Journal of Molecular Biology
Glycan Microarray Analysis of the Hemagglutinins from Modern and Pandemic Influenza Viruses Reveals Different Receptor Specificities
Introduction
Influenza is an acute viral disease of the respiratory tract that affects millions of people each year. Sixteen serotypes of hemagglutinin (HA) (H1–16) and nine (N1-9) of neuraminidase (NA) have been identified in mammalian and avian influenza A viruses. All serotypes circulate in the avian population and, consequently, birds are believed to act as the main reservoir for influenza A viruses. In the last century, viruses with only three of these HAs (H1-3) and two NAs (N1-2) have adapted to humans to produce pandemic strains, H1N1 in 1918, H2N2 in 1957 and H3N2 in 1968 by presumed re-assortment of the HA and NA gene segments between avian and human viruses via infection of a common host.1 Of these, the 1918-19 influenza outbreak was by far the most virulent, killing approximately 50 million people worldwide.2 Thus, the perennial interest in why the 1918 virus was so devastating and whether a similar pandemic could occur again, especially in light of the recent cases of avian influenza in humans.
The primary event in influenza viral infection is binding of the major antigenic viral coat protein, the HA, to glycan cell surface receptors on epithelial cells of either the respiratory tract (humans) or intestine (birds). Receptor binding specificity is primarily distinguished by recognition of the terminal sialic acid and its linkage to the vicinal galactose of carbohydrates in these tissues. Avian viruses preferentially bind to receptors with an α2-3 linkage, whereas human-adapted viruses are specific for the α2-6 linkage.3, 4 The subtle switch from α2-3 to α2-6 receptor specificity is thought to be a critical step in the adaptation of avian viruses to a human host, and appears to limit most avian influenza viruses from directly crossing the species barrier into the human population. Until recently, it was thought that avian viruses could only cross the avian-human species barrier via an intermediate host, such as a pig, which possesses tracheal cell surface receptors (α2-3 and α2-6) susceptible to both avian and human influenza viruses. Thus, the pig would constitute an ideal host for viral replication and genetic re-assortment,1, 5 in what has been termed the “mixing vessel” theory. Recent evidence, however, suggests that extremely high doses of avian virus can infect humans directly. Since 1997, seven outbreaks of avian influenza in humans have occurred, involving H5, H7 and H9 serotypes, resulting in high mortality rates for those infected6, 7, 8, 9, 10, 11 (see the Centers for Disease Control (CDC‡) and the World Health Organization (WHO§) websites for current information). α2-3-Linked sialic acids have now been found on ciliated cells of the human airway epithelium, which could explain why these bird viruses have infected humans, especially when challenged in doses high enough to counter the inhibitory effects of respiratory mucins that contain α2-3-linked sialic acids.12, 13 Consequently, many believe that the emergence of a future deadly influenza virus pandemic strain is inevitable.
Previous analysis of H3 serotypes strongly linked HA1 residues 226 and 228 within the receptor binding site (RBS) to receptor specificity.14 Leu226 and Ser228 were found in human H3 viruses, whereas Gln226 and Gly228 mapped to avian viruses. However, in 1918 and other human H1 HAs, avian consensus residues are mostly found at these two positions, so that until recently, the molecular basis for switching receptor specificity from avian to human was less clear. Remarkably, the only difference in 1918 H1 New York HA (A/New York/1918; 18NY) from an avian HA consensus RBS is a single Glu190Asp mutation (H3 numbering) (Table 1). South Carolina HA (A/South Carolina/1918; 18SC) has an additional Gly225Asp substitution,15 which is sufficient to switch receptor preference from α2-3 to α2-6 in cell-based assays.16
Here, we report the use of a cell-independent glycoarray assay to probe the binding specificities of recombinantly expressed HAs, including the two known 1918 HAs, as well as an 18NY variant where Asp190 was mutated back to the avian consensus sequence (Asp190Glu). In addition, HAs with a Lys222Leu mutation were constructed to assess its effect on binding specificity. These single mutations within the RBS were sufficient to induce substantial changes in receptor specificity. Thus, by combining genomic sequence analysis with this new array technology, we are able to define in fine detail influenza receptor specificities for a variety of H1 and H3 HAs.
Section snippets
Results
A variety of human and avian HAs (Figure 1) were expressed according to our strategy that led to the crystal structure of the 1918 influenza virus HA0 (18SC).17 The His-tag at the C terminus was invaluable for HA detection by fluorescence via anti-His-tag antibodies, as outlined below. Binding site specificity was analyzed using a glycan array (glycoarray), that was developed for high-throughput analysis of glycan–receptor interactions.18 Of the 200 glycans currently imprinted on the array, six
Discussion
A number of HA receptor binding studies have been reported previously (see Skehel & Wiley14 for a review), but these have primarily employed cell-based assays to probe sialic acid specificities among different influenza viruses. Such assays involve enzymatic removal of endogenous sialic acid from red blood cells using sialidases, followed by re-sialylation with linkage-specific sialyltransferases. While such assays approximate the natural binding of a virus to the host cell receptors, the
Cloning
Based on H3 numbering (see Figure 1), cDNA corresponding to the ectodomains of A/New York/1/1918 (residues HA1: 11–329; HA2: 1–176; 18NY), A/New York/1/1918 (with HA1 D190E mutation; 18NY-Av), A/New York/1/1918 (with HA1 K222L mutation; 18NY-KL), A/South Carolina/1/1918 (with HA1 K222L mutation; 18SC-KL), human H1: A/Texas/36/1991 (residues HA1: 11–329, HA2: 1–176; HuH1), human H3: A/Moscow/10/1999 (residues HA1: 11–329, HA2: 1–176; HuH3), avian H3: A/duck/Ukraine/1/1963 (residues HA1: 1–329,
Acknowledgements
The work was supported, in part, by NIH grants AI058113 (to I.A.W., P.P., J.K.T.), GM062116 (to J.P., I.A.W.) and GM060938 (to J.P.) and partial support from other NIH grants. We thank Ms Sharon Ferguson and Ms Julia Hoffmann (The Scripps Research Institute) for expert technical assistance. This is publication 17526-MB from The Scripps Research Institute.
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J.S. and O.B. contributed equally to this work.