Glycan analysis and influenza A virus infection of primary swine respiratory epithelial cells: the importance of NeuAc{alpha}2-6 glycans - PubMed (original) (raw)

Glycan analysis and influenza A virus infection of primary swine respiratory epithelial cells: the importance of NeuAc{alpha}2-6 glycans

Allen C Bateman et al. J Biol Chem. 2010.

Abstract

To better understand influenza virus infection of pigs, we examined primary swine respiratory epithelial cells (SRECs, the primary target cells of influenza viruses in vivo), as a model system. Glycomic profiling of SRECs by mass spectrometry revealed a diverse range of glycans terminating in sialic acid or GalαGal. In terms of sialylation, α2-6 linkage was more abundant than α2-3, and NeuAc was more abundant than NeuGc. Virus binding and infection experiments were conducted to determine functionally important glycans for influenza virus infection, with a focus on recently emerged swine viruses. Infection of SRECs with swine and human viruses resulted in different infectivity levels. Glycan microarray analysis with a high infectivity "triple reassortant" virus ((A/Swine/MN/593/99 (H3N2)) that spread widely throughout the North American swine population and a lower infectivity human virus isolated from a single pig (A/Swine/ONT/00130/97 (H3N2)) showed that both viruses bound exclusively to glycans containing NeuAcα2-6, with strong binding to sialylated polylactosamine and sialylated N-glycans. Treatment with mannosamine precursors of sialic acid (to alter NeuAc/NeuGc abundances) and linkage-specific sialidases prior to infection indicated that the influenza viruses tested preferentially utilize NeuAcα2-6-sialylated glycans to infect SRECs. Our data indicate that NeuAcα2-6-terminated polylactosamine and sialylated N-glycans are important determinants for influenza viruses to infect SRECs. As NeuAcα2-6 polylactosamine glycans play major roles in human virus infection, the importance of these receptor components in virus infection of swine cells has implications for transmission of viruses between humans and pigs and for pigs as possible adaptation hosts of novel human influenza viruses.

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Figures

FIGURE 1.

FIGURE 1.

MALDI-TOF MS profiles of the permethylated _N_-linked glycans derived from SRECs. For complete annotation of the spectrum, see

supplemental Table S1

. Data were obtained from the 50% acetonitrile fraction, and all molecular ions are present in sodiated form ([M + Na]+).

FIGURE 2.

FIGURE 2.

Flow cytometry-based quantification of virus infectivity levels in SRECs. The data are mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.01 compared with infectivity level of each of the other viruses.

FIGURE 3.

FIGURE 3.

Glycan microarray analysis of Sw/MN and Sw/ONT viruses. Sw/MN (A) and Sw/ONT (B) binding to glycans was performed on microarray version 4.1 from the Consortium for Functional Glycomics. Results shown are the average of four replicate spots ± S.E. after the highest and lowest readings of six were excluded, with the highest value set to 100. As the binding of all asialo- and α2–3-sialylated glycans was below 1.5%, the structures of only five α2–3-sialylated glycans are plotted on the graph for clarity of presentation. For complete glycan sequences and relative luciferase units of viruses binding to all glycans see

supplemental Table S4

.

FIGURE 4.

FIGURE 4.

ManNGc and ManNAc treatment of SRECs followed by virus infection. SRECs were grown in the presence of ManNGc and/or ManNAc for 2 days prior to infection. Precursor molecules were solubilized in DMSO and added to a final concentration of 0.05% DMSO. A DMSO control showed no change in infectivity (data not shown). The data shown are the means ± S.E. of three independent experiments performed in triplicate. *, p < 0.01 compared with untreated cells.

FIGURE 5.

FIGURE 5.

Partial MALDI-TOF MS profiles of the permethylated _N_-linked glycans derived from SRECs after digestion with sialidase S or sialidase A. Data were obtained from the 50% acetonitrile fraction and all molecular ions are present in sodiated form ([M + Na]+). Sialylated species are annotated in red (see

supplemental Table S1

).

FIGURE 6.

FIGURE 6.

Sialidase treatment of SRECs prior to virus infection. The data shown are the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.01 compared with untreated cells.

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