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

Abstract

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.

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.