images

Figure 2 | Characterization of the receptor-binding properties of isolated viruses. a, Binding of VN1203 mutants to sialylglycopolymers in solid-phase binding assays. A human virus (K173/PR8), an avian virus (VN1203/PR8) and mutant VN1203/PR8 viruses were compared for their ability to bind to sialylglycopolymers containing either α2,3–linked (blue) or α2,6-linked (red) sialic acids. b, Identification of mutations that confer binding to human-type receptors. c, Binding of VN1203 mutant viruses to human respiratory tissues. K173/PR8, VN1203/PR8 and mutant VN1203/PR8 viruses were incubated with human tissue sections and then stained with either anti-K173 antiserum (green) or anti-VN1203 HA antibodies (green). All sections were subsequently incubated with labelled secondary antibodies and Hoechst dye (blue). d, Characterization of the receptor-binding properties of N158D/N224K/Q226L, N158D/N224K/Q226L/T318I and T318I viruses. The direct binding of virus to sialylglycopolymers containing either α2,3–linked (blue) or α2,6-linked (red) sialic acids was determined as described in panel a.

To assess the effect of enhanced α2,6-glycan recognition on the attachment of viruses to human respiratory tracts, sections of tracheal and lung tissues were exposed to K173/PR8 (human-type receptor binder), VN1203/PR8 (avian-type receptor binder) and mutant VN1203/PR8 viruses (Fig. 2c). Because the N186K/M230I, S227N/G228A, Q226L/E231G, E119G/V152I/N224K/Q226L and N224K/Q226L mutants exhibited appreciablebinding to Siaα2,6Gal (Fig. 2a, b), the attachment of these mutants was also tested. On tracheal sections, theK173/PR8 virus bound extensively to ciliated epithelial cells (Fig. 2c and Supplementary Fig. 3), whereas the VN1203/PR8 virus bound poorly. By contrast, on lung sections, both viruses bound extensively to the alveolar epithelial surface (both type I and II pneumocytes; Fig. 2c and Supplementary Fig. 4). The binding patterns of these viruses correlate with the distribution of Siaα2,3 Gal (that is, avian-type receptors; present in lung epithelia) and Siaα2,6Gal (that is, human-type receptors; present in both trachea and lung epithelia) on the tissues, as observed with lectin staining’18 (Supplementary Fig. 5). Like the human K173/PR8 virus, the E119G/V152I/N224K/Q226L and N224K/Q226L mutants exhibited strong binding to the ciliated epithelial cells of the trachea (Fig. 2c and Supplementary Fig. 3). By contrast, the N186K/M230I, S227N/G228A and Q226L/E231G mutants displayed little-to-no binding to tracheal epithelia (Fig. 2c), despite their binding to Siaα2,6Gal (Fig. 2a). A number of sialylated oligosaccharides with differing branching patterns and chain lengths are thought to be present on the cell surface19. We therefore speculate that the mutants can recognize a short glycan structure such as Siaα2,6Galβ1,4GlcNAc, but may not recognize longer, more complex glycan structures, which arepossibly required forbindingto human tracheal epithelium. On the other hand, all mutants bound to alveolar epithelial cells (both type I and II pneumocytes; Fig. 2c and Supplementary Fig. 4). When the tissue sections were pre-treated with Arthrobacter ureafaciens sialidase (which cleaves all non-reducing terminally branched and unbranched sialic acids), virus binding to the tissues was substantially reduced (Supplementary Fig. 6a-c), confirming the sialic acid binding specificity of the virus. These data indicate that alterations in the receptor specificity of the E119G/V152I/N224K/Q226L and N224K/Q226L



The National Academies | 500 Fifth St. N.W. | Washington, D.C. 20001
Copyright © National Academy of Sciences. All rights reserved.
Terms of Use and Privacy Statement