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 low levels of 9-O-and 7,9-O-Ac detected on HEK-293, A549, and MDCK 183 cells by HPLC could be due to the heterogeneity of the population, as this method 184 measures total Sia for all cells in the population. However, it is likely that even on higher The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/650341 doi: bioRxiv preprint 4B, C, D). This agrees with previous findings that loss of CasD1 leads to loss of both 204 7,9-O-and 9-O-Ac modifications in haploid HAP1 cells (7). HPLC analysis of total Sia 205 showed a significant decrease in 9-O-Ac expression compared to wild-type (WT) cells, 206 however very low levels (<1%) were still detectable, despite a lack of staining with the 207 HE-Fc probes (Fig. 4E) . This could be due to exogenous Sia from the fetal bovine 208 serum in the growth media, as is the case for Neu5Gc which is also detectable at 209 similarly low levels on cells even though the gene for Neu5Gc synthesis, CMAH, is not 210 functional in humans or canines (Supplemental showed significantly higher levels of CasD1 mRNA compared to WT cells, indicating 218 that the CasD1 expression plasmid was being transcribed at high levels ( Fig. 4E) . 219