FIGURE 4.

HD5 colocalizes with BKV. A, subconfluent Vero cells plated on glass coverslips were challenged with AF488-BKV (m.o.i. 16) at 37 °C for 1 h in MEM with or without serum. Cells were washed with MEM, restored with complete media, and virus allowed to internalize by incubating cells at 37 °C for 4 h. Cells were fixed and stained for HD5 using HD5-specific antisera and AF568-labeled goat anti-rabbit antibodies. Representative images were obtained using immunofluorescence confocal microscopy. B, in a cell-free assay, PBS containing 2.5 μg of HD5, 6142 HAU of BKV, HD5, and BKV together, and HD5 and BKV in 750 mm NaCl supplemented PBS were incubated at 1 h at 4 °C. This was overlaid on a gradient containing 80 and 30% nycodenz and centrifuged at 209,000 × g for 5 h. Fractions of 75 μl were collected and immobilized onto two polyvinylidene difluoride membranes. Membranes were probed for either HD5 or BKV and binding was detected using AF680-labeled goat anti-rabbit antibody.