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 . Taken together, all of the results indicated that the attachment of EV71 to cell surface receptor should be assisted with sialic acids. Conclusions Based on our findings, we concluded that cell Selleck BAY 1895344 surface sialylation was important for EV71 infection to RD and SK-N-SH cells. Although the glycan epitopes for EV71 was still unclear, these evidences sufficiently Erastin in vivo supported
that sialylation of cell surface glycoproteins could assist the attachment of EV71 to host cells. In addition, we also demonstrated that SCARB2 was a sialylated glycoprotein. Interactions between SCARB2 with EV71 were decreased after desialylation. Our findings not only demonstrated the important role of sialic acid in EV71 infection to RD and SK-N-SH cells, but also opened a new direction for anti-EV71 drug discovery. Finally, identification and characterization of glycans or proteins which interact with EV71 are now in progress. Methods Virus amplification and purification RD and SK-N-SH cells (ATCC, Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) contained 10% fetal bovine serum (FBS), 2.0 mM L-glutamine, 100 IU of penicillin, and 100 μg of streptomycin. The infectious clone of mouse-adapted EV71 MP4, 4643 and EV71-GFP were obtained from Dr. Jen-Ren Wang. GFP was located in the replicon between P2 and P3 nonstructural regions. In vitro transcription of the linear plasmid of MP4, 4643 and EV71-GFP were performed by kit (Promega) and purified mRNA was transfected to RD cells (for
MP4 and EV71-GFP) or SK-N-SH Olopatadine (for 4643). Virus was amplified in RD cells or SK-N-SH and cultivated using DMEM with 2% FBS at 37°C for 16 to 24 hours. To prepare virus stocks, viruses will be propagated for one more passage in cells. Working stocks contain 108 PFU per ml. The culture medium and cells were collected for purification when CPE was observed. All of the viruses were precipitated with poly ethylene glycol (PEG) and purified by sucrose gradient ultracentrifugation. Preparation of EV71 specific monoclonal antibody (1 G3) The hybridoma cells which produced monoclonal antibody (1 G3) against EV71 VP1 protein region was a gift from Dr. Chun-Keung Yu. The hybridoma cells (106) were injected intraperitoneally into MM-102 mw 10-weeks old BALB/c mice after pristane injection. Ascites was collected and the 1 G3 monoclonal antibody was purified by protein-A affinity column on AKTA prime plus (GE Healthcare).