PEPTIDE FOR SUPPRESSING CORONAVIRUS AND USE THEREOF

Abstract

The present invention relates to: a therapeutic composition for coronavirus comprising, as an active ingredient, one peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6, and SEQ ID NO: 8 that binds to a coronavirus N-protein, a coronavirus-derived spike protein, or a fragment of the spike protein; and a composition that binds to a coronavirus N-protein comprising, as an active ingredient, the coronavirus-derived spike protein or the fragment of the spike protein. It is suggested that the peptides of the present invention, based on the understanding and targeting of the interaction of the coronavirus S protein and N protein of the present invention, have an effect that can be helpful in the treatment of coronaviruses including MERS-CoV, SARS-CoV-2, SARS-CoV, and HCoV-OC43.

Claims

1. A peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6 and SEQ ID NO: 8 that binds to the coronavirus N-protein.

2. The peptide according to claim 1, wherein the peptide is the peptide of SEQ ID NO: 4.

3. The peptide according to claim 1, wherein the coronavirus is Severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, Severe acute respiratory syndrome coronavirus 2, or Human coronavirus OC43 (HCoV-OC43).

4. The peptide according to claim 1, wherein the peptide further comprises a peptide for cell penetration.

5. The peptide according to claim 4, wherein the peptide for cell penetration is a peptide selected from the group consisting of HIV Tat peptide; Pep-1 peptide; oligo-lysine; oligoarginine; and a mixed peptide of oligo-lysine and oligoarginine.

6. A composition for treatment of a coronavirus comprising a coronavirus-derived spike protein or a spike protein fragment thereof as an active ingredient.

7. The composition according to claim 6, wherein the spike protein fragment is a domain after transmembrane of the spike protein.

8. The composition according to claim 6, wherein the coronavirus-derived spike protein or spike protein fragment thereof is a protein of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or a fragment thereof.

9. The composition according to claim 6, wherein the coronavirus is Severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, Severe acute respiratory syndrome coronavirus 2, or Human coronavirus OC43 (HCoV-OC43).

10. The composition according to claim 6, wherein the peptide further comprises a peptide for cell penetration.

11. The composition according to claim 10, wherein the peptide for cell penetration is a peptide selected from the group consisting of HIV Tat peptide; Pep-1 peptide; oligo-lysine; oligoarginine; and a mixed peptide of oligo-lysine and oligoarginine.

12. A composition for binding to a coronavirus N-protein comprising a coronavirus-derived spike protein or a spike protein fragment thereof as an active ingredient.

13. The composition according to claim 12, wherein the spike protein fragment is a domain after transmembrane of the spike protein.

14. The composition according to claim 12, wherein the coronavirus-derived spike protein or spike protein fragment thereof is a protein of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 or a fragment thereof.

15. The composition according to claim 12, wherein the coronavirus is Severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, Severe acute respiratory syndrome coronavirus 2, or Human coronavirus OC43 (HCoV-OC43).

16. The composition according to claim 12, wherein the peptide further comprises a peptide for cell penetration.

17. The composition according to claim 16, wherein the peptide for cell penetration is a peptide selected from the group consisting of HIV Tat peptide; Pep-1 peptide; oligo-lysine; oligoarginine; and a mixed peptide of oligo-lysine and oligoarginine.

Description

DESCRIPTION OF DRAWINGS

[0068] FIGS. 1 to 4 are diagrams showing the expression of S, M and N proteins in MERS-CoV-infected cells at 72 h. Cell lysates were prepared from Vero cells and MERS-CoV-infected Vero cells, and 50 μg (FIGS. 1, 2, 4) and 25 μg (FIG. 3) of protein containing cell lysates were separated by 4-12% gradient SDS-PAGE and analyzed by Western blotting with the indicated antibodies. The exposure times for signal detection were 60 s (FIGS. 1, 2, 4) and 5 s (FIG. 3), respectively,

[0069] FIGS. 5 to 8 are diagrams showing the interaction of MERS-CoV S protein and M protein with MERS-CoV N protein, (FIGS. 5 and 7) with the identification of S protein (FIG. 5) and M protein (FIG. 7) binding proteins, cell lysates were prepared from Vero cells and MERS-CoV-infected Vero cells. Cell lysates (150 μg protein) were immunoprecipitated with anti-S mAb (FIG. 5) or anti-M mAb (FIG. 7), then separated by 4-12% gradient SDS-PAGE and was stained with Coomassie Brilliant Blue G-250. The indicated (arrowhead) protein bands on the gel were digested with trypsin, and the treated peptides were analyzed by ESI-TOF MS/MS. HC, heavy chain. LC, light chain. (FIGS. 6, and 8) To show the association of S protein and M protein with N protein, cell lysates were prepared from Vero cells and MERS-CoV-infected Vero cells. Lysates were immunoprecipitated with anti-S mAb (FIG. 6) or anti-M mAb (FIG. 8) and immune complexes were subjected to Western blotting with the described antibodies. The loading of immunoprecipitated samples for N protein (anti-N Ab) analysis was half the amount for analysis of others (anti-S mAb, anti-M mAb) (FIGS. 6, and 8). The exposure times for signal detection were 120 s (anti-S mAb, anti-M mAb) and 5 s (anti-N Ab), respectively,

