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.

Due to the heterogeneity and low expression of 7,9-O-and 9-O-Ac in WT cells, we sought to engineer cells with more homogeneous and higher levels of these modifications. We overexpressed the human CasD1 (CasD1-OX) in the ΔCasD1 variants of MDCK-NBL2, A549, and HEK-293 cells, expecting that expression of CasD1 under a strong promoter in the ΔCasD1 background would increase the consistency of the synthesis and display of 9-O-and 7,9-O-acetyl Sia relative to the WT cells. CasD1-OX cells showed significantly higher levels of CasD1 mRNA compared to WT cells, indicating that the CasD1 expression plasmid was being transcribed at high levels ( Fig. 4E) . However, only a modest increase in average 9-O-and 7,9-O-Ac expression across the population in HEK-293 cells was seen by flow cytometry, whereas fluorescence microscopy showed heterogeneity of expression in the population ( Fig. 4B to D). The transfected MDCK and A549 CasD1-OX cells showed recovery of 9-O-and 7,9-O-Ac synthesis when analyzed by flow cytometry and immunofluorescence microscopy, but the levels were not as high as those seen in WT cells ( Fig. 4B to D). HPLC analysis confirmed the expression levels for HEK-293, A549, and MDCK cells (Fig. 4F ). In addition, heterogeneity was still seen in these populations for 9-O-Ac. This suggests that 7,9-Oand 9-O-Ac expression is not directly regulated by the levels of CasD1 gene expression but may be affected by posttranslational regulation of CasD1 activity or removal of the modifications by SIAE or other enzymes. In addition, these modifications are not regulated the same across individual cells, as evidenced by the population heteroge- Effects of Modified Sialic Acids on Influenza Viruses ® neity. SIAE transcripts were consistently expressed in HEK-293 WT, ΔCasD1, and CasD1-OX cell populations by qPCR analysis, although SIAE mRNA levels in CasD1-OX cells were lower than those seen in WT (Fig. 4E ).