Loss and gain of N-linked glycosylation sites in globular headand stem of HA found in A/H3N2 flu fatal and severe casesduring 2013 Tunisia flu seasonal survey

Awatef El Moussi • Mohamed Ali Ben Hadj Kacem •

Amine Slim

Received: 20 May 2013 / Accepted: 10 October 2013

� Springer Science+Business Media New York 2013

Abstract Glycosylation on the globular head of the

hemagglutinin (HA) protein of influenza virus acts as an

important target for recognition and destruction of virus by

innate immune proteins of the collectin family. In the

current study, we have characterized the dynamic amino

acid changes at N-linked glycosylation sites of full length

sequences of HA genes of 5 A/H3N2 Tunisian strains

isolates from mild, severe, and fatal cases. Compared to the

reference strain, A/Perth/16/2009 substitutions in potential

N-glycosylation sites were observed in 5 HA genes at five

different positions (45, 124, 128, 144, and 145) generating

the losses and gains of N-linked glycosylation sites. Also

the mutation N145S was presented in the receptor-binding

site of all segments analyzed. Point mutations in several

positions in the gene encoding the H3 of Tunisian strains

were shown to ablate a glycan attachment site and also loss

of a potential glycosylation site. The relation between these

mutations and virulence of influenza A/H3N2 virus needed

to be verified in the further experiments.

Keywords Influenza A/H3N2 � N-linked

glycosylation site � Receptor-binding site � Virulence

Abbreviations

HA Hemagglutinin

NGS N-linked glycosylation sites

RBS Receptor-binding site

S Serine

T Threonine

D Aspartic acid

G Glycine

K Lysine

N Asparagine

Introduction

Influenza A virus escapes from host immune responses by

changing the antigenicity of hemagglutinin (HA) and

neuraminidase (NA) both gradually (antigenic drift) and

abruptly (antigenic shift) [1]. Although the escape from host

immune responses occurs through changes in amino acids at

antigen that is recognized by antibody [2–4], it has been

proposed that the attachment of an oligosaccharide to the

N-glycosylation sites (NGS), which is the asparagine resi-

due of the sequon, in the globular head region also con-

tributes to the escape. This is based on observations in

experimental studies that some NGS attached to the globular

head region interfere with the binding of antigen to antibody

by masking the surface of HA [5, 6]. After the emergence of

influenza A viruses in the human population, the number of

NGS in the globular head region of HA has increased con-

tinuously for several decades. It has been speculated that the

addition or/and the loss of NGS to the globular head region

of HA has conferred selective advantages to the virus by

preventing the binding of antibodies to antigenic sites. NGS

in the fibrous stem are involved in the fusion activity of HA

[7]. Structural complexity of NGS is positively and nega-

tively correlated with HA-receptor binding specificity and

affinity, respectively [8]. The trend toward accumulation of

sites for NGS can be seen in both the H3N2 and H1N1

lineages [9]. NGS are usually conserved among influenza A

viruses [10]. However, NGS in the globular head of H3N2

A. El Moussi (&) � M. A. Ben Hadj Kacem � A. Slim

National Influenza Centre-Tunis, Microbiology Laboratory,

Charles Nicolle’s Hospital, Bvd 9 avril, Tunis, Tunisia

e-mail: awatefbio@ymail.com

123

Virus Genes

DOI 10.1007/s11262-013-0993-0

virus, the number of, which was 2 in 1968, increased up to 6

or 7 in 2013-present for H3N2 virus [11]. It has been

reported that removal of NGS of H3N2 influenza viruses led

to a farther increase in virulence, characterized by enhanced

virus replication, pulmonary inflammation, and vascular

leak [12]. In the present study, we have analyzed the HA

genes of Influenza A/H3N2 virus identified during

2012–2013 and compared them with the reference and

vaccine strains in order to identify the dynamic change of

amino acid in the NGS. Also we show the association

between the changes in specific domain of HA and the

severity of the infection of this virus.

Materials and methods

Clinical samples from five patients with confirmed influ-

enza A/H3N2 infection were obtained and analyzed

Table 1 Clinical information

of patients infected by influenza

A/H3N2 viruses

Influenza A/H3N2

strains

Date of

sampling

Accession

numbers

Clinical

information

Age

(year)

Sex

A/Tunisia/1199/2013 13/02/2013 KC999473 Mild case 36 Female

A/Tunisia/1987/2013 06/02/2013 KC999477 Severe case

(care unit)

1 Male

A/Tunisia/2334/2013 23/02/2013 KC999474 Severe case

(care unit)

20 Female

A/Tunisia/2494/2013 29/01/2013 KC999475 Fatal case 27 Male

A/Tunisia/2635/2013 29/01/2013 KC999476 Mild case 3 Female

Fig. 1 Amino acid comparison between the HA1 domains of H3N2

isolates and the reference and vaccine strains (A/Perth/16/2009 and

A/Victoria/361/2011) showing the substitutions in the potential

N-linked glycosylation sequons (NXS/T) presented at the positions:

45, 124, 128 and 144 resulting by add or loss of N-glycosylation site.

