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.

There is currently only sporadic information about the expression of modified Sia on commonly used cell lines or an understanding of how that compares to the expression in animal tissues. We examined cell lines that are widely used in many experimental systems: A549 human type II alveolar epithelial cells, HEK-293 human kidney derived cells (possibly embryonic adrenal precursor cells [34] ), and MDCK canine kidney epithelial cells. In addition, we tested MDCK type I and type II cells that were previously subcloned by others from the ATCC MDCK line (MDCK-NBL2) and which have been extensively characterized (35) (36) (37) . We used probes derived from porcine torovirus (PToV) and bovine coronavirus (BCoV) hemagglutinin-esterase proteins (HE) that were fused to human IgG1 Fc and which had the esterase active site inactivated (HE-Fc). The PToV HE-Fc probe recognizes 9-O-Ac, while BCoV HE-Fc recognizes primarily 7,9-O-Ac, although with a low affinity for 9-O-Ac (10, 11) . As observed by immunofluorescence microscopy, the different forms were evident at variable levels, with between 10 and 70% of the cells of each type showing staining under standard culture conditions (Fig. 2) . MDCK-NBL2 and MDCK type I cells showed both strong surface and internal staining for 7,9-O-and 9-O-Ac forms, while both were mostly found in intracellular locations in A549 and HEK-293 cells, with an occasional cell showing bright surface staining. MDCK type II cells showed staining only for 9-O-Ac and none for 7,9-O-Ac, indicating that these modifications are regulated independently. In HEK-293 and A549 cells, both 7,9-O-and 9-O-Ac appeared to be localized within the Golgi compartment, as confirmed by costaining with the Golgi marker GM130 (Fig. 3) . Similar localization differences between some cell lines have been seen previously using the ICV HEF as a probe (38) . In our studies, there was inherent variability within populations in terms of both level of staining and localization. For example, in MDCK-NBL2 not all cells were positive for 9-O-Ac, whereas in MDCK type I some cells retained more 9-O-Ac and 7,9-O-Ac internally, while others displayed more of the modified Sia on their surfaces ( Fig. 2A) . This heterogeneity was consistent between different passages of each cell line.