A Single Amino Acid Substitution in 1918 Influenza
http://www.100md.com
病菌学杂志 2005年第17期
Department of Microbiology, Mount Sinai School of Medicine, One Gustave Levy Place, Box 1124, New York, New York 10029
Department of Molecular Biology
Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Department of Molecular Pathology, Armed Forces Institute of Pathology, Rockville, Maryland 20850
Influenza Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333
ABSTRACT
The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the 2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the 2,6 and the 2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.
TEXT
The hemagglutinin (HA) of influenza virus is a surface protein involved in binding host cell sialic acid in the early stages of infection. In general, human viruses preferentially interact with an N-acetylsialic acid attached to galactose with an 2,6 linkage (SA2,6Gal), whereas avian viruses mostly bind to N-acetylsialic acid attached to galactose with an 2,3 linkage (SA2,3Gal) (2, 14-16, 25). The receptor binding specificity of HA has been implicated in determining the host range of a given influenza virus. In agreement with this hypothesis, human tracheal epithelium was shown to contain mostly SA2,6Gal (3), whereas duck gut epithelium contains mostly SA2,3Gal (9). In addition, as viruses adapt to a new host, the receptor binding can become more restricted to one type of sialic acid linkage (5, 13, 17, 25). These adaptations suggest the existence of selective pressure on receptor utilization depending on the host animal. However, it should be noted that all avian and human cells do not exclusively contain SA2,3Gal and SA2,6Gal, respectively. For example, chicken lung and intestinal epithelial cells contain both types of sialic acid linkages (6).
The 1918 influenza pandemic killed an estimated 50 million people, and the origin of this virulent strain continues to be of interest (10). One hypothesis suggests that the virus crossed the species barrier from birds to humans in part due to critical mutations in the HA (H1 subtype) that changed the binding preference from the avian sialic acid (2,3) linkage to the human (2,6) form (1, 12, 23). Six residues are conserved in the HA1 domains of most avian H1 HAs, implicating these residues in the ability to bind to the avian SA2,3Gal receptor (7, 14). Comparison of the avian HA consensus sequence with HA sequences from the 1918 influenza virus strains shows that only one or two of these conserved residues, depending on the viral isolate, are different (Table 1) (23, 24). An earlier study of H1 HAs from swine influenza viruses shows that these amino acids, 190 and 225, are important for determining the receptor binding specificity (13). Here, we report the receptor binding capability of the A/South Carolina/1/18 virus HA and the A/New York/1/18 virus HA (23), which differ by one amino acid, and assess the importance of the amino acids at positions 190 and 225 in this specificity (amino acid residues have H3 numbering) (28).
We used hemadsorption of erythrocytes on cells expressing influenza virus HA as an assay for receptor binding specificity. The binding depends on the interaction of HA with sialic acid-containing molecules on the surface of red blood cells. In order to study the receptor binding properties of the 1918 HAs, we enzymatically modified chicken red blood cells (CRBCs) to express either SA2,6Gal or SA2,3Gal, as previously described (19, 22). All of the sialic acid was removed from 100 μl of 10% CRBCs using 50 mU Vibrio cholerae neuraminidase (VCNA; Roche) at 37°C for 1 h. Removal of sialic acid was confirmed by loss of hemagglutination of virus-like particles (VLPs) by chicken red blood cells (Fig. 2A). Subsequently, resialylation was performed using 0.5 mU 2,3-(N)-sialyltransferase (Calbiochem) or 2.5 mU 2,6-(N)-sialyltransferase (Calbiochem) and 1.5 mM CMP-sialic acid (Sigma) at 37°C in 50 μl for 30 min or in 125 μl for 60 min, respectively. Confirmation of successful resialylation was assayed using hemagglutination of well-defined human and avian viruses (data not shown). The A/South Carolina/1/18 HA gene was subcloned into the mammalian expression vector pCAGGS (23, 27). The HA corresponding to A/New York/1/18 virus was made by mutation of A716G in the A/South Carolina/1/18 HA that coded for a D225G mutation in the protein (Table 1). Human embryonic kidney cells (293T) were transfected with the A/South Carolina/1/18 HA or the A/New York/1/18 HA using Lipofectamine 2000 (Invitrogen). After 24 h, the 293T cells were treated with 5 mU VCNA to remove sialic acids on the HA which could interfere with the assay, and cells were incubated with 0.5% CRBCs for 30 min at 4°C (20). After a thorough washing, the remaining red blood cells were lysed in 150 μl 20 mM Tris-HCl (pH 7.5), 177 mM NH4Cl for 30 min at 25°C. Absorbance of released hemoglobin at 540 nm was used to quantify the amount of red blood cells bound (18). Controls include HAs cloned from a human H3 virus, A/Moscow/10/99, and an avian H3 virus, A/duck/Ukraine/1/63.
