The index influenza A virus subtype H5N1 isolated from a human in 1997 differs in its receptor-binding properties from a virulent avian influenza virus

Kiyoko Iwatsuki-Horimoto1,2, Rie Kanazawa3, Shunji Sugii3, Yoshihiro Kawaoka1,2,4 and Taisuke Horimoto1,2

1 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan
2 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Saitama 332-0012, Japan
3 Laboratory of Veterinary Microbiology, Division of Veterinary Science, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan
4 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706, USA

Correspondence
Taisuke Horimoto
horimoto{at}ims.u-tokyo.ac.jp


   ABSTRACT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
To gain insight into the events that occur when avian influenza viruses are transmitted to humans, the receptor-binding properties of the index H5N1 influenza virus isolated from a human in 1997 and the A/turkey/Ontario/7732/66 (H5N9) virus were compared, by using a haemadsorption assay. Cells expressing the haemagglutinin (HA) of the human isolate were adsorbed by both chicken red blood cells (RBCs) and human RBCs; those expressing the avian virus HA were only adsorbed by chicken RBCs. These results indicate that human and avian influenza virus H5 HAs differ in their recognition of sialyloligosaccharides on the RBCs of different animal species. Mutational analyses indicated that differences in both the oligosaccharide chains and in the amino acid sequences around the HA receptor-binding site were responsible for this difference in receptor binding. These data further support the concept that alteration in receptor recognition is important for replication of avian viruses in humans.

A figure showing the haemadsorption of the wild-type and selected mutant HAs is available as supplementary data in JGV Online.


   MAIN TEXT
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Influenza A viruses infect a variety of animals, including humans, pigs, horses, marine mammals and birds. There are a number of influenza virus A subtypes, haemagglutinins (HAs), H1 to H15, and neuraminidases (NAs), N1 to N9, all of which are found in birds (Wright & Webster, 2001). Virus subtypes that are not found in humans have the potential to cross the species barrier and possibly cause pandemics (Shortridge, 1995). In 1997, for example, an H5N1 virus was transmitted from birds to humans in Hong Kong, killing six of the 18 people it infected (Claas et al., 1998; Subbarao et al., 1998). The mechanism by which this avian virus infected and caused disease in humans, while other avian viruses have not, remains unknown. However, high HA cleavability, a mutation in the PB2 gene and cytokine dysregulation imposed by the NS1 have been implicated in the virulence of this virus (Hatta et al., 2001; Seo et al., 2002; Cheung et al., 2003).

The receptor-binding preference of influenza viruses correlates with the animal species from which the viruses are isolated; human isolates preferentially bind to the terminal sialic acid of glycoprotein and glycolipid receptors with {alpha}2,6 linkages to galactose (SA{alpha}2,6Gal), whereas avian isolates prefer {alpha}2,3 linkages (SA{alpha}2,3Gal) (Connor et al., 1994; Couceiro et al., 1993; Ito et al., 1998; Rogers & D'Souza, 1989). Alteration in receptor specificity may have occurred when the HA gene of an avian virus was introduced into humans, resulting in the 1957 (Asian influenza) and in the 1968 (Hong Kong influenza) pandemics, since the earliest viruses available from these pandemics recognize SA{alpha}2,6Gal (Matrosovich et al., 2000). Interestingly, the index H5N1 human isolate (A/Hong Kong/156/97) from the 1997 outbreak preferentially recognizes the SA{alpha}2,3Gal receptor (Matrosovich et al., 1999; Ha et al., 2001), suggesting that the change in receptor preference may not be required for primary human infection by avian influenza viruses.

To understand further the bird-to-human transmission of influenza virus that occurred in 1997, we compared receptor specificity between the Hong Kong virus and a virulent avian H5 virus by using an assay that has not previously been used to study the 1997 human isolate.

