(A) Schematic of time-of-addition assay. (B) Time-of-addition assay with D1min, cell-penetrating peptides TAT-D1min and D1min-TAT, and anti-BKV neutralizing antibody P8D11. BKV infected cells were treated with inhibitors at 10-fold over measured EC50 concentrations. Productive delivery of the viral genome to the nucleus is measured by the fraction of RPTE cells expressing BKV TAg by indirect immunofluorescent staining 48 hr post-infection (h.p.i), relative to DMSO-treated samples (mean ± SD, n = 4). (C) Virus cell binding inhibitor assay. BKV was treated with indicated inhibitor at indicated concentrations for 1 hr on ice, adsorbed to cells for 1 hr at 4°C, unbound virus washed away, and remaining cell-associated virus measured by indirect immunofluorescent staining of VP1 (mean ± SD, n = 3). (D) ER-to-cytosol retrotranslocation assay. RPTE cells subjected to a synchronized BKV infection (high MOI), cells were harvested 24 h.p.i, and lysates were fractionated into a supernatant (cytoplasmic) and pellet fraction. Fractions were then analyzed by SDS-PAGE, and VP1 protein and cellular compartment markers were detected by immunoblotting. (E) Representative microscopy images of VP2/3 exposure assay. Minor capsid proteins were detected using a polyclonal antibody able to recognize both VP2 and VP3. Scale bar: 20 μm. (F) Quantification of images exemplified in (E), measuring fraction of VP1 stain co-localizing with VP2/3 stain, averaged per well. Rotated data indicate calculated co-localization between VP1 and VP2/3 stains after rotating VP2/3 images 90° to assess rate of random association between the two (mean ± SD, n = 4). (G) Quantification of VP1 foci in images exemplified in (E), averaged per well (mean ± SD, n = 4 for infected samples, n = 2 for uninfected samples). (H) Quantification of VP2/3 foci in images exemplified in (E), averaged per well (mean ± SD, n = 4 for infected samples, n = 2 for uninfected samples).