SK-N-SH cells were pretreated with neuraminidase, MAA or SNA before infected with EV71 4643. (A) The copy number of EV71 dropped 44% and 59% in neuraminidase treated cells. (B) The copy number of EV71 reduced by 42% and 59% in MAA treated cells. (C) The copy number of EV71 decreased by 31% and 52% in SNA treated cells. **: P < 0.01;

***: P < 0.001 CX-6258 order (two-tailed test). Each of the results was averaged from at least six independent assays. Because it has been reported that lactoferrin, a highly sialylated glycoprotein, can inhibit the infection of EV71 [24, 25], we used another highly sialylated glyco4SC-202 manufacturer protein to confirm these interactions between EV71 with sialic acid. Fetuin and asialofetuin were subjected

to EV71 binding assay. Not surprisingly, pretreated cells with fetuin reduced the attachment of EV71 to RD cells by 12-14% (statistically significant, Figure 7). These findings encouraged us to identify the carbohydrate ligands for EV71 viral particles and VP1 protein (recombinant protein from E. coli) by glycan solution microarray. But, unfortunately, none of the binding signals were observed (Additional file 1 Supplementary information). Figure 7 Fetuin blocks the attachment of EV71 to RD cells. Cells were preincubated with fetuin or asialofetuin and infected with EV71. Asialofetuin showed no effect on virus binding, but the attachment of EV71 to RD cells decreased by 12% to 14% in fetuin preincubated cells. *: P < 0.05; **: P < 0.01 (two-tailed test). Each of the results was averaged from at least seven independent assays. Characterization of SCARB2 sialylation in EV71 infection Based on these P505-15 supplier findings, we tried to look deep inside the relationships of sialylation with viral receptor. By using lectin affinity chromatography (LAC) which contained MAA and SNA-agarose beads, we purified sialylated membrane proteins from RD cell membrane 4-Aminobutyrate aminotransferase extracts. Desialylation was performed with neuraminidase on purified glycoproteins

to remove sialic acids. The desialylated glycoproteins were subjected to immunoprecipitation assay, in which EV71 viral particles were immobilized on protein G agarose beads through anti-EV71 antibody. As shown in Figure 8, the cellular receptor of EV71, SCARB2, was observed in all of the purified and immunoprecipitated protein fractions. Because of the neuraminidase treatment, band in lane 3 was slightly shifted down. In addition, band in lane 4 was slightly shifted up owing to the non-reducing treatment of EV71 pulled down fractions. To determine whether sialylation on SCARB2 contribute to its interaction with EV71, the binding of EV71 to recombinant human SCARB2 (hSCARB2, with or without neuraminidase treatment) was analyzed by virus overlay protein binding assay (VOPBA). The result showed that desialylation of hSCARB2 curtailed the binding ability with EV71 (Figure 9).