Non-canonical functions of ATG16L1 in epithelial cells limit lethal infection by influenza A virus (2021)
Figure 6

Fig. 6. Non-canonical autophagy reduces virus replication, endosome fusion and interferon signalling.

A). The WD domain of ATG16L1 reduces IAV replication in MEFs. Cells were infected with IAV and virus titres were estimated at 8 hours post infection by plaque assay. Graphs show three individual experiments with a bar (+/- SD) at the mean and were compared using two-way ANOVA with Bonferroni post-tests ***P<0.001).

B). IAV binding is similar between control and δWD MEFs. Cells were bound with IAV X31 labeled with AF488 on ice for 1 h, and fixed immediately. The cells were stained with anti-HA (H3SKE, red) without permeabilisation to visualise extracellular IAV particles, counter stained with Hoechst (nuclei) and WGA-AF647 to label the cell membrane. Cells were imaged by automated confocal microscopy using a 40x objective, and the maximum intensity projection images were analysed to quantify the number of bound IAV particles per cell. The results and means (horizontal line) of n = 3 independent experiments (50-100 cells per experiment) are shown.

C) IAV infection following acid bypass is comparable between control and δWD MEFs. IAV was bound on ice for 1 h, and warmed at either neutral pH (6.8) or low pH (pH 5.0) for 2 min, followed by incubation in STOP medium containing NH4Cl for 18 hours. Cells were fixed and stained for NP and Hoechst, imaged by automated confocal microscopy and maximum intensity projection images analysed to quantify viral infectivity. The results from N=3 independent experiments (5,000-10,000 cells quantified per experiment), and the means are shown. Scale bar; 500 µm.

D). IAV fusion with endosomes is increased in δWD MEFs. Cells were infected with dual labeled (R18/SP-DiOC18) IAV for 1 h, fixed, counterstained with Hoechst (nuclei blue) and WGA-AF647 (cell perimeter in white). Cells were imaged by automated confocal microscopy and the number of fusion events per cell (represented by number of SP-DiOC18 puncta) was quantified. Fused and non-fused viral particles are shown as green and red spots respectively. The results and means (horizontal lines) of n = 3 independent experiments (30-60 cells per experiment) are shown. Representative cells overlayed with the cell boundary segmented from the WGA staining are shown on the right.

E, F) Numbers of fusion positive cells is increased for δWD MEFs. MEFs from δWD (white circles) or littermate control mice (black circles) were incubated with dual-labelled (SP-DiOC18/R18) IAV at 40C for 45 min and warmed to 37°C for 30 ad 60 minutes. Cells were harvested by trypsinisation, fixed in PFA and analysed by flow cytometry to determine to determine percentage of cells positive for fusion (E) and median fluorescence intensity (MFI) of dequenched SP-DiOC18 signa (F). Graphs show individual replicates (n = 3) with a horizontal line at the mean and were compared using two-way ANOVA with Bonferroni post-tests **P<0.01, ***P<0.001). pH-dependent fusion was assessed by adding bafilomycin A1 to the infection assay (Fig EV5).

(G, H) Endocytosis of IAV increases in δWD MEFs. MEFs from δWD or littermate control mice were incubated with AlexFluor 488-labelled IAV at 40C for 1hr and warmed to 37°C for 15 Cells were fixed and permeabilised and stained with antibody against HA (red). The blue line indicates the plasma membrane of the cell. Green arrows indicate examples of viruses only labelled with green fluorescence. Double-labelled virus particles (green-red) represent non-endocytosed particles bound at the cell surface, whereas green particles represent endocytosed particles. The results and means of n = 3 independent experiments (50-100 cells per experiment) are shown. Student's t-test; p<0.01.The dotted square in the top row panels are enlarged in the bottom row. Scale bars; 10 µm (top row) and 5 µm (bottom row).

(I, J) Interferon signalling increases in δWD MEFs. MEFs from δWD or littermate control mice were infected with IAV. At the indicated time points, mRNA transcripts were evaluated by qPCR for IFIT1 (I) and ISG15 (J). Data from 3 replicates are shown, bars represent the mean ± SD and were compared by two-way ANOVA with Bonferroni post-tests (***P<0.001)

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