Glycans that expressed on cell surface are involved in cell-cell adhesion, leukocyte rolling, cell-extracellular matrix interaction, and microbes’ infection [31
]. Carbohydrates, especially sialic acids, are also reported as receptors for gram positive or negative bacteria, viruses, protozoa, and plant lectins [28
]. For example, sialyl Lewisx
is a ligand for the SabA protein of Helicobacter pylori
]. Cholera toxin of Vibrio chlolerae
specifically binds to the GM1 moiety [35
]. Human influenza virus recognizes α2-6 sialylated glycans and infects host cells subsequently [36
]. Glycosaminoglycan, such as hyaluronic acid and chondroitin sulfate, are confirmed as antiviral agents in preventing Coxsackievirus B5 and dengue virus, respectively [38
]. Further, sialic acid is reported as receptors of many Picornaviridae
Several methods were established to evaluate the attachment and reproduction efficiency of EV71. ELISA assay and flow cytometry provided reliable and reproducible results in quantifying bound EV71 viral particles on cell surface. The binding and subsequent replication of EV71 was detected by measuring the copy number of viral RNA by real-time PCR. In addition, the infection and replication of EV71 could also be confirmed by observing the fluorescence intensity and cytopathic effects in EV71-GFP infected cells. RD is an EV71 highly susceptible cell line which has been applied for viral replication. SK-N-SH cells established from human neuroblastoma were cell model for investigating the EV71 caused neuron toxicity. RD and SK-N-SH cells were infected with EV71 MP4 (mouse adapted strain) and EV71 4643 (human clinical isolates), respectively [40
Since glycosylation was a common and significant feature for cellular and functional receptors of EV71, we first investigated the effects of tunicamycin and benzyl-α-GalNAc (inhibitor for protein N- and O-glycosylation, respectively) in the binding and infection of EV71 to RD cells. Both of the inhibitors decreased the binding of EV71 to RD cells significantly (data not shown). We thus investigated whether the terminal sialic acids on cell surface was involved in EV71 infection to RD and SK-N-SH cells. We found that the expression of cell surface SCARB2 was slightly increased after neuraminidase treatment, and neuraminidase treatment reduced virus binding to RD and SK-N-SH cells in a dose-dependent manner. In addition, the replication of virus was decreased because the binding of EV71-GFP to RD cells was reduced after neuraminidase treatment. These results indicated that sialylation on cell surface should be involved in the attachment and infection of EV71.
As long as there are two major glycosidic linkages between sialic acid with galactose, we applied the lectin competition assay to characterize the binding preference of EV71 to RD and SK-H-SN cells. Not surprisingly, the binding of EV71 was restrained by both lectins on RD and SK-H-SN cells. Both cell surface α2-3- and α2-6-linked sialosides were participated in the binding of EV71 to host cells. The replication of virus was also dropped because the interaction of EV71-GFP to RD cells was blocked by MAA or SNA. These observations can also be found in the infection of other Picornaviridae
viruses such as human rhinovirus 87, encephalomyocarditis virus, and hepatitis A virus [28
]. Then, fetuin/asialofetuin blocking assay was performed and the result indicated that sialylated glycoproteins, such as fetuin, lactoferrin and milk proteins, were inhibitors of EV71 infection [24
]. In order to further identify the carbohydrate epitopes for EV71 infection, viral particles and recombinant viral capsid protein were subjected to carbohydrate solution microarray analysis. But, we could not observe any positive binding signal for viral particles or recombinant VP1 protein. It might be because we don’t have sufficient sialylated epitopes in our microarray library. Further investigations are in progress (collaborate with CFG).
To further characterize the role of sialylation on EV71 cellular receptor, we isolated cell membrane sialylated glycoproteins by lectin affinity chromatography. LAC was a common and useful tool for proteomic and glycomic analysis [41
]. For instance, Butterfield et al. enriched and analyzed abnormal glycoproteins from brain of Alzheimer disease patient by using LAC [41
]. Alvarez-Manilla and colleagues also identified potential glycobiomarkers from embryonic stem cells with LAC technology [43
]. Hence, sialylated membrane proteins were purified with MAA/SNA lectin-agarose column from RD cell membrane extractions. Then, the purified glycoproteins were treated with neuraminidase to remove the effect of sialic acid. The desialylated glycoproteins were subjected to immunoprecipitation assay that pulled down proteins specifically interacted with EV71. Not surprisingly, SCARB2 was observed in western blotting of LAC purified fraction, neuraminidase treated fraction, as well as the EV71 immunoprecipitated fraction. It should be noted that the position of band in lane 4 (EV71 immunoprecipitated fraction) was inconsistent with band in lane 3. This result may come from sample preparation under non-reducing condition which could not break SCARB2-viral protein interactions. These observations prompted us to investigate the binding of EV71 to sialylated and desialylated SCARB2. By using VOPBA, we found that recombinant hSCARB2 lost some of the binding ability to EV71 after desialylation. The same phenomenon have been observed by Yamayoshi et al who found that the interaction of EV71 with recombinant hSCARB2 was moderately decreased after removing N
-glycans from hSCARB2 by enzymatic hydrolysis [46
]. Taken together, all of the results indicated that the attachment of EV71 to cell surface receptor should be assisted with sialic acids.