All the HAs tested in the hemadsorption assay bind CRBCs, which have been described to express both 2,3- and 2,6-linked sialic acid (19). The A/duck/Ukraine/1/63 virus binds preferentially to SA2,3Gal, whereas the human virus, A/Moscow/10/99, preferentially binds to the SA2,6Gal-resialylated CRBCs (Fig. 1A). Like the A/Moscow/10/99 HA, the HA from A/South Carolina/1/18 virus binds better to SA2,6Gal-resialylated CRBCs than to SA2,3Gal-resialylated CRBCs. The receptor binding specificity of the A/South Carolina/1/18 HA for SA2,6Gal is in agreement with a previous report (11). However, the A/New York/1/18 HA which differs from the A/South Carolina/1/18 HA only at position 225 (G225 versus D225) binds equally well to SA2,6Gal- and SA2,3Gal-resialylated CRBCs. The reduced binding to the resialylated CRBCs by all HA constructs in comparison to unmodified blood cells is probably due to the inability to completely replace the density of sialic acid on red blood cells with the purified enzymes (19).
To understand the importance of residue 190 that differs (Table 1) from the avian consensus in the receptor binding pocket of the 1918 HA, we mutated the aspartic acid at this position in the A/New York/1/18 HA to a glutamic acid (1918 "avian" HA). In the hemadsorption assay, this construct had reduced binding to the SA2,6Gal-resialylated CRBCs, as shown in Fig. 1A. The 1918 "avian" HA bound approximately 70% less SA2,6Gal-resialylated CRBCs than the A/South Carolina/1/18 HA and had a binding profile most similar to that of the avian A/duck/Ukraine/1/63 HA. We do not believe this was solely due to reduced protein expression or a nonfunctional mutant, because the 1918 "avian" HA bound well to unmodified CRBCs and to SA2,3Gal-resialylated CRBCs. In addition, quantification by Western blot showed a less than 1.3-fold difference in protein expression (Fig. 1B).
Virus-like particles (VLPs) afforded another method for studying the receptor binding properties of the 1918 HA. Production of VLPs was carried out by transfection of 293T cells with 1 μg each of the pCAGGS plasmid expressing B/Yamagata/1/73 neuraminidase, matrix, and nonstructural protein 1 with A/New York/1/18 HA, A/South Carolina/1/18 HA, or 1918 "avian" HA (21). The B/Yamagata/1/73 viral proteins were used because they gave the highest titer for released VLPs. The VLPs produced equal hemagglutination units in assays with untreated CRBCs. In hemagglutination assays with 2,3Gal-resialylated CRBCs, the A/New York/1/18 HA and the 1918 "avian" HA VLPs hemagglutinated the CRBCs (Fig. 2B, left panel), whereas only the A/New York/1/18 HA and A/South Carolina/1/18 HA VLPs hemagglutinated the 2,6Gal-resialylated CRBCs (Fig. 2B, right panel). These data emphasize the fact that the consensus 1918 "avian" HA preferentially binds to 2,3Gal-resialylated CRBCs, whereas the A/South Carolina/1/18 HA has a low affinity for these CRBCs. Clearly, these data show the importance of positions 190 and 225 in the 1918 HA in determining its receptor binding specificity.
In conclusion, using hemadsorption and hemagglutination assays, we show that the HA surface glycoprotein from the influenza virus causing the 1918 pandemic preferentially binds SA2,6Gal sialic acid corresponding to the human receptor and that one strain, A/New York/1/18, also binds the SA2,3Gal sialic acid of the avian receptor. This experimentally confirms the prediction made by Reid et al. based on sequence comparisons of the 1918 HA sequence with other HAs (24). Only a single amino acid change in the HA (D190E) from the A/New York/1/18 strain to the avian consensus sequence is required to reduce binding to the human receptor. Two structures of the 1918 HA (cleaved [HA1/HA2] and uncleaved [HA0]) were recently determined by X-ray crystallography (8, 26). The structure of the 1918 HA in complex with receptor analogs has not yet been solved, but the structure of a similar viral HA from A/swine/Iowa/30, in complex with both the avian and human receptor analogs, is available (8). By analogy to the crystal structure of the human receptor in complex with the swine HA, the authors propose that the aspartic acid at position 190 in the 1918 influenza HA makes contacts with the GlcNAc and galactose of the SA2,6Gal sugar moiety stabilizing the receptor-HA interaction. Our results support this notion and indicate that the presence of the larger amino acid, glutamate, in the avian HA receptor-binding pocket may be too bulky to accommodate the 2,6 sugar moiety.
