Expression of 9-O- and 7,9-O-acetyl modified sialic acid in cells and their effects on influenza viruses

Karen N. Barnard; Brian R. Wasik; Justin R. LaClair; Wendy S. Weichert; Brynn K. Lawrence; Colin R. Parrish

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. Here we examine and more closely define the expression and distribution of 9-O-128 acetyl and 7,9-O-acetyl Sia on cells in culture, define the effects of SIAE and CasD1 on 129 display of these Sia modifications, and perform an initial examination of the effects of 130 these modified Sia on infection by IAV and ICV. We used CRISPR-Cas9 for gene O-Ac, while BCoV HE-Fc recognizes primarily 7,9-O-Ac but also has a low affinity for 9-150 O-Ac (10, 11). By immunofluorescence microscopy, the different forms were present at 151 variable levels, with 10 to 70% of the cells showing staining under standard culture 152 conditions ( Fig. 2A,B) . MDCK-NBL2 and MDCK-type I cells showed both strong surface 153 and internal staining for 7,9-O-and 9-O-Ac forms, while both were mostly found in 154 intracellular locations in A549 and HEK-293 cells, with an occasional cell showing bright 155 surface staining. MDCK-type II cells showed staining only for 9-O-Ac and none for 7, O-Ac, indicating that these modifications are regulated independently. In HEK-293 and 157 All rights reserved. No reuse allowed without permission.