MERS-CoV and SARS-CoV-2 replication can be inhibited by targeting the interaction between the viral spike protein and the nucleocapsid protein

Abstract

Background: The molecular interactions between viral proteins form the basis of virus production and can be used to develop strategies against virus infection. The interactions of the envelope proteins and the viral RNA-binding nucleocapsid (N) protein are essential for the assembly of coronaviruses including the Middle East respiratory syndrome coronavirus (MERS-CoV). Methods: Using co-immunoprecipitation, immunostaining, and proteomics analysis, we identified a protein interacting with the spike (S) protein in the cells infected with MERS-CoV or SARS-CoV-2. To confirm the interaction, synthetic peptides corresponding to the C-terminal domain of the S protein (Spike CD) were produced and their effect on the interaction was investigated in vitro. In vivo effect of the Spike CD peptides after cell penetration was further investigated using viral plaque formation assay. Phylogeographic analyses were conducted to deduce homology of Spike CDs and N proteins. Results: We identified a direct interaction between the S protein and the N protein of MERS-CoV that takes place during virus assembly in infected cells. Spike CD peptides of MERS-CoV inhibited the interaction between the S and N proteins in vitro. Furthermore, cell penetration by the synthetic Spike CD peptides inhibited viral plaque formation in MERS-CoV-infected cells. Phylogeographic analyses of Spike CDs and N proteins showed high homology among betacoronavirus lineage C strains. To determine if Spike CD peptides can inhibit the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we used the same strategy and found that the SARS-CoV-2 Spike CD peptide inhibited virus replication in SARS-CoV-2-infected cells. Conclusions: We suggest that the interaction between the S protein and the N protein can be targeted to design new therapeutics against emerging coronaviruses, including SARS-CoV-2.

Keywords: MERS-CoV; SARS-CoV-2; nucleocapsid protein; spike protein; targeting.

Figure 1

Interaction of MERS-CoV S and M proteins with MERS-CoV N protein. (A and C) Identification of proteins that bind S protein (A) and M protein (C). Lysates of uninfected and MERS-CoV (0.1 MOI)-infected Vero cells were prepared. The lysates (150 μg protein) were immunoprecipitated with anti-MERS-CoV S mAb (A) or anti-MERS-CoV M mAb (C), resolved by 4-12% gradient SDS-PAGE, and stained with Coomassie brilliant blue G-250. The indicated (arrowhead) protein band was digested with trypsin, and the digested peptides were analyzed by ESI-TOF MS/MS (A). HC, heavy chain. LC, light chain. (B and D) Association of S protein and M protein with N protein. Lysates of uninfected and MERS-CoV-infected Vero cells were prepared and immunoprecipitated with anti-MERS-CoV S mAb (B) or anti-MERS-CoV M mAb (D). (E) β-actin in the lysates from uninfected and MERS-CoV-infected Vero cells was used as a control. The immunocomplexes were subjected to western blotting with the indicated antibodies. The loading amount of immunoprecipitated sample used for the analysis of the N protein (anti-MERS-CoV N Ab) was half of that used for the analysis of the other proteins (anti-MERS-CoV S mAb, anti-MERS-CoV M mAb) (B and D). The exposure time for signal detection was 120 sec for anti-MERS-CoV S mAb and anti-MERS-CoV M mAb and 5 seconds for anti-MERS-CoV N Ab. Anti-S mAb, anti-MERS-CoV S mAb. Anti-M mAb, anti-MERS-CoV M mAb. Anti-N Ab, anti-MERS-CoV N Ab.

Figure 2

Interaction of the cytoplasmic domain of MERS-CoV S protein with MERS-CoV N protein. (A) Schematic diagram of the MERS-CoV S protein and the sequences of the cytoplasmic domain. RBD, receptor-binding domain; FP, fusion peptide; HR1 and HR2, heptad repeat regions 1 and 2; TM, transmembrane; CD, C-terminal domain; Spike CD, C-terminal domain of the S protein. Spike CD-Full, Spike CD-F, Spike CD-M, and Spike CD-B denote the synthetic peptide sequences. (B) Immunoprecipitation analysis. Lysates were prepared from uninfected and MERS-CoV-infected Vero cells. Immunocomplexes obtained using each biotinylated synthetic peptide were subjected to western blotting with anti-MERS-CoV N Ab. The right column represents the relative band intensities of the N protein. (C) Competition between Spike CD peptides and MERS-CoV Spike CD for interaction with MERS-CoV N protein. Cell lysates were prepared from MERS-CoV-infected Vero cells. The lysates were incubated with Spike CD-F, Spike CD-M, or Spike CD-B peptide for 2 h at 37 °C and then with biotinylated Spike CD-Full peptide (Spike CD-Full-Biotin) for 2 h at 37 °C. Immunocomplexes from Streptavidin beads were subjected to western blotting analysis with anti-MERS-CoV N protein antibody. The right column represents relative band intensities of the N protein.

