Distinct Roles for Sialoside and Protein Receptors in Coronavirus Infection

Qing, Enya; Hantak, Michael; Perlman, Stanley; Gallagher, Tom

We next determined whether virus binding to sialic acids elevates protein receptordependent virus entry. Here, authentic JHM HEϪ -CoV particles were bound for 2 h to DBT (murine), HeLa-mCEACAM (human), or BHK (hamster) cells, with or without prior cell exposure to neuraminidase. Infection was then measured by quantifying viral firefly luciferase (Fluc) reporter expression. The neuraminidase pretreatments decreased (E) HeLa cells were formalin fixed, rinsed, and incubated for 2 h at 4°C with VLPs bearing A59 or JHM S. Rluc input multiplicities were equal for all VLP inoculations. Media were removed, cells were lysed, and cell-associated Rluc activities were quantified. Data are presented after subtracting background ("No S") Rluc-positive (Rluc ϩ ) VLP levels, with each data point representing averages from independent experiments (N ϭ 8 [A59] and 7 [JHM]; n ϭ 4 technical replicates per experiment). Error bars present standard errors (SE) from the mean. Statistically significant deviations from background ("No S") binding were assessed by unpaired Student's t test and are indicated as follows: ns, not significant; *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ****, P Ͻ 0.0001. (F) HeLa or HeLa-mCEACAM cells were fixed and treated with vehicle (PBS) or neuraminidase (NA) for 3 h at 37°C. JHM-CoV VLPs were then added for 2 h at 4°C, cell-associated Rluc activities were quantified, and data are presented after subtracting background ("No S") Rluc ϩ VLP levels. Error bars present standard errors (SE) from the mean. Statistically significant deviations were assessed by unpaired Student's t test and are indicated as follows: ns, not significant; *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001; ****, P Ͻ 0.0001.