Pneumonia resulting from infection is one of the leading causes of death worldwide. Pulmonary infection by the respiratory syncytial virus (RSV) is a large burden on human health, for which there are few therapeutic options1. RSV targets ciliated epithelial cells in the airways, but how viruses such as RSV interact with receptors on these cells is not understood. Nucleolin is an entry coreceptor for RSV2 and also mediates the cellular entry of influenza, the parainfluenza virus, some enteroviruses and the bacterium that causes tularaemia3,4. Here we show a mechanism of RSV entry into cells in which outside-in signalling, involving binding of the prefusion RSV-F glycoprotein with the insulin-like growth factor-1 receptor, triggers the activation of protein kinase C zeta (PKCζ). This cellular signalling cascade recruits nucleolin from the nuclei of cells to the plasma membrane, where it also binds to RSV-F on virions. We find that inhibiting PKCζ activation prevents the trafficking of nucleolin to RSV particles on airway organoid cultures, and reduces viral replication and pathology in RSV-infected mice. These findings reveal a mechanism of virus entry in which receptor engagement and signal transduction bring the coreceptor to viral particles at the cell surface, and could form the basis of new therapeutics to treat RSV infection.
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We thank J. S. McLellan for providing input into the work and for the recombinant purified prefusion-RSV-F, DS-Cav1 and NCL proteins; C. M. Overall for discussion about the project, and Q. Kieser for technical support. This work was funded by grants from Women’s and Children’s Health Research Institute (WCHRI), Li Ka Shing Institute of Virology (LKSIOV), The Lung Association of Canada, and the Canadian Institutes of Health Research. C.D.G. was supported by studentships from The Lung Association – Alberta & NWT and The Canadian Lung Association.
The authors declare no competing interests.
Peer review information Nature thanks Jeffrey Bergelson, Barney Graham and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Fig. 1 NCL interacts with the RSV-F glycoprotein and is expressed abundantly in cells.
a, Western blot of anti-RSV-F (left) and anti-NCL (right) co-immunoprecipitations in RSV-infected 1HAEo− cells. Data shown are representative of at least 4 independent experiments. b, Microscale thermophoresis analysis of the affinity of NCL for RSV-FDS-Cav1 and RSV-G glycoproteins. The dissociation constant (Kd = 118 ± 25 nM) is stated as mean ± s.d. of n = 2 independent experimental repeats. c, Top, flow cytometry analysis of cell-surface NCL during RSV infection. Data are mean ± s.d., n = 4 biological replicates, *P < 0.05 measured by one-way ANOVA, Tukey’s post hoc analysis. Data shown are representative of 3 independent repeats. Bottom, western blot of total NCL in 1HAEo− cells during RSV entry. Data shown are representative of more than 5 independent repeats. d, RSV-infected (red) 1HAEo− cells 24 h after addition of the virus. Images are representative of 4 independent repeats. e, NCL mRNA transcript levels in RSV-infected 1HAEo− cells, normalized to the highest NCL expression and HPRT housekeeping gene. One-way ANOVA and Tukey’s post hoc test failed to measure significance; NS, P > 0.05, n = 3 technical replicates as representative of 2 independent repeats. f, Violin plot of data from the single-cell RNA sequencing analysis of relative NCL expression levels in the various structural cell types of adult human lungs. Dots indicate expression values for individual cells and the shape of the graph is the overall distribution of the data for each cell type, n = 1,572 individuals.
a, Single-cell RNA-sequencing analysis of gene expression in lung structural cells. Dimension reduction using principal component analysis and t-SNE of genes with high variability between cells showed that each cluster was related to a specific cell type on the basis of canonical gene expression markers related to cellular function. b, Violin plots of gene expression data points. Dots indicate expression values for individual cells and the shape of the graph is the overall distribution of the data for each cell type, n = 1,572 individuals.
a, Mean fluorescence intensity of surface NCL, measured by imaging flow cytometry. Linear regression analysis compared the slope of the line to 0. NCL 0–60 min: F = 10.3, slope = 32.19 (95% CI: 10.34–54.05), d.f. = 12, P = 0.0075. The line marks the mean of n = 3 biological replicates and is representative of 4 independent repeats. b, Confocal immunofluorescence images of permeabilized NHBE cells stained for nuclei (DAPI; blue), NCL (green) and RSV (red). Arrowheads indicate the colocalization of RSV and NCL. The time after infection is shown in white. These data are duplicated in part in Fig. 1c. The insets show expanded views of the viral particles indicated by the arrowheads. Data is representative of multiple images from 3 independent repeats. All RSV infections were synchronized on ice for 1 h before warming, t = 0 represents the time at which the cells are transferred to 37 °C from ice.
