Cell surface sialylation affects binding of enterovirus 71 to rhabdomyosarcoma and neuroblastoma cells

Abstract

Background: Enterovirus 71 (EV71) is a major causative agent of hand-foot-and-mouth disease (HFMD), and infection of EV71 to central nerve system (CNS) may result in a high mortality in children less than 2 years old. Although there are two highly glycosylated membrane proteins, SCARB2 and PSGL-1, which have been identified as the cellular and functional receptors of EV71, the role of glycosylation in EV71 infection is still unclear.

Results: We demonstrated that the attachment of EV71 to RD and SK-N-SH cells was diminished after the removal of cell surface sialic acids by neuraminidase. Sialic acid specific lectins, Maackia amurensis (MAA) and Sambucus Nigra (SNA), could compete with EV71 and restrained the binding of EV71 significantly. Preincubation of RD cells with fetuin also reduced the binding of EV71. In addition, we found that SCARB2 was a sialylated glycoprotein and interaction between SCARB2 and EV71 was retarded after desialylation.

Conclusions: In this study, we demonstrated that cell surface sialic acids assist in the attachment of EV71 to host cells. Cell surface sialylation should be a key regulator that facilitates the binding and infection of EV71 to RD and SK-N-SH cells.

Figure 1

The attachment and infection of EV71 to RD cells are affected by neuraminidase treatment. Cells were pretreated with neuraminidase followed by infection with EV71 MP4. The bound virus was analyzed by ELISA, flow cytometry and real-time PCR. The binding of virus to RD cells treated with different units of neuraminidase was reduced by 20% and 32% measured by ELISA (A), by 27% and 29% measured by flow cytometry (B), and by 20% and 27% measured by real-time PCR (C). The replication of EV71 dropped by 49% and 66% in neuraminidase treated cells measured by analyzing the copy number of EV71 RNA using real-time PCR after 24 hours incubation (D). **: P < 0.01; ***: P < 0.001 (two-tailed test). Each of the results was averaged from at least six independent assays.

Figure 2

The infection and replication of EV71 to RD cells are affected by neuraminidase treatment investigated with EV71-GFP infection. Cells pretreated with or without neuraminidase (5 mU and 25 mU) were infected with or without EV71-GFP. The cell number, CPE, and fluorescence intensity were observed by fluorescence microscope. After 48 hours, higher fluorescence intensity was found in untreated cells than neuraminidase pretreated cells.

Figure 3

The expression of RD cell surface SCARB2 with or without neuraminidase treatment measured by flow cytometry. Cell surface SCARB2 was nearly the same after 25 mU of neuraminidase treatment.

Figure 4

The attachment and infection of EV71 to RD cells are affected by sialic acid specific lectin treatment. Cells were preincubated with MAA (maackia amurensis) or SNA (sambucus nigra) followed by infection with EV71 MP4. The bound EV71 was analyzed by ELISA and real-time PCR, and the subsequent replication of EV71 in RD cells was detected by real-time PCR analysis. The binding of virus to RD cells treated with different concentrations of MAA was reduced by 19% and 45% measured by ELISA (A) and by 37% and 68% measured by real-time PCR (C). The replication of EV71 dropped 38% and 59% after MAA treatment measured by real-time PCR after 24 hours incubation (E). The virus binding of SNA treated cells reduced by 18% and 38% measured by ELISA (B), and by 28% and 45% measured by real-time PCR (D). The replication of EV71 dropped 30% and 58% after SNA treatment measured by RT-PCR after 24 hours incubation (F). **: P < 0.01; ***: P < 0.001 (two-tailed test). Each of the results was averaged from at least six independent assays.

Figure 5

The infection and replication of EV71 to RD cells are affected by lectin treatment investigated with EV71-GFP infection. Cells preincubated with or without MAA/SNA were infected with or without EV71-GFP. The cell number, CPE, and fluorescence intensity were observed by fluorescence microscope. After 48 hours, higher fluorescence intensity was found in untreated cells than neuraminidase pretreated cells.

Figure 6

The attachment and infection of EV71 4643 to SK-N-SH cells are affected by neuraminidase or sialic acid specific lectin treatment. 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 (two-tailed test). Each of the results was averaged from at least six independent assays.

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.

Figure 8

Western blotting of glycoprotein fractions with anti-SCARB2 antibody. Lane 1: RD cell membrane extracts; Lane 2: MAA/SNA lectin affinity chromatography purified sialylated glycoproteins; Lane 3: desialylated glycoproteins; Lane 4: desialylated glycoproteins immunoprecipitated with EV71 MP4. All of the purified proteins were subjected to western blotting and stained by anti-SCARB2 antibody. SCARB2 could be observed in all of the fractions. Band in lane 3 was slightly shifted down after neuraminidase treatment. But, owing to non-reducing condition, band in lane 4 was slightly shifted up compared to band in lane 3.

Figure 9

Interactions between recombinant hSCARB2 with EV71 are reduced after desialylation. The binding is detected by Viral-Overlaying Protein Binding Assay (VOPBA) with anti-EV71 antibody and HRP conjugated anti-mouse antibody on LAS-3000.