Although sequence information for an avian H1 influenza virus that may have been the direct precursor of the 1918 pandemic strain does not exist, partial sequence of an avian H1 subtype HA from 1917 shows conservation over the receptor binding domain (4). The present work suggests that minimal changes in the receptor binding domain of an avian HA may have been enough to broaden its binding targets to include the major sialic acid receptor expressed on human respiratory epithelium.
ACKNOWLEDGMENTS
We thank James Paulson for his kind gift of 2,6 sialyltransferase.
This work was partially supported by NIAID grant AI058113-01 (I.A.W., A.G.-S., C.F.B., J.K.T., P.P.). I.A.W. and J.S. were supported in part by NIGMS grant GM062116-04. This work was supported in part by NIAID grant 5R01AI050619-04 to J.K.T. D.Z. was supported by NIH training grant A1007647.
REFERENCES
Brownlee, G. G., and E. Fodor. 2001. The predicted antigenicity of the haemagglutinin of the 1918 Spanish influenza pandemic suggests an avian origin. Philos. Trans. R Soc. Lond. B Biol. Sci. 356:1871-1876.
Connor, R. J., Y. Kawaoka, R. G. Webster, and J. C. Paulson. 1994. Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology 205:17-23.
Couceiro, J. N., J. C. Paulson, and L. G. Baum. 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.
Fanning, T. G., R. D. Slemons, A. H. Reid, T. A. Janczewski, J. Dean, and J. K. Taubenberger. 2002. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J. Virol. 76:7860-7862.
Gambaryan, A. S., J. S. Robertson, and M. N. Matrosovich. 1999. Effects of egg-adaptation on the receptor-binding properties of human influenza A and B viruses. Virology 258:232-239.
Gambaryan, A. S., A. B. Tuzikov, N. V. Bovin, S. S. Yamnikova, D. K. Lvov, R. G. Webster, and M. N. Matrosovich. 2003. Differences between influenza virus receptors on target cells of duck and chicken and receptor specificity of the 1997 H5N1 chicken and human influenza viruses from Hong Kong. Avian Dis. 47:1154-1160.
Gambaryan, A. S., A. B. Tuzikov, V. E. Piskarev, S. S. Yamnikova, D. K. Lvov, J. S. Robertson, N. V. Bovin, and M. N. Matrosovich. 1997. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6'-sialyl(N-acetyllactosamine). Virology 232:345-350.
Gamblin, S. J., L. F. Haire, R. J. Russell, D. J. Stevens, B. Xiao, Y. Ha, N. Vasisht, D. A. Steinhauer, R. S. Daniels, A. Elliot, D. C. Wiley, and J. J. Skehel. 2004. The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303:1838-1842.
Ito, T., J. N. Couceiro, S. Kelm, L. G. Baum, S. Krauss, M. R. Castrucci, I. Donatelli, H. Kida, J. C. Paulson, R. G. Webster, and Y. Kawaoka. 1998. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J. Virol. 72:7367-7373.
Johnson, N. P., and J. Mueller. 2002. Updating the accounts: global mortality of the 1918-1920 "Spanish" influenza pandemic. Bull. Hist. Med. 76:105-115.
Kobasa, D., A. Takada, K. Shinya, M. Hatta, P. Halfmann, S. Theriault, H. Suzuki, H. Nishimura, K. Mitamura, N. Sugaya, T. Usui, T. Murata, Y. Maeda, S. Watanabe, M. Suresh, T. Suzuki, Y. Suzuki, H. Feldmann, and Y. Kawaoka. 2004. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature 431:703-707.
Laver, W. G., N. Bischofberger, and R. G. Webster. 2000. The origin and control of pandemic influenza. Perspect. Biol. Med. 43:173-192.
