K⁺ channel modulation arrests BUNV trafficking in endosomes.

(A) Cells were treated with TEA (10 mM) for 30 min (or left untreated) and infected with SYTO82/DiD-BUNV for a further 4 hrs in the presence/absence of TEA. EGF-488 was added for the final 15 min of infection and cells were fixed 4 hpi. Confocal images were taken and the EGF-488 fluorescence channel removed in the representative images showing only SYTO82 and DiDvbt (n≥40). Scale bar = 10 μM. (B)(i) Cells treated with TEA (10 mM) or left untreated as in A, were infected with SYTO82/DiD-BUNV and fixed at 2, 4 or 8 hpi. EGF-488 (2 μg/ml) was added for 15 min prior to fixation as in A, with the representative images showing only SYTO82 and DiDvbt channels (n>65 cells). Scale bar = 10 μM. (ii) As in (i) but cells were treated with Qd (200 μM) and fixed 8 hpi (n>65 cells). (iii) The number of SYTO82/DiD-BUNV virions per cell were quantified using images from (i) and (ii) for n>65 cells and normalised to the untreated (no-drug) control. (C) A549 cells were infected with SYTO82/DiD-BUNV for 1 hour at 4°C and treated with cytopainter to label lysosomes. Cells were warmed to 37°C for 1 hr, virus/dye removed by washing and cells incubated for up to 8 hpi. Representative live cell images are shown (≥80 cells). Scale bar = 10 μM. (ii) The number of SYTO82/DiD-BUNV virions co-localising with cytopainter positive puncta were calculated and the % of co-localised puncta presented in (ii) (* = p≤0.05).