We cloned the cDNA of the HA genes from the index human isolate in the Hong Kong 1997 outbreak, A/Hong Kong/156/97 (HK/97; H5N1) (Claas et al., 1998; Subbarao et al., 1998), and from the virulent avian virus, A/turkey/Ontario/7732/66 (Ty/Ont; H5N9) (Horimoto & Kawaoka, 1994), into the pCAGGS/MCS expression vector, which contains the chicken {beta}-actin promoter (Kobasa et al., 1997; Niwa et al., 1991). When COS-7 cells were transfected with these plasmids (1 µg plasmid per well of a six-well plate) HAs were expressed on the cell surface as detected by immunostaining with a pool of anti-H5 monoclonal antibodies (61B2, 61E2, 81E5; the latter two react with the HA2 portion of the HA; Horimoto et al., 2004) (see supplementary figure in JGV Online). A haemadsorption assay was performed by using human and chicken red blood cells (RBCs). We found that both human and chicken RBCs adsorbed cells expressing HK/97 HA, but only chicken RBCs adsorbed cells expressing Ty/Ont HA (Fig. 3). These data suggest that receptor recognition differs between these two viruses. However, both RBCs bound to cells expressing HAs from other virulent avian viruses including A/turkey/Ireland/1378/85 (H5N8) HA (data not shown). We therefore focused on Ty/Ont HA to analyse further the properties of the HK/97 HA. Human and chicken RBCs contain both SA{alpha}2,3Gal and SA{alpha}2,6Gal glycoconjugates (Ito et al., 1997; Medeiros et al., 2001); however, our results indicate a quantitative and/or qualitative difference in the relative proportions of these cell surface molecules in the RBCs of humans compared to chickens.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 3. The effect of glycosylation and amino acid substitution on haemadsorption activity. The result is assessed by counting numbers of haemadsorption-positive and -negative cells in five randomly selected microscopic fields; – represent no haemadsorption; + represent more than 50 % haemadsorption-positive cells; ± represent ~ 10 % haemadsorption-positive cells (for details see supplementary figure in JGV Online).

 
To understand the molecular basis for the species-specific difference in haemadsorption between the two HAs, we compared their amino acid sequences and found that Ty/Ont HA contained two additional N-linked potential glycosylation sites at positions 131 and 158 (using the H3 numbering system; 142 and 170 in H5, respectively) that were not present in HK/97 HA. Both of these additional sites are located around the receptor-binding site (Weis et al., 1988) (Fig. 1). Since terminal sialic acids on oligosaccharides near the receptor-binding site are known to affect receptor recognition (Ohuchi et al., 1995), Ty/Ont HA-expressing cells were treated with Vibrio cholerae sialidase (10 mU ml-1 at 37 °C for 1 h). Following this treatment, human RBCs were able to adsorb Ty/Ont HA-expressing cells (data not shown), indicating that desialylation of the oligosaccharides in Ty/Ont HA is essential for its interaction with human RBCs, but not with chicken RBCs.



View larger version (51K):
[in this window]
[in a new window]
 
Fig. 1. Globular head portion of H5 HA (Ha et al., 2002; Protein DataBank 1JSM) illustrating the amino acid residues examined in this study. The arrow points to the receptor-binding site. Oligosaccharide side chains of Ty/Ont HA near the receptor-binding site at positions 131 and 158 are shown schematically. Amino acids at positions 138 and 227 are highlighted in white.

