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.

One candidate for control of these modifications is SIAE. When we knocked SIAE out of HEK-293 and A549 cells, we saw an increase in 9-O-Ac that was still retained in the Golgi compartment, but there was no increase in 7,9-O-Ac. Expressing CasD1 from a plasmid in ΔSIAE HEK-293 and A549 cells resulted in an additional small increase in 9-O-Ac that was still Golgi compartment associated, but with little increase in expression on the cell surface. Similar to the CasD1-OX cells, no increase in 7,9-O-Ac was seen in either ΔSIAE and ΔSIAEϩCasD1 cell lines by HPLC analysis. Both ΔSIAEϩCasD1 lines had altered growth rates compared to WT cells: HEK-293 ΔSIAEϩCasD1 had delayed growth rates, while A549 ΔSIAEϩCasD1 had increased growth rates. This suggests that dysregulation of SIAE and CasD1 by gene manipulation affects cell metabolism and growth in a cell-type-specific manner, possibly through the buildup of glycoproteins in the Golgi or through dysregulation of the sialic acid recycling pathway. These effects on cell growth could have implications for cancer and organismal development, since these variant Sia are involved in both these processes (15-17, 20-23, 49) . Further research is needed to determine how 7,9-O-and 9-O-Ac expression is regulated, how they are transported within the ER and Golgi bodies, and what roles they play in cell growth. Of particular interest will be disentangling the individual regulation of 7,9-O-Ac expression compared to 9-O-Ac, since there do seem to be differences in how they are regulated in cells in the present study versus previously reported expression in animal tissues (10, 11) .