PMC

Ultrastructure of BKV reveals HD5-induced aggregation of viral particles. 768 HAU of purified BKV was treated with 50 μg/ml HD5, HBD1, or an equal volume of H₂O and incubated at 4 °C for 1 h. Virus was then immobilized on a carbon-coated formvar grid, washed with water, and stained with 2% uranyl acetate. Using transmission electron microscopy, BK virions were visualized (magnification ×39,000). The lower panels show that BK virions accumulate in dense aggregations when treated with HD5 (magnification ×112,000).

Pretreatment of BKV with HD5 inhibits infection. Purified BKV at the indicated m.o.i. was incubated with 50 μg/ml HD5 or the same volume of H₂O for 1 h at 4 °C in MEM containing 5% FBS, serum-free MEM, or PBS. Vero cells were then infected with the HD5/BKV mixture at 37 °C for 1 h. Unbound virus was removed by washing and complete media was added to cells. Cells were fixed and the percentage of the total cells expressing V-Ag determined at 72 h post-infection. The graph shows the average of five experiments. Error bars represent the standard deviation.

Incubation of HD5 and BKV inhibits viral binding. A, AF488-BKV (m.o.i.: 16) was incubated with H₂O(black line) or 50 μg/ml of the indicated defensins (dotted line) in MEM, MEM containing 5% FBS, or PBS at 4 °C for 1 h. Vero cells in suspension were then incubated with the BKV mixture at 4 °C for 1 h and viral binding was evaluated using flow cytometry. Filled histograms represent cells in the absence of AF488-BKV. B, a 1:100 dilution of purified BKV was incubated with H₂O or 50 μg/ml HD5 in the indicated media at 4 °C for 1 h. This preparation was 2-fold serially diluted across a U-bottom plate containing PBS. The HA plate was incubated with 0.5% erythrocytes in PBS at 4 °C for 16 h. A representation image of a HA plate is shown. C, Vero cells in suspension were treated with 50 μg/ml HD5 (dotted line) or an equal volume of H₂O(bold line) in the indicated solutions at 4 °C for 1 h. Cells were washed and incubated with AF488-BKV (m.o.i. 16), biotintylated-SNA (lectin specific for α(2,6)-linked sialic acid), or biotintylated-MALII (lectin specific for α(2,3)-linked sialic acid) at 4 °C for 1 h. For SNA and MALII samples, cells were washed and incubated with AF488-labeled streptavidin. Fluorescence intensity was measured using flow cytometry. Filled histograms represent unstained cells.

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.

HD5 inhibits SV40 and JCV infection. Subconfluent Vero cells or SVG-A were infected with SV40 (m.o.i. 2) or JCV (m.o.i. 5), respectively, in 2% FBS containing MEM at 37 °C for 1 h in the absence or presence of defensins at the indicated concentrations. After infection, cells were washed twice with MEM to remove unbound virus, and complete media containing defensins was added to cells for the duration of the incubation. Cells were fixed at 48 (Vero) and 72 h (SVG-A) post-infection and stained for V-Ag. Approximately 8,000 cells were screened for V-Ag expression. The graph shows the average of three experiments each of which has been normalized to infected cells without defensin treatment. Error bars represent the standard deviation. The bottom panel shows representative images of infected cells at the 50 μg/ml defensin concentration (magnification ×100). V-Ag-expressing cells are in green and Evans blue cytoplasmic labeling in red.

HNP1, HD5, and HBD2 block BKV infection of Vero cells. A, Vero cells plated at subconfluent density were infected with BKV (m.o.i. 4) in 2% FBS containing MEM at 37 °C for 1 h. Indicated concentrations of defensins were present during infection. Cells were washed to remove unbound virus and 5% FBS containing MEM with the same concentrations of defensins was added to the cells and remained present for the duration of the experiment. Cells were fixed at 72 h and infection was determined by scoring the number of cells expressing the viral protein V-antigen (V-Ag). The graph represents the average of three experiments each of which have been normalized to infected cells without defensin treatment. Error bars represent the standard deviation. B, representative images of BKV-infected Vero cells are shown (magnification ×100). V-Ag-expressing cells are in green and Evans blue cytoplasmic labeling in red. C, Vero cells were infected with purified BKV (m.o.i. 4) in 2% FBS containing MEM at 37 °C for 1 h. Unbound virus was removed by washing and complete media added to cells. At the indicated times, 50 μg/ml HNP1 or HD5 was added to culture media. At the 0-h time point, defensins were added during and also directly following infection. Cells were stained and scored for V-Ag expression at 72 h post-infection. The graph shows the average of three experiments, each of which have been normalized to infected cells without defensin treatment. Error bars represent the standard deviation.