 
To obtain direct evidence that receptor recognition is affected by oligosaccharide side chains near the HA receptor-binding site, we constructed Ty/Ont HA mutants, Ty/Ont HA.131- (Asn-131->Asp) and Ty/Ont HA.158- (Ser-160->Ala), lacking the potential glycosylation sites at positions 131 and 158, respectively (QuikChange XL site-directed mutagenesis kit, Stratagene). We were unable to generate a double mutant lacking both sites, therefore we substituted the sequence that encodes amino acids 123–133 of Ty/Ont HA.131- with the corresponding sequence from HK/97-HA to create Ty/Ont-HA.131/158-, which lacks both potential glycosylation sites. This substitution resulted in the alteration of three amino acids at positions 124, 125 and 129. Although these amino acids do not interact directly with receptor molecules (Weis et al., 1988), we are unable to predict the indirect effects of these amino acid differences on receptor binding. The mobilities of these mutant HAs in SDS-PAGE were faster than that of the wild-type HA, confirming loss of glycosylation (Fig. 2A). Both Ty/Ont HA mutants that lacked only one glycosylation site were haemadsorbed by chicken, but not human, RBCs analogous to wild-type Ty/Ont HA; however, the mutant that lacked both glycosylation sites was adsorbed by human RBCs, albeit to a more limited extent than wild-type HK/97 HA (Fig. 3). All of the mutants bound to human RBCs after sialidase treatment. We also constructed HK/97 HA mutants (HK/97 HA.131+, 158+ and 131/158+) that contained additional glycosylation sites (Asp-131->Asn and/or Ala-160->Thr substitutions) (Fig. 2B). Two of these mutants, HK/97 HA.131+ and 158+, both of which contained one additional glycosylation site, were haemadsorbed by both human and chicken RBCs, whereas mutant HK/97 HA.131/158+, which possessed two additional glycosylation sites, was not adsorbed by chicken RBCs (Fig. 3). HK/97 HA.158+ resembled A/Hong Kong/486 HA, which contains a potential glycosylation site at position 158. We therefore tested the haemadsorption properties of this strain and found no difference between its HA and that of HK/156. Overall, these data suggest that differences in glycosylation near the HA receptor-binding site do not completely explain the difference in receptor recognition between HAs, although oligosaccharides near the receptor-binding site can alter binding affinity and/or specificity, as revealed by the HK/97 HA mutants.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2. Confirmation of the deletion or addition of glycosylation sites in the HA mutants. The HAs on the surface of plasmid-transfected COS-7 cells were biotin-labelled using a commercial kit (Amersham Pharmacia). The cells were lysed, reacted with anti-H5 antibodies and then incubated with protein A beads. The immunoprecipitates were resolved by 10 % SDS-PAGE under reducing conditions.

 
We then focused on the amino acid sequence in the HA receptor-binding site (amino acids 98, 134–138, 153, 155, 183, 190, 194, 195 and 224–229 in the H3 numbering system; Garcia et al., 1996; Claas et al., 1998) and found differences in the site between the two HA species. Recent reports have shown that a single amino acid change in this site can alter the haemagglutination phenotype with chicken RBCs among H3 human viruses (Nobusawa et al., 2003). We found two amino acid differences in the receptor-binding site between HK/97 HA and Ty/Ont HA at positions 138 and 227 (150 and 239 in the H5 numbering system, respectively). The amino acid at position 227 can alter pathogenicity of H5N1 virus, possibly affecting receptor binding (Hatta et al., 2001). We therefore substituted one or both amino acids at these positions from HK/97 HA to Ty/Ont HA (from Ala to Ser at 138, and/or from Ser to Asn at 227, respectively). However, these substitutions did not alter the haemadsorption phenotype (Fig. 3; HK/97 HA.A138S, S227N and A138S/S227N).

We next tested whether changing the oligosaccharide side chains and making the amino acid substitutions in the receptor-binding site affected HA receptor-binding specificity. We constructed HK/97 HA mutants that had the two additional glycosylation sites (HK/97 HA.131/158+) as well as one or both of the amino acid substitutions at positions 138 and/or 227 (Fig. 3; 131/158+//A138S, 131/158+//S227N and 131/158+//A138S/S227N). HK/97 HA mutants with the glycosylation sites did not exhibit the haemadsorption phenotype shown by Ty/Ont HA when only one of the amino acid substitutions was introduced (HK/97 HA.131/158+//A138S and 131/158+//S227N). However, when both amino acid substitutions were made, the HK/97 HA with the additional glycosylation sites (HK/97 HA.131/158+//A138S/S227N) was adsorbed by chicken (to a limited extent probably due to suboptimal binding affinity) but not human RBCs, a haemadsorption phenotype similar to that of Ty/Ont HA. This mutant bound to human RBCs after sialidase treatment. Thus, we conclude that both glycosylation and the amino acid sequence in the receptor-binding site cooperatively determine receptor-binding specificity of HA.

