Fig. 7: Secretion and spread of viral progenies is facilitated by secretory autophagosomes.

a Schematic for separation of infectious Zika virus populations over sucrose-step gradients. b Infectivity and vRNA in fractions collected from the gradient were measured by viral titration and RT qPCR. Data are presented as log₁₀[pfu/ml] and Zika RNA copies/ml. Peak I; heavier fraction, Peak II; lighter fraction. c Zika E protein was detected by ELISA in the two peak fractions upon treatment ±1% NP-40. Error bars represent mean ± s.d., n = 4. d Supernatants collected from Lyn^−/− cells were separated on sucrose gradients as described in a, and measured by plaque assay and RT qPCR. e Wild-type and Lyn^−/− Huh7 cells were infected with Zika at MOI 2 for 18 h, and subcellular structures were visualised by transmission electron microscopy. Scale bars, 200 nm. f DMVs and lysosomes were quantified on blinded images; n = 20 cells/condition. Data are represented as mean ± s.e.m. of five biological replicates; ***P < 0.001 (compared with wild type by ANOVA followed by one-sided Dunnett’s test). g Extracellular populations of venus-labelled Zika virus MR766 strain were separated over sucrose gradient described in a. Huh7 cells were infected with Fractions I and II, and spread of venus Zika was measured by FACS. Box indicates gating strategy on GFP+cells. h. Depletion of indicated proteins was performed by stable shRNA transductions in Huh7 cells, and verified by immunoblotting. i.–l. Cells described in h were cultured in medium supplemented with 10 mM BSA-conjugated fatty acids and infected with Zika at MOI 2 for 48 h, and measured for infectivity using plaque assays (grey bars) and extracellular vRNA using RT qPCR (red bars). Data represent mean ± s.d. of four independent experiments. **P < 0.01; ***P < 0.001 (compared with wild type by ANOVA followed by one-sided Dunnett’s test). m, n Secretion of either Zika (left panel) or Dengue (right panel) VLPs in shRNA-depleted cells described in g. VLP secretion was calculated as thev ratio of extracellular to total VLPs. Data are presented as a percentage of the control (wild-type) value (mean ± s.d. of three (Zika) and four (Dengue) independent experiments); ***P < 0.001 (compared with control by ANOVA followed by one-sided Dunnett’s test).