Journal: bioRxiv
Article Title: Dual roles for influenza A protein PA-X: limiting inflammatory response and disrupting MHC I antigen presentation in human respiratory epithelium
doi: 10.64898/2026.01.30.702929
Figure Lengend Snippet: A-B) Analysis of scRNAseq data from ALI cultures that were infected with Perth H3N2 WT or ΔX at MOI of 0.1, or mock infected as shown in (n = 2, from 2 separate donors). A) Top ten gene sets identified by gene set enrichment analysis of all differentially expressed genes (DEGs) in infected cells at 1 day post infection (DPI), with the top 10 gene sets shown. Influenza A virus-positive cells were defined as cells of any subtype with at least 1% reads mapping to the influenza transcriptome. Variable genes were found using FindAllMarkers with a cutoff of |log2FC| > 0.25 p-adjusted < 0.05, and enriched gene set cutoffs were p-adjusted < 0.05 (n = 2, from 2 separate donors, Donors 1 and 2). B) Heatmaps of DEGs in the antigen processing and presentation gene set (GO:0019882) at 1 and 3 DPI, separated by infected and bystander cells from each condition. C-D) ALI cultures were infected with Perth H3N2 WT or ΔX virus, mock-infected, or pre-treated with IFN-α at 2 ng/µL for 1 hour. Cells were collected at 3 DPI and stained for major histocompatibility complex I (MHC I) and influenza A virus nucleoprotein (IAV NP). C) Diagram of the flow cytometry staining method, where the same anti-HLA-ABC coupled to different fluorophores was used before and after permeabilization to detect surface and intracellular MHC I, respectively, gating strategy for NP+ infected and NP-bystander cells (after gating for live, single cells), and representative histogram results for MHC I HLA-ABC surface and intracellular expression. D) HLA-ABC median fluorescence intensity at 3 DPI from flow cytometry analysis plotted as fold-change relative to mock-infected ALI cultures (n = 4, from 4 separate donors, Donors 2-5). Two-way ANOVA, followed by Šídák’s multiple comparisons test, ns = p > 0.05, *= p ≤ 0.05, **= p ≤ 0.01, ***= p ≤ 0.001. E-G) Calu-3 cells were mock-infected or infected with Perth H3N2 WT or ΔX at an MOI of 0.1 for 18 hours. Surface MHC I was stripped using a citric acid buffer, then cells were collected at multiple timepoints over the course of 6 hours. Surface MHC I was measured by flow cytometry. E) Schematic of acid strip time course and MHC I surface trafficking. F-G) Log2 fold change of surface MHC I median fluorescence intensity over a 6-hour time course in all live mock-infected cells (F) and influenza A virus NP positive WT and ΔX-infected cells (G). Linear regression was performed for each condition, and the p value was calculated to compare Perth H3N2 WT and ΔX NP+ cells. Dashed lines represent the line of best fit, and the shaded regions indicate the 95% confidence interval.
Article Snippet: Cells were washed and then stained with Influenza A Virus NP antibody (clone HB-65; Bioxcell BE0159) pre-conjugated with Alexa Fluor 680.
Techniques: Infection, Virus, Staining, Immunopeptidomics, Flow Cytometry, Expressing, Fluorescence, Stripping Membranes
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![ALI cultures were infected with Perth H3N2 WT or ΔX at MOI of 0.1, or mock infected and RNA was collected for scRNAseq as described in . The scRNAseq data were analyzed to identify infected cells. A) Integrated UMAP of mock, Perth H3N2 WT and Perth H3N2 ΔX infected ALI cultures colored by the percent of reads that mapped to influenza A viral genes in each cell. B-C) Bar graphs with percent influenza A viral reads per cell in each assigned cell type in WT and ΔX infected cultures at 1 (B) and 3 DPI (C). D) Bar graph showing the percentage of infected cells that belong to each epithelial cell type in influenza-positive cells in WT and ΔX infected cultures vs. the composition of the cultures derived in mock-infected samples. Influenza-positive cells were defined as cells having at least 1% reads mapping to the influenza transcriptome. The two donors were aggregated for the analysis in all panels. The two timepoints [1 and 3 days post infection [DPI]) were also aggregated for the analysis in panel A.](https://bio-rxiv-images-cdn.bioz.com/dois_ending_with_29/10__64898_slash_2026__01__30__702929/10__64898_slash_2026__01__30__702929___F3.large.jpg)