Here we have shown a difference in the haemadsorption phenotype with human RBCs between the index H5N1 human isolate in 1997 and a virulent avian virus. This difference relates to the affinity of these two viruses for SA{alpha}2,3Gal-containing receptors on human cells, and may be explained by a mechanism similar to that recently reported for H3N2 viruses (Nobusawa et al., 2003). The H3N2 human viruses, isolated after 1992, do not agglutinate chicken RBCs and also have reduced binding activity to SA{alpha}2,6Gal-containing sialyloligosaccharides in MDCK cells. However, these viruses bind tightly to MDCK cells that have been desialylated and then resialylated with N-acetyl-D-neuraminyl-({alpha}2,6)-D-galactopyranosyl-({beta}1,4)-N-acetyl-D-glucosamine, suggesting that the asialo portion of the sialyloligosaccharides may be responsible for receptor differentiation. Identification and characterization of SA{alpha}2,3Gal-containing receptors on human cells together with binding analysis of human and avian isolates to these molecules would further our understanding of the receptor specificity changes that occur when viruses are transmitted from birds to humans.


   ACKNOWLEDGEMENTS
 
We thank Sue Watson for scientific editing. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministries of Education, Culture, Sports, Science and Technology, Japan, by CREST (Japan Science and Technology Corporation) and by Public Health Service research grants from the National Institute of Allergy and Infectious Diseases. K. I.-H. was supported by a JSPS Research Fellowship for Young Scientists.


   REFERENCES
Top
ABSTRACT
MAIN TEXT
REFERENCES
 
Cheung, C. Y., Poon, L. L. M., Lau, A. S., Luk, W., Lau, Y. L., Shortridge, K. F., Gordon, S., Guan, Y. & Peiris, J. S. M. (2002). Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease? Lancet 360, 1831–1837.[CrossRef][Medline]

Claas, E. C., Osterhaus, A. D. M. E., van Beek, R., De Jong, J. C., Rimmelzwaan, G. F., Senne, D. A., Krauss, S., Shortridge, K. F. & Webster, R. G. (1998). Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet 351, 472–477.[CrossRef][Medline]

Connor, R. J., Kawaoka, Y., Webster, R. G. & Paulson, J. C. (1994). Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology 205, 17–23.[CrossRef][Medline]

Couceiro, J. N. S. S., Paulson, J. C. & Baum, L. G. (1993). Influenza virus strains selectively recognize sialyloligosaccharides on human respiratory epithelium: the role of the host cell in selection of hemagglutinin receptor specificity. Virus Res 29, 155–165.[CrossRef][Medline]

Garcia, M., Crawford, J. M., Latimer, J. W., Rivera-Cruz, E. & Perdue, M. L. (1996). Heterogeneity in the haemagglutinin gene and emergence of the highly pathogenic phenotype among recent H5N2 avian influenza viruses from Mexico. J Gen Virol 77, 1493–1504.[Abstract]

Ha, Y., Stevens, D. J., Skehel, J. J. & Wiley, D. C. (2001). X-ray structures of H5 avian and H9 swine influenza virus hemagglutinins bound to avian and human receptor analogs. Proc Natl Acad Sci U S A 98, 11181–11186.[Abstract/Free Full Text]

Ha, Y., Stevens, D. J., Skehel, J. J. & Wiley, D. C. (2002). H5 avian and H9 swine influenza virus haemagglutinin structures: possible origin of influenza subtypes. EMBO J 21, 865–875.[Abstract/Free Full Text]

Hatta, M., Gao, P., Halfmann, P. & Kawaoka, Y. (2001). Molecular basis for high virulence of Hong Kong H5N1 influenza A viruses. Science 293, 1840–1842.[Abstract/Free Full Text]

Horimoto, T. & Kawaoka, Y. (1994). Reverse genetics provides a direct evidence for a correlation of hemagglutinin cleavability and virulence of an avian influenza A virus. J Virol 68, 3120–3128.[Abstract]

Horimoto, T., Fukuda, N., Iwatsuki-Horimoto, K. & 7 other authors (2004). Antigenic differences between H5N1 human influenza viruses isolated in 1997 and 2003. J Vet Med Sci (in press).

