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ATG9A impacts viral inclusions independently of Rab11a–vRNP interaction.

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posted on 2023-11-20, 18:46 authored by Sílvia Vale-Costa, Temitope Akhigbe Etibor, Daniela Brás, Ana Laura Sousa, Mariana Ferreira, Gabriel G. Martins, Victor Hugo Mello, Maria João Amorim

(A–C) Cells (GFP-Rab11a WTlow or GFP-Rab11a DNlow) were treated with siRNA non-targeting (siNT) or targeting ATG9A (siATG9A) for 48 h and then infected or mock-infected with PR8 virus for 10 h, at an MOI of 3. (A) Viral production was determined by plaque assay and plotted as PFU per milliliter (mL) ± SEM. Data represent 6 replicates from a single experiment. Two independent experiments were performed. Statistical analysis was done by one-way ANOVA, followed by a Kruskal–Wallis test (*p < 0.05; ***p < 0.001). (B) The protein level of ATG9A, lamin B, GFP, and Rab11a before infection were quantified by western blotting. The levels of ATG9A were plotted as the relative expression to lamin B level ± SEM. Expression was normalized to siNT from mock-infected cells. The data are a pool from 3 independent experiments. Statistical analysis was done by unpaired t test between siNT vs. siATG9A conditions of each condition (Rab11a WT vs. DN mock; ***p < 0.01). (C) Localisation of Rab11a (magenta) and PDI (gray) at 10 h postinfection was determined by immunofluorescence using antibody staining. Viral inclusions/Rab11a are highlighted by white boxes. Cell periphery and nuclei (blue, Hoechst staining) are delineated by yellow and white dashed lines, respectively. Mock-infected cells can be found in S1B Fig. Bar = 10 μm. (D–F) Cells (A549) were treated with siRNA non-targeting (siNT) or targeting ATG9A (siATG9A) for 48 h and then infected or mock-infected with PR8 virus for 8 h, at an MOI of 3. (D) The localisation of host Rab11a (green) and viral NP (magenta) proteins at 8 h postinfection was determined by immunofluorescence using antibody staining. Viral inclusions/vRNPs are highlighted by white boxes. Cell periphery and nuclei (blue, Hoechst staining) are delineated by yellow and white dashed lines, respectively. Bar = 10 μm. Experiments were performed twice. (E) Colocalization between Rab11a and NP in the images acquired in (D) was determined using the Colocalization Threshold analysis tool (Image J, NIH) and plotted as the Pearson R value. At least 20 cells, pooled from 2 independent experiments, were analyzed per experimental condition. Red bar represents the median of values. Statistical analysis was done by Mann–Whitney test (n.s., not significant). (F) The roundness and circularity of Rab11a structures in the images acquired in (D) were determined using the Shape Descriptor tool (Image J, NIH) and plotted against each other. The maximum value of roundness and circularity (1) corresponds to a circular structure, whereas the minimum value represents a linear structure (0). Approximately 30 cells, from 2 independent experiments, were analyzed per condition. Statistical analysis was done by Mann–Whitney test (***p < 0.001). The frequency distribution of roundness and circularity of structures marked by Rab11a is shown in S4C and S4D Fig. All the values of individual and pooled experiments are provided in S1 Data File. GFP, green fluorescent protein; IAV, influenza A virus; MOI, multiplicity of infection; NP, nucleoprotein; PFU, plaque-forming unit; SEM, standard error of the mean; vRNP, viral ribonucleoprotein.

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