Matrosovich, M., A. Tuzikov, N. Bovin, A. Gambaryan, A. Klimov, M. R. Castrucci, I. Donatelli, and Y. Kawaoka. 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.
Matrosovich, M. N., A. S. Gambaryan, S. Teneberg, V. E. Piskarev, S. S. Yamnikova, D. K. Lvov, J. S. Robertson, and K. A. Karlsson. 1997. Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology 233:224-234.
Matrosovich, M. N., A. S. Gambaryan, A. B. Tuzikov, N. E. Byramova, L. V. Mochalova, A. A. Golbraikh, M. D. Shenderovich, J. Finne, and N. V. Bovin. 1993. Probing of the receptor-binding sites of the H1 and H3 influenza A and influenza B virus hemagglutinins by synthetic and natural sialosides. Virology 196:111-121.
Matrosovich, M. N., S. Krauss, and R. G. Webster. 2001. H9N2 influenza A viruses from poultry in Asia have human virus-like receptor specificity. Virology 281:156-162.
Mochalova, L., A. Gambaryan, J. Romanova, A. Tuzikov, A. Chinarev, D. Katinger, H. Katinger, A. Egorov, and N. Bovin. 2003. Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs. Virology 313:473-480.
Murrell, M., M. Porotto, T. Weber, O. Greengard, and A. Moscona. 2003. Mutations in human parainfluenza virus type 3 hemagglutinin-neuraminidase causing increased receptor binding activity and resistance to the transition state sialic acid analog 4-GU-DANA (Zanamivir). J. Virol. 77:309-317.
Nobusawa, E., H. Ishihara, T. Morishita, K. Sato, and K. Nakajima. 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.
Ohuchi, M., A. Feldmann, R. Ohuchi, and H. D. Klenk. 1995. Neuraminidase is essential for fowl plague virus hemagglutinin to show hemagglutinating activity. Virology 212:77-83.
Paragas, J., J. Talon, R. E. O'Neill, D. K. Anderson, A. Garcia-Sastre, and P. Palese. 2001. Influenza B and C virus NEP (NS2) proteins possess nuclear export activities. J. Virol. 75:7375-7383.
Paulson, J. C., and G. N. Rogers. 1987. Resialylated erythrocytes for assessment of the specificity of sialyloligosaccharide binding proteins. Methods Enzymol. 138:162-168.
Reid, A. H., T. G. Fanning, J. V. Hultin, and J. K. Taubenberger. 1999. Origin and evolution of the 1918 "Spanish" influenza virus hemagglutinin gene. Proc. Natl. Acad. Sci. USA 96:1651-1656.
Reid, A. H., T. A. Janczewski, R. M. Lourens, A. J. Elliot, R. S. Daniels, C. L. Berry, J. S. Oxford, and J. K. Taubenberger. 2003. 1918 Influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg. Infect. Dis. 9:1249-1253.
Rogers, G. N., and B. L. D'Souza. 1989. Receptor binding properties of human and animal H1 influenza virus isolates. Virology 173:317-322.
Stevens, J., A. L. Corper, C. F. Basler, J. K. Taubenberger, P. Palese, and I. A. Wilson. 2004. Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus. Science 303:1866-1870.
Tumpey, T. M., A. Garcia-Sastre, A. Mikulasova, J. K. Taubenberger, D. E. Swayne, P. Palese, and C. F. Basler. 2002. Existing antivirals are effective against influenza viruses with genes from the 1918 pandemic virus. Proc. Natl. Acad. Sci. USA 99:13849-13854.
Winter, G., S. Fields, and G. G. Brownlee. 1981. Nucleotide sequence of the haemagglutinin gene of a human influenza virus H1 subtype. Nature 292:72-75.(Laurel Glaser, James Stev)
Department of Molecular Biology
Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Department of Molecular Pathology, Armed Forces Institute of Pathology, Rockville, Maryland 20850
Influenza Branch, Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333
ABSTRACT
The receptor binding specificity of influenza viruses may be important for host restriction of human and avian viruses. Here, we show that the hemagglutinin (HA) of the virus that caused the 1918 influenza pandemic has strain-specific differences in its receptor binding specificity. The A/South Carolina/1/18 HA preferentially binds the 2,6 sialic acid (human) cellular receptor, whereas the A/New York/1/18 HA, which differs by only one amino acid, binds both the 2,6 and the 2,3 sialic acid (avian) cellular receptors. Compared to the conserved consensus sequence in the receptor binding site of avian HAs, only a single amino acid at position 190 was changed in the A/New York/1/18 HA. Mutation of this single amino acid back to the avian consensus resulted in a preference for the avian receptor.
