Fig 1.
Decoration of the sialylated envelope of IAVs with probes for photo-crosslinking host proteins involved in virus entry.
(A) Schematic representative of metabolic conversion of Ac4ManNAz into terminal sialic acid on sialylated envelope for generation of azido-displayed IAV (IAV-N3), followed by click conjugation with DIBO-DAZ-Biotin for labeling IAVs with photo-crosslinking probe. Schematic created using BioRender (https://Biorender.com) (B) Immunofluorescent staining depicting A549 cells infected with IAV or azido-displayed IAV particles (MOI = 50) pre-treated with DIBO-488 for 1h on ice. HA (green), Nuclei (DAPI, blue). Scale bars represent 50 μm. (C) Western blotting characterization of purified IAV particles generated in the presence or absence of Ac4ManNAz, subsequently conjugated with DIBO-Biotin. Detection using HA/NA/Biotin antibodies. (D) Presentation of various crosslinked candidate proteins, with TfR1 specifically highlighted in red, employing a volcano plot. Proteins showing a ratio of 2 or higher, relative to the control group, were considered for subsequent analysis. This dataset encompasses information derived from three independent experiments. (E) Analysis of photo-crosslinked products from A549 cells infected with IAV (MOI = 50) pre-labeled with DIBO-DAZ-Biotin or left untreated. Cells were incubated with virus for 1 h on ice, then shifted to 37°C for 5 min. Samples were either exposed to UV irradiation (+) or not (−) for 20 min. Crosslinked proteins were enriched using streptavidin agarose, separated by 8% SDS-PAGE, and detected by western blotting with anti-HA, anti-NA, or anti-TfR1 antibodies. The observed mobility shift of TfR1 in the UV-treated group indicates successful photo-crosslinking between TfR1 and viral components.
Fig 2.
Elucidation of host TfR1 as an IAV entry mediator critically for triggering viral endocytosis.
(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.
Fig 3.
Exploration of TfR1-interacting viral components, the binding manner and photo-crosslinked sites.
(A) Identification of viral components interacting with TfR1 through co-immunoprecipitation assay and western blotting. Lysates from A549 cells at 2 hours post-infection with IAV (MOI = 20) were co-immunoprecipitated using anti-TfR1 antibody along with anti-viral antibodies. (B) Characterization of TfR1’s affinity with IAV particles and its constituents using Surface Plasmon Resonance (SPR) experiments. IAV particles or viral proteins were immobilized on CM5 chips and interacted with TfR1 protein to measure the binding kinetics and affinity constants. (C) Identification of crosslinked sequences within TfR1 upon photo-activation using DIBO-DAZ-Biotin-labeled IAV particles. TfR1 protein was exposed to UV irradiation after incubation with the photo-crosslinking probe-labeled IAVs. UPLC-MS analysis was conducted to detect lost peptides (due to the covalent linkage of glycans and probe) within TfR1 marked in blue or red, with a control using IAV-N3 (-) instead of DIBO-DAZ-Biotin labeled IAVs (+). (D) Schematic representation of a cleavable photo-crosslinking probe featuring an acid-susceptible diphenyl silane moiety between DIBO and Diazirine, facilitating the addition of a group with a precise molecular mass of 72.05750 in crosslinked TfR1 peptides upon photo-activation and folic acid treatment. (E) Identification of photo-crosslinked sequences and specific crosslinked sites within TfR1 via high-resolution mass spectrometry (HRMS), according to the precise molecular marker after acid-cleavage. TfR1 protein was exposed to UV irradiation after incubation with the cleavable photo-crosslinking probe-labeled IAV or its mutant version NA-N219A. UPLC-HRMS was utilized to identify the crosslinked peptides post-SDS-PAGE separation. (F) Structural representation of NA binding within the TfR1 protein (PDB: 6Q23 and 1SUV) derived from blind docking calculations. The final docked conformations of the NA tetramer (cyan) bound to the TfR1 dimer (green) were based on most suitable conformation and crosslinked sites. The proposed binding sites of NA occupy the apical domain of TfR1, considering a 3 Å resolution X-ray structure. TfR1’s extracellular domain is categorized into apical domain (A), helical (H) domain, and protease-like domain (P), with primary transferrin binding in H and P domains. The NA tetramer binds to the apical domain of the TfR1 dimer, proximal to the crosslinked peptide of TfR1.
Fig 4.
Exploration of TfR1-M1 interaction and implication for viral uncoating.
