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Neuropilin-1 is a host factor for SARS-CoV-2 infection

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Science  13 Nov 2020:
Vol. 370, Issue 6518, pp. 861-865
DOI: 10.1126/science.abd3072
  • Fig. 1 NRP1 Interacts with S1 and enhances SARS-CoV-2 infection.

    (A) HEK293T cells transduced to express ACE2 were transfected to express GFP or GFP-tagged S1 and lysed after 24 hours. The lysates were subjected to GFP-nanotrap, and the immune isolates were blotted for ACE2 and NRP1 (N = 3 independent experiments). (B) HEK293T cells were cotransfected to express GFP-tagged S1 or GFP-S1 ΔRRAR and mCherry or mCherry-tagged NRP1 and subjected to GFP-nanotrap (N = 5 independent experiments). Two-tailed unpaired t test; P = 0.0002. (C) HeLawt+ACE2 and HeLaNRP1 KO+ACE2 cells were infected with SARS-CoV-2. Cells were fixed at 6 or 16 hpi and stained for N protein (magenta) and Hoechst (cyan), and virus infectivity was quantified (N = 3 independent experiments). Two-tailed unpaired t test; P = 0.00002 and 0.00088. Scale bar, 200 μm. (D) Caco-2 cells expressing shRNA against NRP1 or a nontargeting control (SCR) were infected with SARS-CoV-2 and fixed at 7 or 16 hpi. The cells were stained for N protein (magenta) and Hoechst (cyan), and infectivity was quantified (N = 3 independent experiments). Two-tailed unpaired t test; P = 0.0005 and 0.00032. Scale bar, 500 μm. (E) Caco-2 shSCR or shNRP1 cells were inoculated with a multiplicity of infection (MOI) = 50 of SARS-CoV-2 and incubated in the cold for 60 min, and fixed. A two-step antibody staining procedure was performed with antibodies against S and N to distinguish external (green) and total (red) virus particles, and the binding of particles per cell was quantified for >3300 particles per condition (N = 3 independent experiments). Two-tailed unpaired t test; P = 0.6859. (F) Caco-2 shSCR or shNRP1 cells were bound with SARS-CoV-2 as in (E), followed by incubation at 37°C for 30 min. The cells were fixed and stained as in (E). Viral uptake was quantified for >4200 particles per condition (N = 3 independent experiments). Two-tailed unpaired t test; P = 0.00079. Scale bars [(E) and (F)], 10 μm and 200 nm (magnified panels). The square regions were enlarged. The bars, error bars, and circles and triangles represent the mean, SEM (B) and SD [(C) to (F)], and individual data points, respectively. ***P < 0.001, ****P < 0.0001. ns, not signficant.

  • Fig. 2 Molecular basis for CendR binding of SARS-CoV-2 S1 with NRP1.

    (A) Binding of NRP1 b1 with native (green line) and mutant (orange line) form of S1 CendR peptide (corresponding to residues 679 to 685) by ITC at two different pH conditions (N = 3 independent experiments). All ITC graphs represents the integrated and normalized data fit with 1-to-1 ratio binding. (B) (Left) NRP1 b1–S1 CendR peptide complex superposed with NRP1 b1–VEGF-A fusion complex (PDB ID: 4DEQ). Bound peptides are shown in stick representation. RMSD, root mean square deviation. (Right) Enlarged view highlighting the binding of S1 CendR peptide b1. Key binding residues on b1 are shown in stick representation. Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; N, Asn; P, Pro; R, Arg; S, Ser; T, Thr; W, Trp; and Y, Tyr. (C). HEK293T cells were cotransfected with combinations of GFP-tagged S1493-685 and S1493-685 R685D, and mCherry or mCherry-NRP1 b1, and subjected to mCherry-nanotrap (N = 5 independent experiments). Two-tailed unpaired t test; P < 0.0001. (D). HEK293T cells were cotransfected with combinations of GFP-tagged S1493-685 and mCherry, mCherry-NRP1 b1 or mCherry-NRP1 b1 T316R mutant, and subjected to mCherry-nanotrap (N = 5 independent experiments). Two-tailed unpaired t test; P < 0.0001. (E) HeLaNRP1KO + ACE2 cells transfected with GFP, NRP1 wt-GFP, or NRP1 T316R-GFP constructs were infected 24 hours later with SARS-CoV-2. At 16 hpi, the cells were fixed and stained for SARS-CoV-2-N, and viral infection was quantified in the GFP-positive subpopulation of cells (N = 3 independent experiments). The percentage of infection was normalized to that of GFP-transfected cells. Two-tailed unpaired t test; P = 0.002. The bars, error bars, and circles represent the mean, SEM [(C) and (D)] and SD (E), and individual data points, respectively. **P < 0.01, ****P < 0.0001. ns, not signficant.