Immunofluorescence images taken 30 min after the infection of 1HAEo− cells with RSV type-A and RSV type-B clinical isolates obtained from nasopharyngeal swabs, showing the colocalization of NCL (green) with RSV (red). Data shown are representative of multiple images from 5 different isolates for each RSV type. All RSV infections were synchronized on ice for 1 h before warming, t = 0 represents the time at which the cells are transferred to 37 °C from ice.
a, Violin plot of data from the single-cell RNA sequencing analysis of relative EGFR expression levels in the various structural cell types of adult human lungs. EGFR is expressed primarily in basal cells. Dots indicate expression values for individual cells and the shape of the graph is the overall distribution of the data for each cell type, n = 1,572 individuals. b, c, Cells were treated with the cellular inhibitors tested in Figs. 1e, 2a. Solid lines indicate the mean of n = 3 biological replicates and the dotted line indicates viability of control cells (100%). Data shown are representative of 2 independent repeats. d, The efficacy and specificity of pre-treatments of 1HAEo− cells with MEK1/2 kinase (UO126), PI3K (LY294002) and p38 MAPK (SB203580, BIRB 796) was determined in an entry time course of RSV infection. Protein levels in cell lysates were measured by western blot. SOD-1 or β-actin were used as loading controls. Data shown are representative of 4 independent experiments.
Extended Data Fig. 6 IGF1R expression in mosquito and human cells confers infectivity and binding to RSV.
a, Foci of RSV infection in 1HAEo− cells pre-treated with inhibitors of cell-surface receptors. The results for treatment with inhibitors of other receptors are presented in Figs. 1e, 2a. **P < 0.01, ***P < 0.001 by one-way ANOVA and Tukey’s post hoc analysis. Data shown are the mean of n = 4 biological replicates and are representative of at least 2 independent experiments. b, RSV was incubated with 1HAEo− cells for 60 min on ice and imaged by confocal microscopy. Arrowheads indicate the colocalization of RSV particles and IGF1R. Data shown are representative of multiple images of 3 independent repeats. c, Example flow cytometry plots of RSV–GFP-infected C636 mosquito cells transfected with human IGF1R. d, Plot summarizing the data from the flow cytometry experiments shown in c, indicating the percentage of cells infected by RSV. Bars indicate the mean of n = 3 biological replicates that are representative of 2 independent repeats; ***P < 0.001 by one-way ANOVA, with Tukey’s post hoc analysis. e, Western blot of IGF1R expression in wild-type 1HAEo− cells and IGF1R knockout 1HAEo− cells that were gene-edited using CRISPR–Cas9. IGF1R expression was rescued in knockout cells using lentiviral transduction (KO + IGF1R). Data is representative of 2 independent repeats. f, RSV-FDS-Cav1 was bound to 1HAEo− cells or IGF1R knockout cells on ice, then stained with a fluorescent antibody and quantified by a plate reader. Bars represent the mean of n = 5–7 biological replicates, representative of 2 independent repeats; *P < 0.05 by one-way ANOVA, Tukey’s post hoc analysis.
Extended Data Fig. 7 PKCζ is activated by RSV during entry and silencing the expression of IGF1R and PRKCZ does not affect RSV activation of ERK1/2 MAPK or PI3K.
a, Foci of RSV infection in human fibroblasts with genetic TLR-signalling deficiencies. Data are mean ± s.d. of n = 3 biological replicates; *P < 0.05, **P < 0.01 by one-way ANOVA and Tukey’s post hoc analysis. b, Imaging flow cytometry experiments, performed 0 and 60 min after RSV infection, determined the effects of cellular inhibitors on the change in colocalization of RSV and NCL in 1HAEo− cells (ΔNCL–RSV patching). Lines represent the mean of n = 3 biological replicates as a representative of 3 independent repeats. No treatment differed significantly from the control according to a one-way ANOVA with Dunnett’s post hoc test, P > 0.05. c, Western blot analysis of PRKCZ knockdown, IGF1R knockdown, or scramble (−) siRNA knockdown 1HAEo− cell lysates infected with RSV. SOD-1 was a loading control. Blots are representative of 3 independent repeats. d, PKCζ is activated during RSV entry, as seen by a spike in activity 10 min after infection. 1HAEo− cells were either infected with RSV or mock-infected, PKCζ was immunoprecipitated and activity measured in an in vitro kinase assay. PKCζ activity is reported as nmol ATP converted to ADP. See also Fig. 2g. Means of n = 2 biological replicates were compared with one Student’s two-tailed t-test per timepoint, taking into account false discovery rate using the method of Benjamini, Krieger and Yekutieli. **P < 0.01. RSV infections in b–d were synchronized on ice for 1 h before warming; t = 0 represents the time at which the cells are transferred to 37 °C from ice.