Also it showed substitutions at positions 145 and 198 of RSB of HA

Virus Genes

123

(Table 1). Viral RNA was extracted from 140 ll of throat

swab specimens using the QIAmp Viral RNA Mini kit�

(QIAGEN). In order to detect and assign the A/H3N2

strains isolated from patients, a real-time PCR CDC pro-

tocol was used [13]. RT-PCR assays were set up in order to

amplify full length H3 gene by reverse transcription-PCR

using the One-step RT-PCR kit� (QIAGEN). The purified

PCR products were obtained using the QIAquick PCR

Purification Mini kit� (QIAGEN). Nucleotide sequences

were determined using BigDye Terminator (version 3.1)

cycle sequencing standard kit (Applied Biosystems) and

the Applied Biosystems Sequencer (3130 Genetic Ana-

lyzer). Sequences were assembled and aligned with the

vaccine strain: A/Victoria/361/2011 and the reference

strain A/Perth/16/2009 using the SeqScape (version 2.6)

and MEGA version 4.0 programs [14]. Potential NGS were

predicted using nine artificial neural networks with the

NetNGlyc server 1.0 [15]. The N-glycosite prediction tool

at Los Alamos [16] was used to visualize the fraction of

isolates possessing certain glycosylation sites along the

aligned sequences. The nucleotide sequence data from this

study were deposited in the GenBank in the NIH genetic

sequence database, accession numbers listed in Table 1.

Results

We analyzed 5 full-length H3 in the globular head and stem

of HA sequences from mild, severe, and fatal cases. Sub-

sequent analysis of N-linked glycosylation sites (N-X-S/T)

was found in conserved sites at N8, N22, N38, N483, and

N485 in the fibrous stem of HA; and at N63, N133, N165,

and N246 in the globular head of HA among H3N2 viruses

studied. Substitutions in potential NGS (amino acids Asn-

X-Ser/Thr, where X is not Asp or Pro) were observed in 5

HA genes at 5 different positions (45, 124, 128,144, and

145) (Fig. 1). The amino acid substitution generating a

potential NGS (NNS) was observed at amino acid site 45

(S to N) in two viruses isolated from fatal and severe cases

(A/Tunisia/2494/2013 and A/Tunisia/1987/2013). In addi-

tion, compared to the reference strain A/Perth/16/2009, we

noted a loss of NGS at the position 45 in three segments

isolated from two severe cases and one mild case. Also we

observed in these strains an addition of NGS (NSS) at the

position 144 (K to N). Interestingly, an amino acid sub-

stitution generated of a loss of NGS was observed at amino

acid site 124 (serine to asparagine) in one strain (A/Tuni-

sia/2494/2013) isolated from 27 year old fatal case who

suffered from severe pneumonia with acute respiratory

syndrome. Moreover, a substitution of amino acid (threo-

nine to alanine) generating a loss of NGS at the position

128 was detected in virus (A/Tunisia/1987/2013) isolated

from 1-year-old severe case hospitalized in the care unit

suffering from severe pneumonia. Also a substitution (K to

D) at position 144 was observed in three strains analyzed

generating a loss of NGS.

Discussion

Collectins are a family of collagenous lectin molecules that

are calcium-dependent carbohydrate binding proteins pre-

viously shown to bind enveloped viruses [17–19]. The

function of these proteins is believed to be as a first line of

defense against both bacterial and viral pathogens by bind-

ing to carbohydrate moieties on the pathogen surface. In

support of this concept, children with a deficiency in man-

nose binding lectin are more prone to a variety of serious

infections [20, 21]. In fact, the NGS of HA are involved in

several functions, such as folding of ectodomain, fusion

activity of HA, shielding of antigenic sites, proteolytic

activity of HA, recognition by collections, and receptor

binding. Attachment of oligosaccharide side chains to

asparagines residues of the nascent polypeptide chain is a

common co-translational modification affecting both struc-

tural and functional features of the respective glycoproteins

[22, 23]. With the HA protein of influenza viruses, there is

considerable variation in NGS located in the area of the

globular head domain, while those linked to the stem of the

molecule are highly conserved [24]. Here we report the gain

and the lacking of the conserved NGS (compared to refer-

ence strain: A/Perth/16/2009) at the position 45. The asso-

ciation between the loss of the NGS in this position and the

virulence of the virus is the most documented [25].The loss

has been detected in viruses isolated from two severe cases

at position 45 from the stem of the HA and at positions 122

and 126 from the globular head of the HA isolated from one

fatal case and one severe case, respectively. The mutant HA

with loss of NGS was isolated from patients with a clinical

complication (pneumonia). In an experimental approach,

deletion of HA glycosylation sites from influenza A virus,

led to increased virulence in mice [26]. These observations

suggest that theses mutations may play a role direct or

indirect of the pathogenicity of this virus. In fact, the results

obtained in the present study may reflect those obtained in a

previous study where the loss of potential NGS on the head

of HA is a critical factor modulating the virulence of

influenza viruses for mice [26].