  • Fig. 3 Selective inhibition of the S1-NRP1 interaction reduces SARS-CoV-2 infection.

    (A) Enzyme-linked immunosorbent assay of anti-NRP1 monoclonal antibodies (mAb#1, mAb#2, mAb#3) at 3 μg/ml using plates coated with NRP1 b1b2 wild type, b1b2 mutant (S346A, E348A, T349A), or bovine serum albumin (BSA), used as a control (N = 3 independent experiments). Binding is represented as arbitrary units of absorbance at 655 nm. Two-tailed unpaired t test; P = 0.0207, 0.2430, 0.0007. (B) Cells were first treated with anti-H11N3 (100 μg/ml) (Ctrl) mAb, mAb#1, mAb#2, or mAb#3 for 1 hour before infection with SARS-CoV-2. Cells were fixed at 16 hpi and stained for N protein (magenta) and Hoechst (cyan) (N = 3 independent experiments). Two-tailed unpaired t test; P = 0.015, 0.36, 0.0003. Scale bar, 500 μm. (C) HEK293T cells were cotransfected with combinations of mCherry or mCherry-b1 and GFP-tagged S1493-685 and subjected to mCherry-nanotrap with or without coincubation with mAb#3 (N = 3 independent experiments). Two-tailed unpaired t test; P = 0.0143. (D) NRP1 b1–S1 CendR peptide complex superimposed with NRP1 b1–EG00229 inhibitor complex (PDB ID:3I97). Key binding residues on b1, bound peptides, and EG00229 are shown in stick representation. (E) ITC analysis of EG00229 binding to b1 domain of NRP1 at two different pH conditions. Preincubation with EG00229 blocks S1 CendR peptide binding (orange line), and the CendR peptide can reduce binding of EG00229 (green line) (N = 3 independent experiments). All ITC graphs represent the integrated and normalized data fit with 1-to-1 ratio binding. (F). Cells were first treated with 100 μM EG00229 or dimethyl sulfoxide before infection with SARS-CoV-2. Cells were fixed at 7 and 16 hpi and stained for N protein (magenta) and Hoechst (cyan) (N = 3 independent experiments). The square regions were enlarged. Scale bars, 500 μm and 100 μm (magnified panels). Two-tailed unpaired t test; P = 0.0059 and 0.0013. The bars, error bars, and circles and triangles represent the mean, SEM (C) and SD [(A), (B), and (F)], and individual data points, respectively. *P < 0.05, **P < 0.01, ***P < 0.001.

Supplementary Materials

  • Neuropilin-1 is a host factor for SARS-CoV-2 infection

    James L. Daly, Boris Simonetti, Katja Klein, Kai-En Chen, Maia Kavanagh Williamson, Carlos Antón-Plágaro, Deborah K. Shoemark, Lorena Simón-Gracia, Michael Bauer, Reka Hollandi, Urs F. Greber, Peter Horvath, Richard B. Sessions, Ari Helenius, Julian A. Hiscox, Tambet Teesalu, David A. Matthews, Andrew D. Davidson, Brett M. Collins, Peter J. Cullen, Yohei Yamauchi

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S5
    • Tables S1 to S3
    • References
    MDAR Reproducibility Checklist

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