TEXT
The hemagglutinin (HA) of influenza virus is a surface protein involved in binding host cell sialic acid in the early stages of infection. In general, human viruses preferentially interact with an N-acetylsialic acid attached to galactose with an 2,6 linkage (SA2,6Gal), whereas avian viruses mostly bind to N-acetylsialic acid attached to galactose with an 2,3 linkage (SA2,3Gal) (2, 14-16, 25). The receptor binding specificity of HA has been implicated in determining the host range of a given influenza virus. In agreement with this hypothesis, human tracheal epithelium was shown to contain mostly SA2,6Gal (3), whereas duck gut epithelium contains mostly SA2,3Gal (9). In addition, as viruses adapt to a new host, the receptor binding can become more restricted to one type of sialic acid linkage (5, 13, 17, 25). These adaptations suggest the existence of selective pressure on receptor utilization depending on the host animal. However, it should be noted that all avian and human cells do not exclusively contain SA2,3Gal and SA2,6Gal, respectively. For example, chicken lung and intestinal epithelial cells contain both types of sialic acid linkages (6).
The 1918 influenza pandemic killed an estimated 50 million people, and the origin of this virulent strain continues to be of interest (10). One hypothesis suggests that the virus crossed the species barrier from birds to humans in part due to critical mutations in the HA (H1 subtype) that changed the binding preference from the avian sialic acid (2,3) linkage to the human (2,6) form (1, 12, 23). Six residues are conserved in the HA1 domains of most avian H1 HAs, implicating these residues in the ability to bind to the avian SA2,3Gal receptor (7, 14). Comparison of the avian HA consensus sequence with HA sequences from the 1918 influenza virus strains shows that only one or two of these conserved residues, depending on the viral isolate, are different (Table 1) (23, 24). An earlier study of H1 HAs from swine influenza viruses shows that these amino acids, 190 and 225, are important for determining the receptor binding specificity (13). Here, we report the receptor binding capability of the A/South Carolina/1/18 virus HA and the A/New York/1/18 virus HA (23), which differ by one amino acid, and assess the importance of the amino acids at positions 190 and 225 in this specificity (amino acid residues have H3 numbering) (28).
We used hemadsorption of erythrocytes on cells expressing influenza virus HA as an assay for receptor binding specificity. The binding depends on the interaction of HA with sialic acid-containing molecules on the surface of red blood cells. In order to study the receptor binding properties of the 1918 HAs, we enzymatically modified chicken red blood cells (CRBCs) to express either SA2,6Gal or SA2,3Gal, as previously described (19, 22). All of the sialic acid was removed from 100 μl of 10% CRBCs using 50 mU Vibrio cholerae neuraminidase (VCNA; Roche) at 37°C for 1 h. Removal of sialic acid was confirmed by loss of hemagglutination of virus-like particles (VLPs) by chicken red blood cells (Fig. 2A). Subsequently, resialylation was performed using 0.5 mU 2,3-(N)-sialyltransferase (Calbiochem) or 2.5 mU 2,6-(N)-sialyltransferase (Calbiochem) and 1.5 mM CMP-sialic acid (Sigma) at 37°C in 50 μl for 30 min or in 125 μl for 60 min, respectively. Confirmation of successful resialylation was assayed using hemagglutination of well-defined human and avian viruses (data not shown). The A/South Carolina/1/18 HA gene was subcloned into the mammalian expression vector pCAGGS (23, 27). The HA corresponding to A/New York/1/18 virus was made by mutation of A716G in the A/South Carolina/1/18 HA that coded for a D225G mutation in the protein (Table 1). Human embryonic kidney cells (293T) were transfected with the A/South Carolina/1/18 HA or the A/New York/1/18 HA using Lipofectamine 2000 (Invitrogen). After 24 h, the 293T cells were treated with 5 mU VCNA to remove sialic acids on the HA which could interfere with the assay, and cells were incubated with 0.5% CRBCs for 30 min at 4°C (20). After a thorough washing, the remaining red blood cells were lysed in 150 μl 20 mM Tris-HCl (pH 7.5), 177 mM NH4Cl for 30 min at 25°C. Absorbance of released hemoglobin at 540 nm was used to quantify the amount of red blood cells bound (18). Controls include HAs cloned from a human H3 virus, A/Moscow/10/99, and an avian H3 virus, A/duck/Ukraine/1/63.
