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.

Production of CasD1 knockout and CasD1-overexpressing cells. To better understand the control of expression of 7,9-O-and 9-O-Ac, glycoengineered cell lines were created by manipulating the expression of CasD1 and SIAE genes. To do this, we knocked out CasD1 via CRISPR-Cas9 editing or overexpressed CasD1 via transfection of an expression plasmid. Knockout variants of CasD1 (ΔCasD1) were prepared from MDCK-NBL2, A549, and HEK-293 cells using CRISPR-Cas9 targeting of early exons in the CasD1 gene (Fig. 4A ). ΔCasD1 clones were confirmed by examining for 7,9-O-and 9-O-Ac display and then by PCR and sequencing of the genomic region surrounding the deletion in modified Sia-negative clones (Fig. 4A ). For all cell types, ΔCasD1 variants showed loss of both 7,9-O-and 9-O-Ac display when probed with the different specific HE-Fc probes via flow cytometry and immunofluorescence microscopy ( Fig. 4B to D). , MDCK type I, and MDCK type II cells. Cells were probed with HE-Fcs probes derived from BCoV and PToV, which recognize 9-O-Ac and 7,9-O-Ac, respectively. Cells were permeabilized (perm) using Carbo-Free blocking reagent with 0.001% Tween 20, while nonpermeabilized cells (non-perm) received only Carbo-Free block. All cells were imaged at ϫ60; nuclei were stained with DAPI. Scale bar, 10 m. (B) Representative flow cytometry graphs showing distribution of positive staining for HEK-293, A549, and MDCK-NBL2 cell lines. BCoV and PToV HE-Fcs probes were used and permeabilization (perm) and nonpermeabilization (non-perm) methods were as in immunofluorescence assay staining.