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 determine the role of SIAE activity in regulating 7,9-O-and 9-O-Ac display, SIAE knockout (ΔSIAE) cells were generated from WT HEK-293 and A549 cells by targeting early exons in the SIAE gene (Fig. 5A ). Complete knockout of SIAE was confirmed both by genotyping to show deletions in both alleles and by loss of mRNA by qPCR ( Fig. 5A and D) . ΔSIAE cells showed a small but significant increase in surface display and a large increase in internal display of 9-O-Ac based on flow cytometry, but they did not appear to show any changes in the surface or internal display of 7,9-O-Ac ( Fig. 5B and C) . HPLC analysis also showed a small increase of 9-O-Ac in HEK-293 cells, but no 7,9-O-Ac was detected in either cell line (Fig. 5E ). When the overexpression CasD1 plasmid was transfected into ΔSIAE cells (ΔSIAEϩCasD1), the cells showed an increase in both surface and internal 9-O-Ac but no increase in surface or internal display of 7,9-O-Ac by either flow cytometry or immunofluorescence microscopy ( Fig. 5B and C) . HPLC confirmed the flow cytometry results by showing a small increase of 9-O-Ac levels in HEK-293 cells, similar to those seen in CasD1-OX (Fig. 5E) . A549 cells showed a small but nonsignificant increase in 9-O-Ac levels compared to WT by HPLC analysis. Interestingly, HEK-293 ΔSIAEϩCasD1 cells grew more slowly than either ΔSIAE or WT cells (Fig. 5F ). However, A549 ΔSIAEϩCasD1 showed increased growth rates compared to ΔSIAE or WT cells, while ΔSIAE cells had slightly lower growth rates compared to WT. This suggests that 7,9-O-and 9-O-Ac may affect cell metabolism and growth rates and that these effects may be cell type specific. Overall, these results show that SIAE regulates levels of 9-O-Ac and 7,9-O-Ac but that knocking out SIAE only leads to small increases in these modifications. However, there appear to be mechanisms regulating the surface display (Fig. 6C and D) . The C/Ann Arbor and C/Victoria recovered a low level of infectivity in the MDCK CasD1-OX cells, whereas the D/bovine and D/swine strains had a higher level of infectivity recovered in the CasD1-OX cells compared to ICV but still were much lower than in WT MDCK cells. The inability of the ICV and IDV strains to fully recover infectivity may be due to the low surface levels of 9-O-Ac Sia on MDCK CasD1-OX cells. Although the CasD1-OX cells had detectable surface levels of 9-O-Ac, these levels were lower than in WT MDCK cells and could be below the receptor levels required for efficient infection by these viruses. This suggests that ICV and IDV are able to utilize the levels of modified Sia on WT MDCK cells as their primary receptors for binding and infection but cannot infect when they are removed or below a certain necessary threshold.