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

Journal: RSC Medicinal Chemistry

Article Title: De novo design of a stapled peptide targeting SARS-CoV-2 spike protein receptor-binding domain †

doi: 10.1039/d3md00222e

Figure Lengend Snippet: Binding kinetics study of pep39 and the spike protein RBD. (A) The deflection curve for the peptide pep39 immobilized on the cantilever in response to different concentrations of SARS-CoV-2 spike protein. Anti-S1 antibody served as a positive control and H1N1 protein, as a negative control. (B) The association and dissociation between immobilized spike protein RBD and different concentrations of pep39. Recombinant human ACE2 protein used as a positive control and PBS buffer as a reference blank.

Article Snippet: We purchased silicon cantilevers from Nanoworld Inc., and the SARS-CoV-2 spike protein RBD, the antibody for the spike protein RBD and influenza H1N1 hemagglutinin protein were procured from Sino Biological Inc; and 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and sulfo-NHS from ThermoFisher Scientific.

Techniques: Binding Assay, Positive Control, Negative Control, Recombinant

Journal: Nature Communications

Article Title: Human antibody 3E1 targets the HA stem region of H1N1 and H5N6 influenza A viruses

doi: 10.1038/ncomms13577

Figure Lengend Snippet: ( a , b ) Prophylactic efficacy of 3E1 mAb against a lethal challenge with the A/Sichuan/09/2009(H1N1) virus ( a ) or A/CK/NX/S6/2014(H5N6) virus ( b ). The weight loss (left) and survival curves (right) of mice treated with 30, 10, 3 or 1 mg kg −1 of 3E1 mAb or PBS buffer 24 h before lethal challenge by an intranasal inoculation with the H1-SC09 or H5-NX14 virus (at day 0) are shown. ( c , d ) Therapeutic efficacy of 3E1 mAb against a lethal challenge with the A/Sichuan/09/2009(H1N1) ( c ) or A/CK/NX/S6/2014(H5N6) virus ( d ). The weight loss (left) and survival curves (right) of mice treated with PBS buffer (at day 1) or 20 mg kg −1 of 3E1 mAb right after or 1, 2, 3 days after lethal challenge by an intranasal inoculation with the H1-SC09 or H5-NX14 virus (at day 0) are shown. Error bars represent mean±s.d. Data represent a representative experiment from three independent experiments.

Article Snippet: The HAs from the A/Brevig Mission/1/1918(H1N1), A/WSN/1933(H1N1), A/Brisbane/59/2007(H1N1), A/California/07/2009(H1N1), A/Canada/720/2005(H2N2), A/Hong Kong/483/1997(H5N1), A/Vietnam/1203/2004(H5N1), A/Anhui/1/2005(H5N1), A/Sichuan/26221/2014(H5N6), A/Hong Kong/1073/1999(H9N2), A/black-headed gull/Netherlands/1/00(H13N8), A/black-headed gull/Sweden/5/99(H16N3), A/Hong Kong/1/1968(H3N2), A/Aichi/2/1968(H3N2), A/Brisbane/10/2007(H3N2), A/Taiwan/70113/2007(H3N2), A/Netherlands/219/2003(H7N7) and A/Zhejiang/DTID-ZJU01/2013(H7N9) viruses were purchased from Sino Biological Inc. HAs of the A/Washington/05/2011(H1N1) and A/California/04/2009(H1N1) viruses were prepared as described above.

Techniques: