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

Sialic acids (Sia) are widely displayed on the surfaces of cells and tissues. Sia come in a variety of chemically modified forms, including those with acetyl modifications at the C7, C8, and C9 positions. Here, we analyzed the distribution and amounts of these acetyl modifications in different human and canine cells. As Sia or their variant forms are receptors for influenza A and influenza C viruses, we examined the effects of these modifications on virus infections. We confirmed that 9-O-acetyl and 7,9-O-acetyl modified Sia are widely but variably expressed across cell lines from both humans and canines. While they were expressed on the cell surface of canine MDCK cell lines, they were located primarily within the Golgi compartment of human HEK-293 and A549 cells. The O-acetyl modified Sia were expressed at low levels of 1-2% of total Sia in these cell lines. We knocked out and over-expressed the sialate O-acetyltransferase gene (CasD1), and knocked out the sialate O-acetylesterase gene (SIAE) using CRISPR/Cas9 editing. Knocking out CasD1 removed 7,9-O- and 9-O-acetyl Sia expression, confirming previous reports. However, over-expression of CasD1 and knockout of SIAE gave only modest increases in 9-O-acetyl levels in cells and no change in 7,9-O-acetyl levels, indicating that there are complex regulations of these modifications. These modifications were essential for influenza C infection, but had no obvious effect on influenza A infection.