Ito, T., Suzuki, Y., Mitnaul, L., Vines, A., Kida, H. & Kawaoka, Y. (1997). Receptor specificity of influenza A viruses correlates with the agglutination of erythrocytes from different animal species. Virology 227, 493–499.[CrossRef][Medline]

Ito, T., Couseiro, J. N. S. S., Kelm, S. & 8 other authors (1998). Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J Virol 72, 7367–7373.[Abstract/Free Full Text]

Kobasa, D., Rodgeres, M. E., Wells, K. & Kawaoka, Y. (1997). Neuraminidase hemadsorption activity, conserved in avian influenza A viruses, does not influence viral replication in ducks. J Virol 71, 6706–6713.[Abstract]

Matrosovich, M., Zhou, N., Kawaoka, Y. & Webster, R. G. (1999). The surface glycoproteins of H5 influenza viruses isolated from humans, chickens, and wild aquatic birds have distinguishable properties. J Virol 73, 1146–1155.[Abstract/Free Full Text]

Matrosovich, M., Tuzikov, A., Bovin, N., Gambaryan, A., Klimov, A., Castrucci, M. R., Donatelli, I. & Kawaoka, Y. (2000). Early alterations of the receptor-binding properties of H1, H2, and H3 avian influenza virus hemagglutinins after their introduction into mammals. J Virol 74, 8502–8512.[Abstract/Free Full Text]

Medeiros, R., Escriou, N., Naffakh, N., Manuguerra, J.-C. & van der Werf, S. (2001). Hemagglutinin residues of recent human A (H3N2) influenza viruses that contribute to the inability to agglutinate chicken erythrocytes. Virology 289, 74–85.[CrossRef][Medline]

Niwa, H., Yamamura, K. & Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199.[CrossRef][Medline]

Nobusawa, E., Ishihara, H., Morishita, T., Sato, K. & Nakajima, K. (2000). 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 278, 587–596.[CrossRef][Medline]

Ohuchi, M., Feldmann, A., Ohuchi, R. & Klenk, H.-D. (1995). Neuraminidase is essential for fowl plague virus hemagglutinin to show hemagglutinating activity. Virology 212, 77–83.[CrossRef][Medline]

Rogers, G. N. & D'Souza, B. L. (1989). Receptor binding properties of human and animal H1 influenza virus isolates. Virology 173, 317–322.[Medline]

Seo, S. H., Hoffmann, E. & Webster, R. G. (2002). Lethal H5N1 influenza viruses escape host anti-viral cytokine responses. Nat Med 8, 950–954.[CrossRef][Medline]

Shortridge, K. F. (1995). The next pandemic influenza virus? Lancet 346, 1210–1212.[Medline]

Subbarao, K., Klimov, A., Katz, J. & 13 other authors (1998). Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science 279, 393–396.[Abstract/Free Full Text]

Weis, W., Brown, J. H., Cusack, S., Paulson, J. C., Skehel, J. J. & Wiley, D. C. (1988). Structure of the influenza virus hemagglutinin complexed with its receptor, sialic acid. Nature 333, 426–431.[CrossRef][Medline]

Wright, P. F. & Webster, R. G. (2001). Orthomyxoviruses. In Fields Virology, 4th edn, pp. 1533–1579. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott -Williams & Wilkins.

Received 21 July 2003; accepted 23 December 2003.



This Article
Abstract
Full Text (PDF)
Supplementary figure
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Iwatsuki-Horimoto, K.
Articles by Horimoto, T.
Articles citing this Article
PubMed
PubMed Citation
Articles by Iwatsuki-Horimoto, K.
Articles by Horimoto, T.
Agricola
Articles by Iwatsuki-Horimoto, K.
Articles by Horimoto, T.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
INT J SYST EVOL MICROBIOL MICROBIOLOGY J GEN VIROL
J MED MICROBIOL ALL SGM JOURNALS