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 effect of 7,9-O-and 9-O-Ac on NA cleavage, mouse erythrocytes were incubated with soluble NA, in the form of NA-expressing viruslike particles (VLPs) (48) . Freed Sia was then collected and analyzed using HPLC, and the profile of Sia released by the NA VLPs was compared to Sia composition on mouse erythrocytes released by chemical hydrolysis (Fig. 7C ). The profiles for both N1 and N2 VLPs showed a preferential release of unmodified Neu5Ac compared to modified Sia forms, shown in the increased proportion of this Sia in the N1 and N2 profiles. Compared to the chemical release profile, the N1 VLPs did not release any detectable O-acetylated Sia, while the N2 VLPs were able to release 9-O-Ac (Neu5,9Ac 2 ) but released significantly less 7-O-and 8-O-Ac and were unable to release any 7,9-O-Ac (Neu5,7,9Ac 3 ). Similar to N2 VLPs, commercial NeuA from Arthrobacter ureafaciens, used as an activity control, also had a bias toward unmodified Neu5Ac, with decreased activity against 7-O-, 8-O-, and 7,9-O-Ac. These results indicate that mono O-acetyl modifications, including 7-O-, 8-O-, and 9-O-Ac, reduced the Sia susceptibility to NA cleavage and that diacetyl 7,9-O-Ac was the most resistant to NA cleavage. In addition, there was variability between the ability of the NA VLPs and NeuA to cleave the different Sia forms. Although the presence of these modified Sia on cells was too low to affect IAV infection, it is likely that higher levels present on erythrocytes, or potentially in secreted proteins in mucus, would inhibit infection or the release of virus.