Heat shock protein 90ß in the Vero cell membrane binds Japanese encephalitis virus

Wang, Yuan; Li, Yan; Ding, Tianbing

Immunofluorescence assay (IFA). When HSP90β-JEV binding on the Vero cell surface was confirmed, a JEV infection inhibition test was performed under impermeable conditions using an IFA with the rabbit anti-human HSP90β mAb mentioned above. Well-grown Vero cells were treated with 3.5 mM EDTA instead of trypsin to avoid cell surface protein digestion, washed with PBS, and transferred onto glass coverslips (15 µl, 1x10 6 /ml) in 24-well plates and grown with 10% FCS RPMI-1640 in a humidified incubator with 5% CO 2 at 37˚C. When the confluence reached 50-60%, the cells were washed 3 times with PBS and were fixed in 4% PBS-buffered formaldehyde for 30 min at room temperature. After a PBS wash, the fixed cells were successively incubated with rabbit anti-human HSP90β mAb (two concentrations of 10 µg/ml and 20 µg/ml) at 4˚C for 2 h, titrated JEV (MOI=0.1) at 4˚C overnight, anti-JEV mAb 2H4 (1:10, primary antibody) at 4˚C for 2 h and FITC-conjugated goat anti-mouse IgG (1:100, secondary antibody; Bioworld Technology, Inc., St. Louis Park, MN, USA) at room temperature for 1 h. Immunofluorescence was observed under a BH-60 immunofluorescence microscope (Olympus Corp.) after glycerol mounting.