FIG. 1.

Selective cleavage of α(2,3)- and α(2,6)-linked sialic acids from Vero cells treated with NA and SAS inhibits BKV infection. (A) The removal of α(2,3)- and α(2,6)-linked sialic acids was detected by flow cytometry using a biotinylated α(2,3)-specific lectin, MALII, and a biotinylated α(2,6)-specific lectin SNA. Lectin binding to untreated cells is indicated by dotted lines, and lectin binding to treated cells is indicated by boldface lines. The shaded histogram is the binding of streptavidin conjugated to Alexa Fluor 488 in the absence of added lectins. NA at 0.025 U/ml selectively cleaves α(2,3)-linked sialic acid, as indicated by inhibition of MALII binding and no inhibition of SNA binding (top). NA at 0.800 U/ml cleaves both α(2,3)- and α(2,6)-linked sialic acid (middle). The recombinant enzyme, SAS, selectively cleaves α(2,3)-linked sialic acids (bottom). (B) After Vero cells were treated with phosphate-buffered saline, 0.800-U/ml NA, 0.025-U/ml NA, or 0.125-U/ml SAS as indicated, the cells were infected with BKV. After 48 h of incubation, the percentage of infected cells was determined by cellular expression of the viral protein T-Ag. Error bars represent the standard deviation. All enzyme treatments significantly reduced infection (P < 0.001). The experiment was repeated 10 times with identical results. (C) Representative images of the data shown in panel B (magnification, ×100). (D) NA and SAS have no effect on simian virus 40 (SV40) infection of Vero cells. Green, cells positive for T-Ag expression.