza viruses in a mammalian host. PLoS Pathog. 2009. 5:e1000709.

59. Wang CC, Chen JR, Tseng YC, Hsu CH, Hung YF, Chen SW, et al. Glycans on influenza hemagglutinin affect receptor binding and immune response. Proc Natl Acad Sci U S A. 2009. 106:18137–18142.