Extended Data Figure 9. Effects of ATL3 depletion on virus particle maturation and the secretory pathway.

a, Cells were transduced with lentiviruses encoding for ARF4, ARF5 or non-targeting (NT) shRNAs. 72 h later RNA levels were quantified by RT-qPCR. Shown is the average fold change in viral RNA levels, corrected for HRPT. b-c, Cells were transduced with lentiviruses encoding for ATL2, ATL3 or NT shRNAs. b, After 72 h cells were transfected with a construct encoding for Gaussia luciferase and luciferase secretion was measured over 10 h. Graph shows the average levels of secreted luciferase compared to the 0 h time point. c, 72 h post transduction, cells were transfected with VSV-G_ts045_GFP and 8 h later incubated at 40°C. 16 h later temperature was lowered to 32°C and cells were imaged by confocal microscopy. Graph shows the means and SEM of perinuclear fluorescence intensity distribution for each condition. d-h, A549 cells were transduced with constructs of given specificities and cultured for 48 h. d, Cells were infected with ZV, and 48 h later viral RNA levels were determined by RT-qPCR (left panel). Intracellular viral RNA levels were corrected for HRPT. Titers of infectious virus contained in cell lysates and culture supernatants were determined using a PFU assay (right panel). For both panels, average fold changes are shown. e, Levels of prM and NS1 released from DV infected cells were calculated by quantifying western blots using Fiji software. Values were normalized to NT shRNA transduced cells (horizontal line). f, Cells were infected with DV for 48 h. RNA levels were determined by RT-qPCR; graph shows average fold change. g-h, Extracellular proteins were evaluated using western blot. h, Levels of prM released from DV infected cells were calculated using Fiji software. Values are displayed relative to NT shRNA transduced cells. All graphs show means and SEM derived from an average of 3 independent experiments. Significance was determined relative to NT shRNA transduced cells. * or ** represent p values < 0.05 or 0.01, respectively as determined by one-way ANOVA with a Dunnett’s multiple comparison analysis.