FIG. 2.

Effects of mutations at positions 226 and/or 228 on the hemadsorbing activity of human virus HA. Cos-1 cells were transfected with plasmid expressing HA. At 40 h posttransfection, they were washed twice with PBS containing 10% FCS and incubated with chilled 1% erythrocyte suspensions (chicken [in house], human [type O, St. Jude Children’s Research Hospital blood bank], or horse [Rockland]) in PBS. After a 1-h incubation at 4°C, the cells were washed at least five times with PBS and rinsed with methanol to remove erythrocytes that were nonspecifically bound. The cells were then air dried and stained with a 1:20 dilution of Giemsa stain (Sigma) for 15 min at 4°C. The numbers of hemadsorption-positive cells were recorded by examining five randomly selected microscopic fields that consisted of approximately 500 cells. Experiments were performed three times with similar results.