Production of SYTO82/DiD-BUNV to monitor virus trafficking.

(A) Time course of A549 cells infected with BUNV. Cells were lysed at 3 hr intervals post-infection. Western blot analysis of BUNV-N protein and GAPDH (loading control) are shown (n = 3). (B) A549 cells were infected with BUNV supernatants collected from infected A549 cells at the indicated 3 hr intervals. Infected cells were fixed at 18 hpi and BUNV-N protein was labelled using anti-BUNV-N antibodies alongside Alexa-Fluor 594 nm secondary antibodies. Widefield images taken on the IncuCyte Zoom are shown (n = 3). Scale bar = 200μm (C) Schematic representation of BUNV labelling. A549 cells were infected with BUNV for 18 hrs, then SYTO82 dye was added to label the viral RNA segments until virus supernatants were collected at 24 hrs. Virus supernatants were concentrated, the BUNV envelope labelled with DiDvbt and SYTO82/DiD-BUNV was purified on a 10–30% iodixanol gradient. 1 ml fractions were collected (n = 3). (D) Fractions from SYTO82/DiD-BUNV purification were used to infect A549 cells for 18 hrs. Western blot analysis for BUNV-N was performed to confirm virus infectivity. (E) Cells were infected as in D, fixed, and stained with anti-BUNV-N and Alexa Fluor-488 antibodies. Widefield images were taken on the IncuCyte Zoom (n = 3). Scale bar = 200 μM. (F) A549 cells were infected with SYTO82/DiD-BUNV for 2 hrs and fixed. SYTO82 (em.max 560 nm) and DiDvbt (em.max 665 nm) fluorescent signals were imaged alongside DAPI in fixed cells. (G) Cells were infected with SYTO82/DiD-BUNV for 1 hr at 4°C, then heated to 37°C and infection was allowed to proceed until fixing at 2 hrs, 4 hrs, 8 hrs or 12 hrs. Biotinylated EGF-488 (2 μg/ml) was added for 15 min at 37°C prior to fixing to act as a cell marker. Confocal images were taken for n>80 cells for each time point and the EGF-488 fluorescence channel was removed in the representative images (Scale bar = 10 μM). (H) NH4Cl (10 μM) was added at the indicated timepoints post-BUNV infection and BUNV-N expression assessed by western blot analysis 24 hours post-infection. CTL = no drug included during the timecourse (n = 3).