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Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

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Science  13 Nov 2020:
Vol. 370, Issue 6518, pp. 856-860
DOI: 10.1126/science.abd2985
  • Fig. 1 NRP1 facilitates the cellular entry of SARS-CoV-2 pseudotyped particles.

    (A) Representative images and quantification of SARS-CoV-2 S protein (SARS-2) (blue bars) and vesicular stomatitis virus glycoprotein (VSV-G) pseudotype (gray bars) infectivity in HEK-293T cells transiently expressing control (ctrl) vector, ACE2, NRP1, or TMPRSS2 (TSS2). Data are normalized to the respective infectivity of SARS-2 and VSV-G pseudotype in ACE2-expressing cells. Two-way analysis of variance (ANOVA) was carried out with Tukey’s correction for multiple comparisons. (B) HEK-293T cells transiently expressing ACE2 and TMPRSS2 or NRP1, ACE2, and TMPRSS2 were inoculated with SARS-2 pseudotype. Data are normalized to SARS-2 infectivity in cells expressing ACE2 and TMPRSS2. One-way ANOVA was performed with Tukey’s correction for multiple comparisons. (C) SARS-2 pseudotype infectivity in Caco-2 cells expressing NRP1 or control vector. Data are normalized to the respective infectivity of SARS-2 and VSV-G pseudotype in control cells. Two-way ANOVA was carried out with Sidak’s correction for multiple comparisons. (D and E) HEK-293T cells transiently expressing NRP1, ACE2, and TMPRSS2 were inoculated with SARS-2 pseudotype in the presence of mAb3 antibody against NRP1 [(D), mAb3] or control mAb2 [(D), ctrl Ab] and in the presence of soluble NRP1 wild-type b1b2 domain [(E), wt b1b2] or NRP1 mutant b1b2 domain [(E), mut b1b2]. Data in (E) are normalized to untreated cells expressing NRP1, ACE2, and TMPRSS2. Two-tailed unpaired Student’s t test was performed. All data are represented as means ± SDs from three independent experiments [(A) to (C)] or three biological replicates [(D) and (E)]. *P < 0.05, **P < 0.01, ****P < 0.0001. All images show GFP-positive, infected cells (magenta) and Hoechst stain (cyan). Scale bars, 100 μm.

  • Fig. 2 A blocking antibody against the b1b2 domain of NRP1 reduces infection by wild-type SARS-CoV-2 (SARS-2-wt) but not a mutant with a deletion at the furin-cleavage site (SARS-2-mut).

    (A) Sequence analysis of viruses isolated at different passages (P) from different cell types. The first sequence is the reference from the Wuhan isolate (NC_045512.2). The sequence abundance in each virus population is indicated as a percentage. ND, not determined. §, SARS-2-wt; §§, SARS-2-mut. A, Ala; S, Ser; Y, Tyr; Q, Gln; T, Thr; N, Asn; P, Pro; R, Arg; V, Val. (B) A deletion adjacent to the furin-cleavage site abrogates the enzymatic cleavage of the S protein. Immunoblot analysis was carried out on cell lysates from Vero-E6 cells infected for 16 hours with two viral populations (§ and §§). Numbers indicate protein size (in kilodaltons). (C and D) Representative images and quantification of SARS-2-wt infectivity in HEK-293T cells stably expressing ACE2 (blue bars) or NRP1 (orange bars) compared with nontransfected cells (gray bars). Different virus titers were used. Data are normalized to the infectivity in ACE2-expressing cells at multiplicity of infection (MOI) = 5. Two-way ANOVA was done with Tukey’s correction for multiple comparisons. (E and F) Representative images (E) and quantification (F) of HEK-293T cells stably expressing the indicated combinations of ACE2, TMPRSS2 (TSS2), and NRP1 after inoculation with SARS-2-wt (wt; blue bars) or SARS-2-mut (mut; gray bars). Data are normalized to the respective infectivity in ACE2-expressing cells. Two-way ANOVA was performed with Tukey’s correction for multiple comparisons. (G and H) Caco-2 cell infection in the presence of control mAb2 (ctrl. Ab) or mAb3 blocking antibodies against NRP1 after SARS-2-wt (wt; blue bars) or SARS-2-mut (mut; gray bars) inoculation. Data are normalized to the respective vehicle control (phosphate-buffered saline) sample. Two-way ANOVA was performed with Tukey’s correction for multiple comparisons. Data are means ± SDs from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Magenta, SARS-2-wt– and SARS-2-mut–infected cells; Hoechst stain, cyan. Scale bars, 50 μm.

  • Fig. 3 NRP mediates entry of nanoparticles coated with SARS-CoV-2 (SARS-2) S–derived CendR peptides into cultured cells, olfactory epithelium, and the central nervous system of mice.

    (A) Peptide sequences used for AgNP coating. The peptides mimic SARS-2 S protein after furin cleavage (post) and as controls; S protein before cleavage (pre), in which the terminal amino acid is replaced by alanine (Ala); or with an amide terminus (post amide). X, any amino acid. The arrow indicates the cleavage site. (B and C) Representative images and quantification of the internalization of peptide-coated AgNPs in HEK-293T cells expressing NRP1. Merged images show AgNP-positive cells (magenta) and Hoechst stain (cyan). One-way ANOVA was carried out with Tukey’s correction for multiple comparisons. (D to G) Representative images and quantification of the main olfactory epithelium [(D) and (E), respectively] and cortex [(F) and (G), respectively] 6 hours after intranasal administration of AgNPs coated with SARS2-post and SARS2-post amide peptides. n = 4 replicates for (C); n = 5 (E) and n = 4 (G) mice per condition. Data are means ± SDs. Two-tailed unpaired Student’s t test; *P < 0.05, ***P < 0.001. Magenta, AgNPs; cyan, Hoechst stain; green, NeuN (neuronal marker); yellow, AQP4. Scale bars, 100 μm (B), 20 μm [(D) and (F)].

  • Fig. 4 SARS-CoV-2 infects the olfactory epithelium.

    (A) Costaining of S protein (brown) and NRP1 (purple) in the apical olfactory epithelium (OE) in a noninfected control (left) and in the apical OE (middle) and adjacent mucosa (right) in a COVID-19 patient. LP, lamina propria; HB, horizontal basal cells. (B) Costaining of NRP1 (magenta) or OLIG-2 (magenta) with S protein (yellow) in OE cells. Nuclei are shown in blue. Scale bars, 10 μm.

Supplementary Materials

  • Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

    Ludovico Cantuti-Castelvetri, Ravi Ojha, Liliana D. Pedro, Minou Djannatian, Jonas Franz, Suvi Kuivanen, Franziska van der Meer, Katri Kallio, Tuğberk Kaya, Maria Anastasina, Teemu Smura, Lev Levanov, Leonora Szirovicza, Allan Tobi, Hannimari Kallio-Kokko, Pamela Österlund, Merja Joensuu, Frédéric A. Meunier, Sarah J. Butcher, Martin Sebastian Winkler, Brit Mollenhauer, Ari Helenius, Ozgun Gokce, Tambet Teesalu, Jussi Hepojoki, Olli Vapalahti, Christine Stadelmann, Giuseppe Balistreri, Mikael Simons

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

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    • Materials and Methods
    • Figs. S1 to S11
    • Table S1
    • References
    MDAR Reproducibility Checklist

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