Extended Data Fig. 8 Examination of RSV entry fusion and colocalization using RSV–DiD on 1HAEo− cells expressing NCL–GFP.
a, 1HAEo− cells were infected with DiD-labelled RSV (red) on ice, fixed, stained for RSV-F (green) and imaged using fluorescence microscopy. b, Immunoprecipitation using an anti-RSV-F antibody of RSV-infected HEK-293T cells transiently expressing either NCL–GFP or GFP. NCL (left) and GFP (right) were detected by western blot. c, d, Living 1HAEo− cells expressing NCL–GFP (c) or GFP alone (d) were infected with RSV-DiD (red) and imaged over time (the time (in min) after infection is indicated above the panels). Arrowheads denote fusing RSV particles, as indicated by an increase in DiD fluorescence (see schematic in Fig. 2h). DiD–RSV was used in Fig. 2c, i, j, and Supplementary Videos 1, 2. All RSV infections in this figure were synchronized on ice for 1 h before warming, t = 0 represents the time at which the cells are transferred to 37 °C from ice. Selected images are representative of multiple fusion events in at least 4 independent experimental repeats.
a, Overlaid flow cytometry histograms of untreated (red) and IGF-1 treated (blue) HEK-293T cells expressing NCL–GFP. b, Quantification of the histograms shown in a, showing the percentage of cells expressing NCL on the cell surface. Data are derived from 3 independent experiments, line indicates median value with dots showing individual data points. Significance was determined by Student’s two-tailed t-test; *P = 0.045. c–f, ALI culture inhibitor data is representative of multiple images from 2 different donors and 1 experimental repeat. c, ALI cultures stained for IGF1R (pink) and cilia (β-tubulin, cyan). The arrowhead marks the plane of XZ displayed as XY in the bottom panel. d, Images of RSV-infected ALI cultures, pretreated with the IGF1R inhibitor PQ401, were quantified using Volocity software. The number of objects containing both RSV and IGF1R (left, n = 4) or RSV and NCL (right, n = 4) were compared using Student’s two-tailed t-test; **P < 0.01. e, f, RSV-infected ALI cultures from Fig. 3d (PKCζ inhibitor) were quantified using Volocity software. e, Mean NCL voxel intensity above (apical, n = 6) and below (basal, n = 6) the cortical actin cytoskeleton was compared using a one-way ANOVA, with Tukey’s test for significance (comparing PKCζ inhibitor to control); **P < 0.01. f, Pearson’s correlation coefficient of colocalization between NCL and β-tubulin of cilia was compared using Student’s two-tailed t-test, **P < 0.01. g, BALB/c mice (n = 32) were inoculated with RSV intranasally, treated with PKCζ inhibitor 1 day after infection, then euthanized 3 days later. Lungs were inflated with formalin, wax-embedded and sectioned. RSV infection was assessed by immunohistochemistry and the detected of activated DAB substrate (brown stain). Example images are shown, and quantification is presented in Fig. 3e.
Extended Data Fig. 10 Schematic showing the binding of RSV to its receptor, and subsequent recruitment of the co-receptor.
RSV-F on the surface of the virion binds to IGF1R on the cell surface. This activates the PKCζ signalling pathway, recruiting nucleolin from the nucleus through the cytoplasm to the cell membrane, where it facilitates fusion of the viral and the host membranes thus enabling RSV replication to proceed.
This file contains the uncropped western blots.
Cell Surface NCL-GFP colocalizes with DiD-labelled RSV particles during viral fusion. Extended focus video of 3-channel detection of DAPI stain of host cell DNA (blue), NCL-GFP (green), and DiD labelled RSV (red). RSV particles were labelled with lipophilic DiD and added to NCL-GFP expressing 1HAE cells on ice for 1 h. The cells were warmed to 37 °C with supplementary 5% CO2 on a Delta Vision OMX microscope and imaged. Each frame of the movie represents a 2 min period between serial stack acquisitions. Data are representative of at least four independent experiments.
Representative examples of NCL-GFP recruitment and fusion by DiD labelled RSV particles. Extended focus video of 3-channel detection of DAPI stain of host cell DNA (blue), NCL-GFP (green), and DiD labelled RSV (red). Each frame of the movies represent a 2 min period between serial stack acquisitions. Data are representative of at least four independent experiments. Scale bar = 5 μm.
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Griffiths, C.D., Bilawchuk, L.M., McDonough, J.E. et al. IGF1R is an entry receptor for respiratory syncytial virus. Nature 583, 615–619 (2020). https://doi.org/10.1038/s41586-020-2369-7
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