Adenovirus type 37 uses sialic acid as a cellular receptor on Chang C cells

Abstract

Epidemic keratoconjunctivitis (EKC) is a severe eye infection caused mainly by adenovirus type 8 (Ad8), Ad19, and Ad37. We have shown that the EKC-causing adenoviruses use sialic acid as a cellular receptor on A549 cells instead of the coxsackie-adenovirus receptor, which is used by most adenoviruses. Recently, Wu et al. (Virology 279:78-89, 2001) proposed that Ad37 uses a 50-kDa protein as a receptor on Chang C conjunctival cells and that this interaction is independent of sialic acid. According to the American Type Culture Collection, this cell line carries HeLa cell markers and should be considered to be a genital cell line. This prompted us to investigate the function of sialic acid as a cellular receptor for Ad37 in Chang C cells. In this study, we demonstrate that enzymatic removal or lectin-mediated blocking of cell surface sialic acid inhibits the binding of Ad37 virions to Chang C cells, as does soluble, virion-interacting sialic acid-containing substances. The binding was Ca2+ or Mg2+ ion independent and mediated by the knob domain of the trimeric viral fiber polypeptide. Moreover, Ad37 virions infected Chang C cells and two other genital cell lines (HeLa and SiHa) as well as a corneal cell line in a strictly sialic acid-dependent manner. From these results, we conclude that Ad37 uses sialic acid as a major receptor in cell lines derived from both genital and corneal tissues.

FIG. 1.

Enzymatic removal of sialic acid from the surface of Chang C cells inhibits attachment of Ad8 and Ad37 virions. Chang C cells (2 × 10⁵) in suspension were treated with or without neuraminidase and incubated with ³⁵S-labeled Ad8 or Ad37 virions (A) or Ad5 or Ad19p virions (B) as described in Materials and Methods. The cell-associated radioactivity was measured and plotted as the amount of virions bound divided by the total number of virions added. The data represent the means from three independent experiments.

FIG. 2.

Ad37 virions attach to the surface of Chang C cells through an interaction between viral fiber knobs and cellular sialic acid. (A) Ad37 virions were prevented from binding to Chang C cells with polyclonal rabbit antiserum raised against the Ad37 fiber knob. ³⁵S-labeled Ad37 virions were incubated with 10-fold dilutions of antiserum and then added to 2 × 10⁵ Chang C cells/sample. Unbound virions were removed by washing, and the cell-associated radioactivity was measured. pre-im, preimmune serum; anti-knob, serum raised against the Ad37 knob. (B) Neuraminidase-treated (neu) or mock-treated (CTRL) Chang C cells were incubated with 5 μg of His-tagged recombinant fiber knobs as indicated and subsequently detected with mouse anti-His tag antibodies, followed by FITC-labeled rabbit anti-mouse antibodies. Cells (10⁴/sample) were counted with a flow cytometer. All data represent the means from three independent experiments.

FIG. 3.

Scatchard analysis of Ad37 binding to Chang C cells. Increasing amounts of ³⁵S-labeled Ad37 virions were incubated with Chang C cells in suspension as described in Materials and Methods. Ratios of bound (cell-associated) virion radioactivity to free virion radioactivity (B/F) were plotted against the number of bound virions per cell. The disassociation constant (K_D = 0.35 nM) was derived from the slope of the line. The number of receptor sites for Ad37 virions (2.9 × 10⁴) was derived from the value extrapolated to intercept the abscissa. The data shown were obtained from three separate experiments.

FIG. 4.

Sialic acid-interacting lectins inhibit attachment of adenoviruses to Chang C cells in a glycosidic bond-specific and serotype-specific manner. Chang C cells (2 × 10⁵) were preincubated with WGA, SNA, or MAA and then further incubated with one of the following ³⁵S-labeled virions: A and C, Ad37; B, Ad5. Unbound virions were removed by washing, and the cell-associated radioactivity was measured. All data represent the means from three independent experiments. CTRL, control.

FIG. 5.

Ad37 virions do not require divalent cations for attachment to Chang C or HCE cells. Chang C or HCE cells were incubated on ice with ³⁵S-labeled Ad37 virions in Tris-HCl (pH 7.4) containing 1% FCS and 10 mM EDTA (A) or various concentrations of CaCl₂ or MgCl₂ (B and C). Unbound virions were removed by washing, and cell-associated radioactivity was measured. The data represent the means from three independent experiments. CTRL, control.

FIG. 6.

Neuraminidase treatment of genital (Chang C, HeLa, SiHa) and corneal (HCE) cells protects from infection by Ad37 (A) but not by Ad5 (B). Adherent cells were preincubated with or without neuraminidase and then with virion dilutions corresponding to 5,000 FFU/sample, permitting attachment to cells but not internalization. Thus, the virion dilutions were adjusted in order to obtain 100 FFU/viewing field in the control wells (nontreated cells). Then, unbound virions were removed by washing and incubated at 37°C, thus permitting internalization of virions in cells. At 40 h postinfection, the cells were rinsed, fixed, stained, and examined in an immunofluorescence microscope (C) as described in Materials and Methods. The results are presented as the means from three independent experiments. CTRL, control.

FIG. 6.

Neuraminidase treatment of genital (Chang C, HeLa, SiHa) and corneal (HCE) cells protects from infection by Ad37 (A) but not by Ad5 (B). Adherent cells were preincubated with or without neuraminidase and then with virion dilutions corresponding to 5,000 FFU/sample, permitting attachment to cells but not internalization. Thus, the virion dilutions were adjusted in order to obtain 100 FFU/viewing field in the control wells (nontreated cells). Then, unbound virions were removed by washing and incubated at 37°C, thus permitting internalization of virions in cells. At 40 h postinfection, the cells were rinsed, fixed, stained, and examined in an immunofluorescence microscope (C) as described in Materials and Methods. The results are presented as the means from three independent experiments. CTRL, control.