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

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.

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 24h. The lysates were subjected to GFP-nanotrap and the immune-isolates were blotted for ACE2 and NRP1 (N=3). (B) HEK293T cells were co-transfected to express GFP-tagged S1 or GFP-S1 ΔRRAR and mCherry or mCherry-tagged NRP1 and subjected to GFP-nanotrap (N=5). Two-tailed unpaired t-test; P= 0.0002. (C) HeLa^wt+ACE2 and HeLa^{NRP1 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). 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 non-targeting 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). 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 MOI=50 of SARS-CoV-2 and incubated in the cold for 60 min, and fixed. A two-step antibody staining procedure was performed using anti-S and -N Abs to distinguish external (green) and total (red) virus particles, and the binding of particles per cell was quantified for over 3300 particles per condition (N=3). 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 over 4200 particles per condition (N=3). Two-tailed unpaired t-test; P=0.00079. Scale bars for (E) and (F) = 10 μm and 200 nm (zoom panels). The square regions were zoomed in. The bars, error bars, circles and triangles represent the mean, SEM (B) and SD (C-F), individual data points, respectively. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.

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 679-685) by ITC at two different pH conditions (N=3). 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. (C). HEK293T cells were co-transfected with combinations of GFP-tagged S1^493-685 and S1^493-685 R685D, and mCherry or mCherry-NRP1 b1, and subjected to mCherry-nanotrap (N=5). Two-tailed unpaired t-test; P <0.0001. (D). HEK293T cells were co-transfected with combinations of GFP-tagged S1^493-685 and mCherry, mCherry-NRP1 b1 or mCherry-NRP1 b1 T316R mutant and subjected to mCherry-nanotrap (N=5). Two-tailed unpaired t-test; P <0.0001. (E) HeLa^NRP1KO + ACE2 cells transfected with GFP, NRP1 wt-GFP or NRP1 T316R-GFP constructs were infected 24 h later with SARS-CoV-2. At 16 hpi the cells were fixed and stained for SARS-CoV-2-N, and viral infection quantified in the GFP-positive subpopulation of cells (N=3). 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-D) and SD (E), individual data points, respectively. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.

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

(A) ELISA 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 BSA, used as control (N=3). 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 pre-treated with 100 μg/mL of anti-H11N3 (Ctrl) mAb, mAb#1, 2 or 3 for 1 h prior to infection with SARS-CoV-2. Cells were fixed at 16 hpi and stained for N protein (magenta) and Hoechst (cyan) (N=3). Two-tailed unpaired t-test; P=0.015, 0.36, 0.0003. Scale bar=500 μm. (C) HEK293T cells were co-transfected with combinations of mCherry or mCherry-b1 and GFP-tagged S1^493-685 and subjected to mCherry-nanotrap with or without co-incubation with mAb#3 (N=3). 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. Pre-incubation with EG00229 blocks S1 CendR peptide binding (orange line), and the CendR peptide can reduce binding of EG00229 (green line). (N=3). All ITC graphs represents the integrated and normalized data fit with 1-to-1 ratio binding. (F). Cells were pre-treated with 100 μM of EG00229 or DMSO prior to infection with SARS-CoV-2. Cells were fixed at 7 and 16 hpi and stained for N protein (magenta) and Hoechst (cyan) (N=3). The square region was zoomed in. Scale bars=500 μm and 100 μm (zoom panel). Two-tailed unpaired t-test; P = 0.0059 and 0.0013. The bars, error bars, circles and triangles represent the mean, SEM (C) and SD (A, B, F) and individual data points, respectively. *P< 0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001.