(A) Time-course monitoring of viral M1 and host TfR1 expression levels in infected A549 cells (MOI = 0.01) conducted via western blotting analysis. The culture medium was refreshed daily, and cells were harvested at specified time points. Equal loading was confirmed by detecting GAPDH. (B) Assessment of the impact of TfR1 on viral M1 protein by co-transfecting the M1 or PB2 plasmid (0.5 μg) with varying concentrations of TfR1 plasmid in 293T cells for 48 hours. Equal loading was confirmed by detecting GAPDH. (C) Determination of the specificity of TfR1-mediated viral protein degradation by individually co-transfecting plasmids from the IAV plasmid complex with the TfR1 plasmid in A549 cells for 48 hours. Equal loading was confirmed by detecting GAPDH. (D) Investigation into the role of the proteasome system in TfR1-mediated degradation of M1 protein. 293T cells were transfected with the IAV plasmid complex along with increased concentrations of TfR1 plasmid in the absence or presence of MG132 (10 μM). Analysis of TfR1 and different IAV proteins’ protein levels was conducted via western blotting 48 hours post-transfection. Equal loading was confirmed by detecting GAPDH. Mean gray value were used to statistical analyze. (E) Exploration of the aggresome systems’ role in TfR1-mediated degradation of M1 protein. 293T cells were co-transfected with TfR1 plasmid and the viral plasmid complex containing M1 and NP genes in the absence or presence of Tubacin (5 μM). Western blotting was performed to analyze TfR1, M1, and NP protein levels 48 hours post-transfection. Equal loading was confirmed by detecting GAPDH. (F) Laser confocal microscopy experiments to analyze cellular colocalization patterns involving HDAC6 (blue, upper) and ubiquitin (blue, lower) with TfR1 (red) and M1 (green) in TfR1/M1-ZsGreen transfected 293T cells. Scale bars, 10 μm. (G) Detection of aggresome (green)/MTOC(Gamma Tubulin, red) in 293T cells co-transfected with TfR1/SLC3A2 and M1 plasmids by laser confocal microscopy experiments using the Aggresome Detection Kit (ab139486). MTOC(Gamma Tubulin, red), M1 (blue), aggresome (green),nucleus(DAPI,cyan). The arrow indicates aggresome. Scale bars, 10 μm. (H) In vitro quantification of truncated TfR1, M1 and their complex protein aggregation by Protein Aggregation Assay Kit (ab234048). Aggregation was determined based on the ΔRFU using a standard curve for calculation. Unpaired t-tests were used for statistical analysis, denoting significance as follows: *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, while “n.s.” indicates non-significance.
Fig 5.
Identification of TfR1 region responsible for triggering matrix protein degradation.
(A) Visualization of the predicted tertiary structure of TfR1 monomer generated by Alpha Fold, highlighting the transmembrane, stalk, and helical domains in blue, green, and purple, respectively. (B) Determination of the critical region in TfR1 responsible for initiating M1 degradation through domain swapping with SLC3A2. Co-transfection of resultant TISE and SITE plasmids with the M1 plasmid in 293T enabled the reevaluation of M1 degradation, validated by western blotting. (C) Investigation into the contribution of TfR1’s transmembrane domain in instigating M1 degradation. Truncated plasmids containing various sequences surrounding the transmembrane domain of TfR1, along with the M1 plasmid, were co-transfected into 293T cells for 48 hours. (D) Assessment of M1 degradation facilitated by various intracellularly truncated versions of TfR1. 293T cells underwent co-transfection with M1 plasmid and truncated TfR1 plasmids, followed by analysis of TfR1 and M1 protein levels through western blotting 48 hours post-transfection. (E) Structurally depicting the binding of M1 (green, PDB: 6Z5L) with the transmembrane and intracellular stop-transfer sequence of TfR1 (purple, AlfaFold2-prediction) based on blind docking calculations. TfR1 (residues 53-67) was proposed to occupy the NTD of M1, forming four hydrogen bonds between Thr5, Leu117, Gln153, Asp156 of M1 and Asn55, Arg61, Lys58 of TfR1, respectively, and a charge interaction between M1’s Glu152 and TfR1’s Lys58.
Fig 6.
Translational potentials of the discovery of mutual degradation of host receptor and viral matrix protein.
(A) Illustration depicting three truncated intracellular versions of TfR1 with an isoleucine zipper-his (ZH) instead of transmembrane domain, exhibiting predominantly homo-dimer and trimer formations, visualized through western blotting using anti-His antibody. (B) Investigation into the impact of the TfR1 transmembrane domain on triggering M1 degradation. 293T cells underwent co-transfection with plasmids expressing M1 and truncated intracellular TfR1 variants featuring an isoleucine zipper-his instead of the transmembrane domain. Protein levels of M1 were assessed via western blotting 48 hours post-transfection. Equal loading was confirmed by detecting GAPDH. (C) Demonstration of the broad-spectrum effect of TfR1-mediated degradation on the viral matrix protein, spanning antigenically distinct influenza strains. 293T cells were co-transfected with TfR1 and M1 protein plasmids derived from different IAV strains. The protein levels of TfR1 and distinct M1 variants were analyzed via western blotting 48 hours post-transfection. Equal loading was confirmed by detecting GAPDH. (D) TfR1-mediated M1 degradation suppresses IAV progeny production in a dose-dependent manner. A549 cells were co-transfected with a total of 1.5 μg plasmid DNA, consisting of 12 IAV packaging plasmids and increasing amounts of TfR1 plasmid, balanced with the control plasmid SLC3A2 to maintain consistent total DNA across conditions. Viral titers were measured by RT-qPCR at 48 h post-transfection and normalized to untransfected controls. (E) Examination of the translational potential of TfR1-mediated degradation beyond influenza viruses. 293T cells were transfected with plasmids expressing TfR1 alongside matrix proteins from different viruses (MeV, RABV, and EBOV), each tagged with flag tag. Protein levels of TfR1 and various matrix proteins (Flag-tagged) were assessed via western blotting 48 hours post-transfection using anti-flag antibody. Equal loading was confirmed by detecting GAPDH. (F) Scheme representative of TfR1 -mediated endocytosis and uncoating. TfR1 functions as a receptor for kickstarting viral endocytosis by interacting its extracellular apical domain with the viral NA, bridging the gap between HA-sialic acid receptor attachment and clathrin-mediated viral endocytosis. Then, TfR1 acts as a trigger for matrix degradation by forming a misfolded protein complex with the viral matrix M1 via its intracellular stop-transfer sequence, filling the void between HA/endosomal fusion and proteasome/aggresome -mediated nucleocapsid uncoating. Unpaired t-tests were used for statistical analysis, denoting significance as follows: *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001, while “n.s.” indicates non-significance.