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.

To look more closely at the expression and roles of 7,9-O-and 9-O-Ac modifications and their effects on viral infections, we prepared glycoengineered cell lines that lacked these modifications or that expressed higher levels of CasD1. A deletion and frameshift in CasD1 completely removed both 9-O-and 7,9-O-Ac expression, confirming this enzyme was responsible for creating both modifications, likely through addition to the C-7 position, from which it migrates to the C-9 position (5, 8, 9) . Adding CasD1 back into the cells by plasmid transfection restored modified Sia expression, but none of the cell clones isolated showed universally higher levels of modified Sia synthesis and population heterogeneity was still seen. The CasD1-transfected HEK-293 cells showed the greatest increase, whereas for A549 and MDCK cells the expression was similar to or lower than that for WT cells. However, even in the HEK-293 CasD1-OX cells, 9-O-Ac still only accounted for ϳ1.5% of total Sia, and 7,9-O-Ac was not detected. This indicates that the levels of these modifications are not only controlled by the expression of CasD1 and could be regulated by other processes. In addition, there is clearly a differential regulation of 7,9-O-Ac compared to 9-O-Ac, as overexpressing CasD1 did not lead to an increase in 7,9-O-Ac, since it was not detectable by HPLC and there was only very low fluorescence via staining with BCoV HE-Fc.