All the HAs tested in the hemadsorption assay bind CRBCs, which have been described to express both 2,3- and 2,6-linked sialic acid (19). The A/duck/Ukraine/1/63 virus binds preferentially to SA2,3Gal, whereas the human virus, A/Moscow/10/99, preferentially binds to the SA2,6Gal-resialylated CRBCs (Fig. 1A). Like the A/Moscow/10/99 HA, the HA from A/South Carolina/1/18 virus binds better to SA2,6Gal-resialylated CRBCs than to SA2,3Gal-resialylated CRBCs. The receptor binding specificity of the A/South Carolina/1/18 HA for SA2,6Gal is in agreement with a previous report (11). However, the A/New York/1/18 HA which differs from the A/South Carolina/1/18 HA only at position 225 (G225 versus D225) binds equally well to SA2,6Gal- and SA2,3Gal-resialylated CRBCs. The reduced binding to the resialylated CRBCs by all HA constructs in comparison to unmodified blood cells is probably due to the inability to completely replace the density of sialic acid on red blood cells with the purified enzymes (19).
To understand the importance of residue 190 that differs (Table 1) from the avian consensus in the receptor binding pocket of the 1918 HA, we mutated the aspartic acid at this position in the A/New York/1/18 HA to a glutamic acid (1918 "avian" HA). In the hemadsorption assay, this construct had reduced binding to the SA2,6Gal-resialylated CRBCs, as shown in Fig. 1A. The 1918 "avian" HA bound approximately 70% less SA2,6Gal-resialylated CRBCs than the A/South Carolina/1/18 HA and had a binding profile most similar to that of the avian A/duck/Ukraine/1/63 HA. We do not believe this was solely due to reduced protein expression or a nonfunctional mutant, because the 1918 "avian" HA bound well to unmodified CRBCs and to SA2,3Gal-resialylated CRBCs. In addition, quantification by Western blot showed a less than 1.3-fold difference in protein expression (Fig. 1B).
Virus-like particles (VLPs) afforded another method for studying the receptor binding properties of the 1918 HA. Production of VLPs was carried out by transfection of 293T cells with 1 μg each of the pCAGGS plasmid expressing B/Yamagata/1/73 neuraminidase, matrix, and nonstructural protein 1 with A/New York/1/18 HA, A/South Carolina/1/18 HA, or 1918 "avian" HA (21). The B/Yamagata/1/73 viral proteins were used because they gave the highest titer for released VLPs. The VLPs produced equal hemagglutination units in assays with untreated CRBCs. In hemagglutination assays with 2,3Gal-resialylated CRBCs, the A/New York/1/18 HA and the 1918 "avian" HA VLPs hemagglutinated the CRBCs (Fig. 2B, left panel), whereas only the A/New York/1/18 HA and A/South Carolina/1/18 HA VLPs hemagglutinated the 2,6Gal-resialylated CRBCs (Fig. 2B, right panel). These data emphasize the fact that the consensus 1918 "avian" HA preferentially binds to 2,3Gal-resialylated CRBCs, whereas the A/South Carolina/1/18 HA has a low affinity for these CRBCs. Clearly, these data show the importance of positions 190 and 225 in the 1918 HA in determining its receptor binding specificity.
In conclusion, using hemadsorption and hemagglutination assays, we show that the HA surface glycoprotein from the influenza virus causing the 1918 pandemic preferentially binds SA2,6Gal sialic acid corresponding to the human receptor and that one strain, A/New York/1/18, also binds the SA2,3Gal sialic acid of the avian receptor. This experimentally confirms the prediction made by Reid et al. based on sequence comparisons of the 1918 HA sequence with other HAs (24). Only a single amino acid change in the HA (D190E) from the A/New York/1/18 strain to the avian consensus sequence is required to reduce binding to the human receptor. Two structures of the 1918 HA (cleaved [HA1/HA2] and uncleaved [HA0]) were recently determined by X-ray crystallography (8, 26). The structure of the 1918 HA in complex with receptor analogs has not yet been solved, but the structure of a similar viral HA from A/swine/Iowa/30, in complex with both the avian and human receptor analogs, is available (8). By analogy to the crystal structure of the human receptor in complex with the swine HA, the authors propose that the aspartic acid at position 190 in the 1918 influenza HA makes contacts with the GlcNAc and galactose of the SA2,6Gal sugar moiety stabilizing the receptor-HA interaction. Our results support this notion and indicate that the presence of the larger amino acid, glutamate, in the avian HA receptor-binding pocket may be too bulky to accommodate the 2,6 sugar moiety.
