Expression of 9-O- and 7,9-O-Acetyl Modified Sialic Acid in Cells and Their Effects on Influenza Viruses

Barnard, Karen N.; Wasik, Brian R.; LaClair, Justin R.; Buchholz, David W.; Weichert, Wendy S.; Alford-Lawrence, Brynn K.; Aguilar, Hector C.; Parrish, Colin R.

Cells and viruses. HEK-293, A549, MDCK-NBL2, MDCK type I, and MDCK type II cells were grown in Dulbecco modified Eagle medium (DMEM) with 5% fetal bovine serum and 50 g/ml gentamicin. HEK-293, A549, and MDCK-NBL2 cells were obtained from the American Type Culture Collection (ATCC). MDCK type I and type II cells were gifts from William Young (University of Kentucky) (35, 37, 54, 55) . Immunofluorescence microscopy and flow cytometry. Cells were stained using probes derived from viral hemagglutinin-esterase proteins fused to human IgG1 Fc (HE-Fc). The porcine torovirus strain 4 HE-Fc (PToV HE-Fc) primarily recognizes 9-O-Ac, and the bovine coronavirus Mebus strain HE-Fc (BCoV HE-Fc) recognizes 7,9-O-Ac and shows low levels of binding to 9-O-Ac (10, 11) . For immunofluorescence microscopy, the cells were seeded onto glass coverslips and incubated overnight at 37°C and 5% CO 2 . Coverslips were fixed in 4% paraformaldehyde (PFA) for 15 min. Coverslips were incubated with Carbo-Free blocking solution (Vector Laboratories) for 1 h at room temperature, with optional permeabilization with 0.001% Tween 20. For staining, HE-Fc probes were precomplexed with Alexa 488-labeled antihuman IgG antibody for 1 h at 4°C and then diluted in Carbo-Free blocking solution to a final concentration of 5 g/ml HE-Fc and a 1:500 dilution of secondary antibody. Cells were stained with HE-Fc/anti-IgG complex for 1 h at room temperature. Coverslips were mounted using Prolong Antifade-Gold with DAPI (4=,6=-diamidino-2-phenylindole; Invitrogen). Cell were imaged using a Nikon TE300 fluorescence microscope. For flow cytometry, cells were seeded onto nonadherent cell culture dishes and incubated overnight at 37°C and 5% CO 2 . Cells were collected using ice-cold phosphate-buffered saline (PBS; HEK-293 and A549 cells) or Accutase (Sigma, MDCK cells) to retain surface glycans and then fixed in 4% PFA for 15 min. The cells were blocked as described above. HE-Fc probes were prepared as described above with final concentrations of 5 g/ml HE-Fc probe and 1:1,200 of anti-IgG. A Guava EasyCyte Plus flow cytometer (EMD Millipore, Billerica, MA) was used to collect data, followed by analysis using FlowJo software (TreeStar, Ashland, OR). Statistical analyses were performed using GraphPad Prism software (v8).