Figure 3

Localization of MERS-CoV R-Spike CD in Vero cells and cytotoxicity of the cell-penetrating peptides. (A) Vero cells were cultured for 24 h and then incubated with R-Spike CD-MERS-CoV-Biotin peptide for 30 min in a 5% CO₂ incubator at 37 °C. The samples were fixed with 4% paraformaldehyde and permeabilized with 0.1% triton X-100. Cell-penetrated R-Spike CD-MERS-CoV-Biotin peptide was detected using Alexa Fluor-488-conjugated Streptavidin (Green) and a Carl Zeiss LSM710 microscope. Nuclei were stained with Hoechst 33258 (Blue). Scale bar, 10 µm. (B) Effect of cell-penetrating peptides on the growth of Vero cells and Calu-3 cells. Vero cells or Calu-3 cells were cultured with the indicated concentrations of cell-penetrating peptides for 3 days. The cells were incubated with CCK-8 solution, and then, soluble formazan was measured using a microplate reader. R-Spike CD-MERS-CoV, the peptide corresponding to the C-terminal domain of the MERS-CoV S protein conjugated with nine D-arginine residues at the N-terminus; R-Spike CD-MERS-CoV-Biotin, a biotinylated R-Spike CD-MERS-CoV peptide; R-Spike CD-SARS-CoV-2, the peptide corresponding to the C-terminal domain of the SARS-CoV-2 S protein conjugated with nine D-arginine residues at the N-terminus; R-CP-1, a nine D-arginine-conjugated control peptide.

Figure 4

Effects of R-Spike CD-MERS-CoV on MERS-CoV production. (A and B) Reduction of MERS-CoV protein production by R-Spike CD-MERS-CoV. (A) Cell lysates were prepared at the indicated time points from Vero cells infected with MERS-CoV (0.1 MOI, with or without R-Spike CD-MERS-CoV). The cell lysates were analyzed by western blotting with the indicated antibodies. (B) Vero cells were infected with MERS-CoV (0.1 MOI, with or without R-Spike CD peptide-MERS-CoV) in serum-free medium. The cells were cultured for 48 h and then analyzed by confocal microscopy after staining with anti-MERS-CoV S mAb and then, Alexa Fluor 488-conjugated goat anti-mouse IgG antibody. Scale bar, 20 μm. (C and D) Inhibition of MERS-CoV plaque formation by R-Spike CD-MERS-CoV. MERS-CoV was mixed with two-fold serially diluted R-Spike CD-MERS-CoV and R-CP-1 (n = 3). The MERS-CoV virus (200 pfu)-peptide mixture was added to Vero cells in a 5% CO₂ incubator at 37˚C. After 1 h of incubation, the medium was removed, and the cultures were replenished with DMEM/F12 containing 0.6% oxoid agar. After 4 days of incubation, plaque formation was verified by staining with crystal violet. (C) A representative picture showing plaque formation. (D) Quantification of the plaques formed by MERS-CoV infection after treatment with each peptide at the indicated concnetrations. Plaque numbers obtained in control plates treated with MERS-CoV virus only were taken as 100%. R-Spike CD-MERS-CoV, the peptide corresponding to the C-terminal domain of the S protein conjugated with nine D-arginine residues at the N-terminus; R-CP-1, nine-D-arginine-conjugated control peptide. *p < 0.05, ***p < 0.001 compared to virus-only controls. These results are representative of two independent experiments.

Figure 5

Phylogenetic analysis of Spike CDs and N proteins of different betacoronavirus species. (A and B) The phylogenetic trees of betacoronaviruses generated using Dayhoff (for Spike CDs) and JTT+G (for N proteins) models of evolution. Support for the topologies was assessed by bootstrap analysis with 1,000 iterations. The phylogenetic positions of (A) Spike CDs and (B) N proteins are shown in relation to representative betacoronaviruses. Relative homology of Spike CDs and N proteins is shown with homology to MERS-CoV/KOR/KNIH/002_05_2015 control taken as 100%. Spike CDs, S protein C-terminal domains.

Figure 6

Interaction of SARS-CoV-2 S protein with N protein and effects of R-Spike CD-SARS-CoV-2 on production of SARS-CoV-2 proteins. (A) Interaction of SARS-CoV-2 S protein with N protein. Lysates were prepared from uninfected and SARS-CoV-2 (0.1 MOI)-infected Vero cells. The lysates were immunoprecipitated with anti-SARS-CoV-2 S mAb (left). The immunocomplexes were subjected to western blotting with anti-SARS-CoV-2 S mAb or anti-SARS-CoV-2 N Ab. The cell lysates were analyzed by western blotting with the indicated antibodies (right). Anti-S Ab, anti-SARS-CoV-2 S Ab. Anti-N mAb, anti-SARS-CoV-2 N mAb. (B-E) Effects of R-Spike CD-SARS-CoV-2 on production of SARS-CoV-2 proteins. Vero cells (B and C) and Calu-3 cells (D and E) were infected with SARS-CoV-2 (0.1 MOI) and then treated with PBS or 2 μM of cell-penetrating peptides (R-Spike CD-SARS-CoV-2 or R-CP-1) at 6 h after virus infection (n = 3) in DMEM medium containing 2% FBS. The cells were cultured for 48 h and then analyzed by confocal microscopy after staining with anti-SARS-CoV-2 S Ab (B and D) or anti-SARS-CoV-2 N mAb (C and E) and then, Alexa Fluor 488-conjugated secondary antibody. Scale bar, 20 μm. These results are representative of two independent experiments.

Figure 7

Effect of R-Spike CD-SARS-CoV peptides on the replication of SARS-CoV-2. (A and B) Vero cells (A) and Calu-3 cells (B) infected with SARS-CoV-2 (0.1 MOI) and then treated with PBS or 2 μM of cell-penetrating peptides (R-Spike CD-SARS-CoV-2 or R-CP-1) at 6 h after virus infection (n = 3). Supernatants of virus-infected cell cultures were collected at 24 h after virus infection. Virus replication was quantified by qRT-PCR analysis of the SARS-CoV-2 RdRP gene (left) and plaque formation assay (right). *p < 0.05, **p < 0.01, ***p < 0.001. These results are representative of two independent experiments.