Journal: PLOS Pathogens

Article Title: TfR1 facilitates influenza virus endocytosis and uncoating by interacting with NA and M1 via extracellular and intracellular domains

doi: 10.1371/journal.ppat.1013511

Figure Lengend Snippet: (A) Western blot analysis examining the impact of TfR1 knockdown in A549 cells on IAV (A/WSN/1933 (H1N1)) 12 hours infection (MOI = 0.5). Equal loading was confirmed by detecting GAPDH. (B) Immunofluorescent staining of A549 cells and cells with TfR1 knockdown 4h post -infection (A/WSN/1933 (H1N1)) (MOI = 0.5). (C) Evaluation of TfR1 knockdown’s inhibitory effect on IAV infection (MOI = 0.5) in A549 cells. Viral RNA levels were quantified by RT-qPCR at 12 hours post-infection across multiple strains: A/WSN/1933 (H1N1), A/PR/8/1934 (H1N1), and A/Aichi/2/1968 (H3N2). (D) Western blot characterization investigates the correlation between TfR1 expression levels in different cell lines and their susceptibility to IAV infection (MOI = 0.5). Equal loading was confirmed by detecting GAPDH. (E) Comparative evaluation of sialic acid versus TfR1 as potential receptors in mediating IAV entry. A549 cells transfected with TfR1/vector plasmid were treated with neuraminidase (50 units/mL) or BSA at 37°C for 2 hours before infection with IAV (A/WSN/1933 (H1N1), MOI = 0.5) ( left panel ). Alternatively, IAV was pretreated with neuraminidase or BSA at 37°C for 2 hours before infecting A549 cells ( right panel ). After 12 hours, infected cells were lysed and quantified by RT-qPCR. (F) Comparative evaluation of TfR1 as a potential receptor mediating IAV attachment and endocytosis. A549 and TfR1-knockdown cells were incubated with either unlabeled IAVs (A/WSN/1933 (H1N1), MOI = 10) or sulfo-NHS-SS-Biotin-labeled IAVs (MOI = 5) at 4°C for 1 hour (to allow attachment) or subsequently shifted to 37°C for 2 hours (to allow endocytosis). After washing, attached viruses were quantified by RT-qPCR, and internalized viruses were measured by flow cytometry. (G) Flow cytometry analysis assessing TfR1 involvement in IAV entry, quantifying internalized biotin-SS-labeled IAVs (A/WSN/1933 (H1N1)) (MOI = 5) in A549 cells, TfR1-knockout A549 cells, and CHO cells. TCEP treatment was applied to cleave disulfide bond and remove the biotin tag on non-internalized IAV particles, followed by fixation, permeabilization and incubation with AF488-conjugated streptavidin. Unpaired t-tests were used for statistical analysis with corresponding p-values. *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, while “n.s.” indicates non-significance.

Article Snippet: Transferrin Receptor/TFR1/CD71 Protein, Human, Recombinant (ECD, His Tag), HPLC-verified (Sino Biological, Cat#11020-H07H), Influenza A H1N1 (A/WSN/1933) Hemagglutinin/ HA Protein (His Tag) (Sino Biological, Cat#11692-V08H), Influenza A H1N1 (A/Puerto Rico/8/34/Mount Sinai) Matrix protein 1/ M1 Protein (His Tag), (Sino Biological, Cat#40010-V07E).

Techniques: Western Blot, Knockdown, Infection, Staining, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Incubation, Labeling, Flow Cytometry, Knock-Out