Sialic Acid-Containing Glycans as Cellular Receptors for Ocular Human Adenoviruses: Implications for Tropism and Treatment

Abstract

Human adenoviruses (HAdV) are the most common cause of ocular infections. Species B human adenovirus type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause epidemic keratoconjunctivitis (EKC). Recently, HAdV-D53, -D54, and -D56 emerged as new EKC-causing agents. HAdV-E4 is associated with both PCF and EKC. We have previously demonstrated that HAdV-D37 uses sialic acid (SA)-containing glycans as cellular receptors on human corneal epithelial (HCE) cells, and the virus interaction with SA is mediated by the knob domain of the viral fiber protein. Here, by means of cell-based assays and using neuraminidase (a SA-cleaving enzyme), we investigated whether ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells. We found that HAdV-E4 and -D56 infect HCE cells independent of SAs, whereas HAdV-D53 and -D64 use SAs as cellular receptors. HAdV-D8 and -D54 fiber knobs also bound to cell-surface SAs. Surprisingly, HCE cells were found resistant to HAdV-B3 infection. We also demonstrated that the SA-based molecule i.e., ME0462, designed to bind to SA-binding sites on the HAdV-D37 fiber knob, efficiently prevents binding and infection of several EKC-causing HAdVs. Surface plasmon resonance analysis confirmed a direct interaction between ME0462 and fiber knobs. Altogether, we demonstrate that SA-containing glycans serve as receptors for multiple EKC-causing HAdVs, and, that SA-based compound function as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs.

Keywords: adenovirus; cellular receptor; epidemic keratoconjunctivitis; pharyngoconjunctival fever; sialic acid; tropism.

Figure 1

Multiple sequence alignment of the fiber knobs of epidemic keratoconjunctivitis (EKC)- and pharyngoconjunctival fever (PCF)- causing HAdVs. EKC- and PCF-causing HAdVs contain highly and partially conserved critical sialic acid (SA)-binding residues, respectively. Residues highlighted in cyan with white texts represent conserved SA-binding residue (Tyr312) among all ocular HAdVs. Residues highlighted in cyan with black texts represent conserved SA-binding residues (Pro317 and Lys345) among all EKC-causing HAdVs fiber knobs and these residues are not conserved (highlighted in pink) in HAdV-B3 and -E4 fiber knobs. Residues at 310 and 344 on HAdV-D37 mediate contacts with SA via water-mediated hydrogen bonds and these residues either partially or not conserved among all ocular HAdVs (highlighted in yellow). The crucial residue (lysine or alanine) at 240 is conserved (highlighted in brown) in the fiber knobs of all ocular HAdVs except for HAdV-E4 (highlighted in green). Amino acid start positions are according to the HAdV-D37 sequence (accession No. DQ900900).

Figure 2

Infection of ocular HAdVs on neuraminidase pre-treated human corneal epithelial (HCE) cells. (A) HAdV-B3, -E4, -D37, -D53, -D56, and –D64 infection of HCE cells pre-treated with and without neuraminidase. (B) Cytopathic effect (CPE) analysis of HAdV-B3 on A549 and HCE cells. Error bars represent mean ± SEM. *** p < 0.001 relative to control.

Figure 3

Effect of neuraminidase treatment on the binding of epidemic keratoconjunctivitis (EKC)- causing HAdVs to human corneal epithelial (HCE) cells. (A) Binding of ³⁵S-labelled HAdV-D37, -D53, and -D64 to HCE cells pre-treated with and without neuraminidase. (B) Binding of HAdV-D8/53, -D37/64, and -D54 fiber knobs to HCE cells pre-treated with and without neuraminidase. Error bars represent mean ± SEM. *** p < 0.001 relative to control.

Figure 4

Effect of ME0462 on epidemic keratoconjunctivitis (EKC)-causing HAdVs binding to and infection of human corneal epithelial (HCE) cells. (A) Binding of ³⁵S-labeled HAdV-D37, -D53, and -D64, pre-incubated with increasing dose-dependent concentrations of ME0462, to HCE cells. (B) HAdV-D37, -D53, and -D64, pre-incubated with ME0462 (80 µM), infection of HCE cells. (C) HAdV-D8/53, -D37/64, and -D54 fiber knobs, pre-incubated with ME0462 (80 µM), binding to HCE cells. Error bars represent mean ± SEM. *** p < 0.001 relative to control.