ATG9A depletion affects viral genome assembly but not viral genome packaging.

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 at an MOI of 3. (A) The vRNA levels for each viral RNA segment (1–8) was expressed relative to GAPDH levels at 8 h postinfection and was determined by real-time RT-qPCR using specific primers as detailed in the Methods section. Data are triplicates from a single experiment. Two independent experiments were performed. Statistical analysis was done by a two-way ANOVA test, followed by a Sidak’s multiple comparisons test (**p < 0.01; ***p < 0.001). (B) The integrated density of NP protein in the cell cytosol at 8 h postinfection was determined by immunofluorescence, using the Analyze Particles function of FIJI (ImageJ, NIH). More than 80 cells, pooled from 4 independent experiments, were analyzed per condition. Statistical analysis was done by Kruskal–Wallis test (***p < 0.001). (C) The levels of the 3 viral surface proteins at 8 h postinfection (HA, hemagglutinin; NA, neuraminidase; M2, matrix protein 2) were determined by flow cytometry using monoclonal antibodies against each viral protein and analyzed as shown. The MFI of each viral protein at the cell surface was plotted for each experimental condition. Statistical analysis was done by one-way ANOVA, followed by a Sidak’s multiple comparisons test (**p < 0.01; ***p < 0.001). Data are a pool of 4 independent experiments performed. (D, E) The vRNA copy number per mL for each viral RNA segment (1–8) and the vRNA-to-PFU ratio at 8 h postinfection was determined by real-time RT-qPCR using specific primers as detailed in the Methods section. Data are triplicates from a single experiment. Two independent experiments were performed. Statistical analysis was done by a two-way ANOVA test, followed by a Bonferroni’s multiple comparisons test (*p < 0.05; ***p < 0.001; n.s., not statistically significant). (F) Scheme illustrates that ATG9A is critical for viral inclusion shape and regulates rate of viral genome assembly but does not affect genome packaging into budding virions. All the values of individual and pooled experiments are provided in S1 Data File. MFI, median fluorescence intensity; MOI, multiplicity of infection; NP, nucleoprotein; PFU, plaque-forming unit; RT-qPCR, quantitative reverse transcription PCR.