Sialic acid functions in enterovirus 70 binding and infection

Abstract

The interaction of viruses with host cell receptors is the initial step in viral infection and is an important determinant of virus host range, tissue tropism, and pathogenesis. The complement regulatory protein decay-accelerating factor (DAF/CD55) is an attachment receptor for enterovirus 70 (EV70), a member of the Picornaviridae, commonly associated with an eye infection in humans known as acute hemorrhagic conjunctivitis. In early work, the EV70 receptor on erythrocytes, responsible for its hemagglutinating activity, was shown to be sensitive to neuraminidase, implying an essential role for sialic acid in virus attachment. Here, we extend these results to show that cell surface sialic acid is required for EV70 binding to nucleated cells susceptible to virus infection and that sialic acid binding is important in productive infection. Through the use of site-directed mutagenesis to eliminate the single N-linked glycosylation site of DAF and of a chimeric receptor protein in which the O-glycosylated domain of DAF was replaced by a region of the HLA-B44 molecule, a role in EV70 binding for the sialic acid residues of DAF was excluded, suggesting the existence of at least one additional, sialylated EV70-binding factor at the cell surface. Treatment of cells with metabolic inhibitors of glycosylation excluded a role for the N-linked oligosaccharides of glycoproteins but suggested that O-linked glycosylation is important for EV70 binding.

FIG. 1.

Neuraminidase pretreatment inhibits EV70 binding to several susceptible cell lines. Cells were incubated in the presence of the indicated concentrations of neuraminidase for 30 min at 37°C. After being washed, cells were incubated for 1 h with ³⁵S-labeled EV70 (33°C) or E11 (37°C) (2,000 to 10,000 cpm). Virus binding data are presented as the means ± standard deviations for binding to treated cells relative to binding to untreated cells (i.e., 0 mU of neuraminidase/ml) from at least three experiments. (A) EV70 and E11 binding to HeLa cells. Untreated cells bound 33 and 67% of input EV70 and E11, respectively. (B) EV70 and E11 binding to U-937 cells. Untreated cells bound 51 and 16% of input EV70 and E11, respectively. (C) EV70 binding to NIH 3T3 cells constitutively expressing human DAF. Untreated cells bound 30% of input EV70. ND, not determined.

FIG. 2.

Neuraminidase pretreatment inhibits EV70 infection of U-937 cells. Cells were incubated in 50 mU of neuraminidase/ml for 30 min at 37°C. After being washed twice with TBS, cells were infected with EV70 at a multiplicity of infection of 5 PFU/cell and incubated at 33°C. Aliquots were removed at the indicated times, and EV70 titers ± standard deviations for duplicate experiments were determined by plaque assay on LLC-MK₂ cells.

FIG. 3.

(A) Elimination of the N-linked carbohydrate of DAF has no effect on EV70 binding. An A-to-G mutation was introduced into DAF cDNA, resulting in an Asn-to-Ser substitution (N61S) in the DAF polypeptide. Vector-only control (vector), wild-type (DAF), and mutant (DAF N61S) constructs were transfected into the EV70 receptor-negative cell line NIH 3T3. ³⁵S-EV70 binding was determined after 48 h and normalized for DAF construct expression as determined by flow cytometry (see Materials and Methods). (B) EV70 binding to both wild-type and mutant DAF is sensitive to neuraminidase. Transfection and virus binding were performed as for panel A, except that cells were incubated in serum-free medium (control) or in serum-free medium containing 50 mU of neuraminidase/ml for 30 min at 37°C before ³⁵S-EV70 binding. For both panels, data are presented as means ± standard deviations for at least three experiments.

FIG. 4.

The O-glycosylated domain of DAF is not directly involved in EV70 binding. CHO cells constitutively expressing wild-type DAF or a chimeric construct consisting of the four CCP domains of DAF fused to the majority of the HLA-B44 molecule (DAF/HLA-B44) and control cells transfected with vector alone were assayed for EV70 binding. Virus binding results were normalized for DAF construct expression level.

FIG. 5.

N-linked glycans are not involved in EV70 attachment. (A) HeLa and U-937 cells, as indicated, were incubated in cell culture medium alone (control) or in cell culture medium containing 0.2 μg of tunicamycin (tunic.)/ml for 24 h at 37°C prior to ³⁵S-EV70 binding. Data are presented as means ± standard deviations for three experiments. (B) Immunoblot analysis of DAF released from tunicamycin-treated and untreated HeLa cells by digestion with PI-PLC. The apparent molecular weights (in thousands) of prestained protein markers are shown at the left.

FIG. 6.

Disruption of O-linked glycosylation inhibits EV70 binding. (A) HeLa cells were incubated in the presence or absence of 3 mM benzyl GalNAc for 48 h at 37°C prior to ³⁵S-EV70 or ³⁵S-E11 binding. (B) Same as panel A with U-937 cells in place of HeLa cells. For panels A and B, data are presented as means ± standard deviations for three experiments. (C) Immunoblot analysis of DAF released from benzyl GalNAc-treated and untreated HeLa cells by digestion with PI-PLC. The apparent molecular weights (in thousands) of prestained protein markers are shown at the left.