Although sequence information for an avian H1 influenza virus that may have been the direct precursor of the 1918 pandemic strain does not exist, partial sequence of an avian H1 subtype HA from 1917 shows conservation over the receptor binding domain (4). The present work suggests that minimal changes in the receptor binding domain of an avian HA may have been enough to broaden its binding targets to include the major sialic acid receptor expressed on human respiratory epithelium.
ACKNOWLEDGMENTS
We thank James Paulson for his kind gift of 2,6 sialyltransferase.
This work was partially supported by NIAID grant AI058113-01 (I.A.W., A.G.-S., C.F.B., J.K.T., P.P.). I.A.W. and J.S. were supported in part by NIGMS grant GM062116-04. This work was supported in part by NIAID grant 5R01AI050619-04 to J.K.T. D.Z. was supported by NIH training grant A1007647.
REFERENCES
Brownlee, G. G., and E. Fodor. 2001. The predicted antigenicity of the haemagglutinin of the 1918 Spanish influenza pandemic suggests an avian origin. Philos. Trans. R Soc. Lond. B Biol. Sci. 356:1871-1876.
Connor, R. J., Y. Kawaoka, R. G. Webster, and J. C. Paulson. 1994. Receptor specificity in human, avian, and equine H2 and H3 influenza virus isolates. Virology 205:17-23.
Couceiro, J. N., J. C. Paulson, and L. G. Baum. 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.
Fanning, T. G., R. D. Slemons, A. H. Reid, T. A. Janczewski, J. Dean, and J. K. Taubenberger. 2002. 1917 avian influenza virus sequences suggest that the 1918 pandemic virus did not acquire its hemagglutinin directly from birds. J. Virol. 76:7860-7862.
Gambaryan, A. S., J. S. Robertson, and M. N. Matrosovich. 1999. Effects of egg-adaptation on the receptor-binding properties of human influenza A and B viruses. Virology 258:232-239.
Gambaryan, A. S., A. B. Tuzikov, N. V. Bovin, S. S. Yamnikova, D. K. Lvov, R. G. Webster, and M. N. Matrosovich. 2003. Differences between influenza virus receptors on target cells of duck and chicken and receptor specificity of the 1997 H5N1 chicken and human influenza viruses from Hong Kong. Avian Dis. 47:1154-1160.
Gambaryan, A. S., A. B. Tuzikov, V. E. Piskarev, S. S. Yamnikova, D. K. Lvov, J. S. Robertson, N. V. Bovin, and M. N. Matrosovich. 1997. Specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: non-egg-adapted human H1 and H3 influenza A and influenza B viruses share a common high binding affinity for 6'-sialyl(N-acetyllactosamine). Virology 232:345-350.
Gamblin, S. J., L. F. Haire, R. J. Russell, D. J. Stevens, B. Xiao, Y. Ha, N. Vasisht, D. A. Steinhauer, R. S. Daniels, A. Elliot, D. C. Wiley, and J. J. Skehel. 2004. The structure and receptor binding properties of the 1918 influenza hemagglutinin. Science 303:1838-1842.
Ito, T., J. N. Couceiro, S. Kelm, L. G. Baum, S. Krauss, M. R. Castrucci, I. Donatelli, H. Kida, J. C. Paulson, R. G. Webster, and Y. Kawaoka. 1998. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J. Virol. 72:7367-7373.
Johnson, N. P., and J. Mueller. 2002. Updating the accounts: global mortality of the 1918-1920 "Spanish" influenza pandemic. Bull. Hist. Med. 76:105-115.
Kobasa, D., A. Takada, K. Shinya, M. Hatta, P. Halfmann, S. Theriault, H. Suzuki, H. Nishimura, K. Mitamura, N. Sugaya, T. Usui, T. Murata, Y. Maeda, S. Watanabe, M. Suresh, T. Suzuki, Y. Suzuki, H. Feldmann, and Y. Kawaoka. 2004. Enhanced virulence of influenza A viruses with the haemagglutinin of the 1918 pandemic virus. Nature 431:703-707.