Studies by Suzuki [27] demonstrated that the gains of

NGS may be involved in antigenic changes of HA in human

H3N2 virus. Glycosylation sites located on the globular

head of HA1 and alterations in this site could presumably

influence other functions mediated by the viral HA [28]. It

has become clear that the addition of glycosylation in many

viruses is also a mechanism for viral evasion and persis-

tence. A/Tunisia/2494/2013 and A/Tunisia/2635/2013

Virus Genes

123

stains gained a NGS at position 144, thereby masking the

supposed ‘‘key’’ site for antigenic change [29]. Bragstad

et al. [30], suggested that the substitutions at predicted NSG

at position 144 in HA antigenic site A might have con-

tributed to the increased infectivity of the reasserted

A/H3N2 viruses of the 2003–2004 season, causing an epi-

demic in Denmark. In summary, our study identified 4

changes (generating 4 losses of potential NGS) out of 10 in

mild cases (4/10, 40 %) and 6 changes (generating 4 losses

and 2 additions of NGS) in severe cases (6/10, 60 %). Our

data highlight the importance of specific sites of NGS on the

H3 HA in determining of possible role to inducting to more

virulence of the virus. Changes in the 140–146 region,

antigenic site A, are characteristic for antigenically distinct

viruses of epidemic significance [28]. Post infection sera

contain antibodies that recognize mainly not only residue

144 but also residue 198 and 157 on site B [31, 32]. We

report the substitution A198S in two viruses (A/Tunisia/

2494/2013 and A/Tunisia/1987/2013) isolated from fatal

and severe cases. This substitution at this position may lead

the virus to escape from host immune system [31, 32]. The

globular head of HA contains the receptor-binding site

(RBS), a shallow pocket of highly conserved amino acids

that interact with sialylated receptors. Here, we report the

substitution of the amino acid asparagine (N) to serine (S) at

the position 145 of RBS in all HA gene sequenced. Several

studies have identified specific residues directly within, as

well as in the vicinity of the RBS, which are critical for

sialic acid binding and receptor specificity [33, 34]. In

particular, amino acid 145 [35, 36] is important in modu-

lating the receptor specificity of H3 subtype viruses. Our

findings imply that the variation on potential NGS and also

on RBS may lead to a further increase in virulence of

influenza A/H3N2. An explanation by additional experi-

mental approach of the role of NGS in disease is an

important concept to better understand the biology of

influenza A viruses in the lung of humans.

Acknowledgments The authors gratefully acknowledge WHO

Centre for influenza in London for the Collaboration and Centers for

disease Control and prevention (CDC). The authors also thank Ines

Laaribi, Dorra Arab Ennigrou, Salma Abid, Mejda Ben Nasr, and

Najoua Jarroudi in the National Influenza Centre-Tunis.

References

1. M.I. Nelson, E.C. Holmes, Nat. Rev. Genet. 8, 196–205 (2007)

2. A.C. McHardy, B. Adams, PLoS Pathog. 5, e1000566 (2009)

3. C.A. Russell, T.C. Jones, I.G. Barr, N.J. Cox, R.J. Garten, V.

Gregory, I.D. Gust, A.W. Hampson, A.J. Hay, A.C. Hurt, J.C. de

Jong, A. Kelso, A.I. Klimov, T. Kageyama, N. Komadina, A.S.

Lapedes, Y.P. Lin, A. Mosterin, M. Obuchi, T. Odagiri, A.D.

Osterhaus, G.F. Rimmelzwaan, M.W. Shaw, E. Skepner, K.

Stohr, M. Tashiro, R.A. Fouchier, D.J. Smith, Science 320,

340–346 (2008)

4. Y. Suzuki, Mol. Biol. Evol. 23, 1902–1911 (2006)

5. J.J. Skehel, D.J. Stevens, R.S. Daniels, A.R. Douglas, M. Knossow,

I.A. Wilson, D.C. Wiley, Proc. Natl. Acad. Sci USA 81,

1779–1783 (1984)

6. W. Wang, B. Lu, H. Zhou, A.L. Suguitan Jr, X. Cheng, K.

Subbarao, G. Kemble, H. Jin, J. Virol. 84, 6570–6577 (2010)

7. S.R. Das, P. Puigbo, S.E. Hensley, D.E. Hurt, J.R. Bennink, J.W.

Yewdell, PLoS Pathog 6, e1001211 (2010)