Laver, W. G., N. Bischofberger, and R. G. Webster. 2000. The origin and control of pandemic influenza. Perspect. Biol. Med. 43:173-192.
Matrosovich, M., A. Tuzikov, N. Bovin, A. Gambaryan, A. Klimov, M. R. Castrucci, I. Donatelli, and Y. Kawaoka. 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.
Matrosovich, M. N., A. S. Gambaryan, S. Teneberg, V. E. Piskarev, S. S. Yamnikova, D. K. Lvov, J. S. Robertson, and K. A. Karlsson. 1997. Avian influenza A viruses differ from human viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology 233:224-234.
Matrosovich, M. N., A. S. Gambaryan, A. B. Tuzikov, N. E. Byramova, L. V. Mochalova, A. A. Golbraikh, M. D. Shenderovich, J. Finne, and N. V. Bovin. 1993. Probing of the receptor-binding sites of the H1 and H3 influenza A and influenza B virus hemagglutinins by synthetic and natural sialosides. Virology 196:111-121.
Matrosovich, M. N., S. Krauss, and R. G. Webster. 2001. H9N2 influenza A viruses from poultry in Asia have human virus-like receptor specificity. Virology 281:156-162.
Mochalova, L., A. Gambaryan, J. Romanova, A. Tuzikov, A. Chinarev, D. Katinger, H. Katinger, A. Egorov, and N. Bovin. 2003. Receptor-binding properties of modern human influenza viruses primarily isolated in Vero and MDCK cells and chicken embryonated eggs. Virology 313:473-480.
Murrell, M., M. Porotto, T. Weber, O. Greengard, and A. Moscona. 2003. Mutations in human parainfluenza virus type 3 hemagglutinin-neuraminidase causing increased receptor binding activity and resistance to the transition state sialic acid analog 4-GU-DANA (Zanamivir). J. Virol. 77:309-317.
Nobusawa, E., H. Ishihara, T. Morishita, K. Sato, and K. Nakajima. 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.
Ohuchi, M., A. Feldmann, R. Ohuchi, and H. D. Klenk. 1995. Neuraminidase is essential for fowl plague virus hemagglutinin to show hemagglutinating activity. Virology 212:77-83.
Paragas, J., J. Talon, R. E. O'Neill, D. K. Anderson, A. Garcia-Sastre, and P. Palese. 2001. Influenza B and C virus NEP (NS2) proteins possess nuclear export activities. J. Virol. 75:7375-7383.
Paulson, J. C., and G. N. Rogers. 1987. Resialylated erythrocytes for assessment of the specificity of sialyloligosaccharide binding proteins. Methods Enzymol. 138:162-168.
Reid, A. H., T. G. Fanning, J. V. Hultin, and J. K. Taubenberger. 1999. Origin and evolution of the 1918 "Spanish" influenza virus hemagglutinin gene. Proc. Natl. Acad. Sci. USA 96:1651-1656.
Reid, A. H., T. A. Janczewski, R. M. Lourens, A. J. Elliot, R. S. Daniels, C. L. Berry, J. S. Oxford, and J. K. Taubenberger. 2003. 1918 Influenza pandemic caused by highly conserved viruses with two receptor-binding variants. Emerg. Infect. Dis. 9:1249-1253.
Rogers, G. N., and B. L. D'Souza. 1989. Receptor binding properties of human and animal H1 influenza virus isolates. Virology 173:317-322.
Stevens, J., A. L. Corper, C. F. Basler, J. K. Taubenberger, P. Palese, and I. A. Wilson. 2004. Structure of the uncleaved human H1 hemagglutinin from the extinct 1918 influenza virus. Science 303:1866-1870.
Tumpey, T. M., A. Garcia-Sastre, A. Mikulasova, J. K. Taubenberger, D. E. Swayne, P. Palese, and C. F. Basler. 2002. Existing antivirals are effective against influenza viruses with genes from the 1918 pandemic virus. Proc. Natl. Acad. Sci. USA 99:13849-13854.
Winter, G., S. Fields, and G. G. Brownlee. 1981. Nucleotide sequence of the haemagglutinin gene of a human influenza virus H1 subtype. Nature 292:72-75.(Laurel Glaser, James Stev)