8. C.C. Wang, J.R. Chen, Y.C. Tseng, C.H. Hsu, Y.F. Hung, S.W.

Chen, C.M. Chen, K.H. Khoo, T.J. Cheng, Y.S. Cheng, J.T. Jan,

C.Y. Wu, C. Ma, C.H. Wong, Proc. Natl. Acad. Sci. USA 106,

18137–18142 (2009)

9. S. Sun, Q. Wang, F. Zhao, W. Chen, Z. Li, PLoS ONE 6, e22844

(2011)

10. D.J. Vigerust, K.B. Ulett, K.L. Boyd, J. Madsen, S. Hawgood,

J.A. McCullers, J. Virol. 81, 8593–8600 (2007)

11. Y. Abe, E. Takashita, K. Sugawara, Y. Matsuzaki, Y. Muraki, S.

Hongo, J. Virol. 78, 9605–9611 (2004)

12. M.D. Tate, E.R. Job, A.G. Brooks, P.C. Reading, Virology 413,

84–92 (2011)

13. WHO: (2009), http://www.who.int/csr/resources/publications/

swineflu/realtimeptpcr/en/

14. K. Tamura, J. Dudley, M. Nei, S. Kumar, Mol. Biol. Evol. 24,

1596–1599 (2007)

15. R. Gupta, E. Jung, S. Brunak (2004), http://www.cbs.dtu.dk/

services/NetNGlyc/

16. M. Zhang, B. Gaschen, W. Blay, B. Foley, N. Haigwood, C.

Kuiken, B. Korber, Glycobiology 14, 1229–1246 (2004)

17. K.A. Ryan-Poirier, Y. Kawaoka, J. Virol. 65, 389–395 (1991)

18. E.C. Crouch, K. Smith, B. McDonald, D. Briner, B. Linders, J.

McDonald, U. Holmskov, J. Head, K. Hartshorn, Am. J. Respir.

Cell Mol. Biol. 35, 84–94 (2006)

19. J. Meschi, E.C. Couch, P. Skolink, K. Yahya, U. Holmskov, R.

Lethlarsen, I. Tornoe, T. Tecle, M.R. White, K.L. Hartshorn, J.

Gen. Virol. 86, 3097–3107 (2005)

20. M. Matrosovich, P. Gao, Y. Kawaoka, J. Virol. 72, 6373–6380

(1998)

21. P.C. Reading, L.S. Morey, E.C. Crouch, E.M. Anders, J. Virol.

71, 8204–8212 (1997)

22. H. Lis, N. Sharon, Eur. J. Biochem. 218, 1–27 (1993)

23. A. Valki, Glycobiology 3, 97–130 (1993)

24. E. Nobusawa, T. Aoyama, H. Kato, Y. Suzuki, Y. Tateno, K.

Nakajima, Virology 182, 475–485 (1991)

25. Y. Kobayashi, Y. Suzuki, J. Virol. 86, 3446–3451 (2012)

26. J.A. Summerfield, S. Ryder, M. Sumiya, M. Thursz, A. Gorchein,

M.A. Monteil, M.W. Turner, Lancet 345, 886–889 (1995)

27. Y. Suzuki, Genes Genet. Syst. 86, 287–294 (2011)

28. P.C. Reading, D.L. Pickett, M.D. Tate, P.G. Whitney, E.R. Job,

A.G. Brooks, Respir. Res. 23, 117 (2009). doi:10.1186/1465-

9921-10-117

29. D.C. Wiley, I.A. Wilson, J.J. Skehel, Nature 289, 373–378 (1981)

30. K. Bragstad, L.P. Nielsen, A. Fomsgaard, Virol. J. 5, 40 (2008).

doi:10.1186/1743-422X-5-40

31. M.L. Wang, J.J. Skehel, D.J. Wiley, J. Virol. 57, 124–128 (1986)

32. L. McLain, S.E. Jones, S.L. Aldridge, N.J. Dimmock, J. Gen.

Virol. 75, 3493–3502 (1994)

33. R.J. Russell, D.J. Stevens, L.F. Haire, S.J. Gamblin, J.J. Skehel,

Glycoconj. J. 23, 85–92 (2006)

34. J.J. Skehel, D.C. Wiley, Annu. Rev. Biochem. 69, 531–569

(2000)

35. U. Holmskov, R. Malhotra, R.B. Sim, J.C. Jensenius, Immunol.

Today 15, 67–74 (1994)

36. X. Sun, A. Jayaraman, P. Maniprasad, R. Raman, K.V. Houser,

C. Pappas, H. Zeng, R. Sasisekharan, J.M. Katz, T.M. Tumpey,

J. Virol. 87, 8756–8766 (2013)

Virus Genes

123