[0070] FIG. 9 is a diagram showing the identification of the MERS-CoV S protein-binding protein. The protein band co-immunoprecipitated with the MERS-CoV S protein was treated on a gel with trypsin, and the peptide was analyzed by ESI-TOF MS/MS. MS/MS analysis of the mass peak (arrow) obtained in the ˜45 kDa band shows the peptide spectrum of the MERS-CoV N protein,

[0071] FIGS. 10 to 12 are diagrams showing the interaction between the cytoplasmic domain of the MERS-CoV S protein and the MERS-CoV N protein (FIG. 10) shows a schematic diagram and the sequence of the cytoplasmic domain of the S protein, 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 MERS-CoV S protein; Spike CD-full, MERS-CoV Spike CD-F, Spike CD-M and Spike CD-B represent synthetic peptide sequences. (FIG. 11) Representing the immunoprecipitation assay, cell lysates were prepared from Vero cells and MERS-CoV-infected Vero cells. Western blot analysis using anti-N Ab was performed on immune complexes obtained from each biotinylated synthetic peptide sequence. The right column shows the relative band intensities of the N protein. (FIG. 12) Showing competition of Spike CD peptide for the interaction of MERS-CoV Spike CD with MERS-CoV N protein, cell lysates were prepared from MERS-CoV-infected Vero cells. Lysates were incubated for 2 h at 37° C. with each of the Spike CD-F, Spike CD-M and Spike CD-B peptides and then with biotinylated Spike CD-Full-Biotin at 37° C. incubated for 2 hours. Western blot analysis using an anti-N protein antibody was performed on the immune complexes obtained by streptavidin-beads. The right column shows the relative band intensities of the N protein,

[0072] FIG. 13 is a diagram showing the location of R-Spike CD in Vero cells. Vero cells were cultured for 24 hours and then incubated with R-Spike CD-biotin peptide at 37° C. for 30 minutes in a 5% CO.sub.2 incubator. Samples were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Cell-permeated R-spike CD-biotin peptide was detected with Alexa flour-488-conjugated streptavidin (green) by Carl Zeiss LSM710. Nuclei were stained with Hoechst 33258 (blue). (It's black and white, but you can't see the blue green . . . .) Scale bar, 10 μm. R-spike CD, a peptide corresponding to the C-terminal domain of the MERS-CoV S protein with nine D-arginines at the N-terminus; R-Spike CD-Biotin, Biotinylated R-Spike CD Peptide,

[0073] FIGS. 14 to 17 are diagrams showing the effect of R-Spike CD on MERS-CoV production, (FIGS. 14 and 15) showing a decrease in MERS-CoV protein production by R-Spike CD, (FIG. 14) Cells lysates were prepared at the indicated time points from Vero cells infected with MERS-CoV (with or without R-Spike CD). Cell lysates were analyzed by Western blotting using the indicated antibodies. (FIG. 15) Vero cells were infected with MERS-CoV (with or without R-Spike CD peptide) in serum-free medium. Cells were incubated for 48 h, then stained with anti-S mAb followed by Alexa Flour 488-conjugated goat anti-mouse IgG antibody and analyzed by confocal microscopy. Scale bar, 20 μm. (FIGS. 16 and 17) To show inhibition of MERS-CoV plaque formation by R-Spike CD, MERS-CoV was mixed with 2-fold serially diluted R-Spike CD and R-CP-1. The MERS-CoV virus-peptide mixture was treated to Vero cells in a 5% CO.sub.2 incubator at 37° C. After incubation for 1 hour, the medium was removed and then supplemented with DMEM/F12 containing 0.6% Oxoid agar. After culturing for 4 days, plaque formation was confirmed by staining with crystal violet. (FIG. 16) Representative photographs showing plaque formation. (FIG. 17) shows the quantification of plaques obtained by MERS-CoV infection after treatment with each peptide from 0 μM to 100 μM, where the number of plaques obtained from control plates treated with only MERS-CoV virus was 100%. R-spike CD, a peptide corresponding to the C-terminal domain of the S protein with 9 D-arginines at the N-terminus; R-CP-1, nine D-arginine-conjugated control peptides.

[0074] FIG. 18 is a diagram showing the effect of R-Spike CD-COVID-19 on SARS-CoV-2 production, showing inhibition of SARS-CoV-2 production by cell-permeable Spike CD-COVID-19 peptide (R-Spike CD-COVID-19), SARS-CoV-2 (0.1 MOI) was incubated at 37° C. with 5% CO.sub.2 incubator. treated with Vero cells. After incubation for 1 hour, the medium was removed and then supplemented with DMEM containing 2% FBS. After incubation for 6 h, the cell-permeable Spike CD-COVID-19 peptide (R-Spike CD-COVID-19), the control cell-penetrating peptide (R-CP-1) and the cell-permeable MERS-CoV spike CD peptide (R-Spike CD-COVID-19)-Spike CD (MERS), each 5 μM were treated respectively. 24 hours after SARS-CoV-2 infection, viral RNA was isolated from the cell culture medium, and cDNA was prepared. The produced virus was quantified by performing real-time RCR using primers for SARS-CoV-2 RNA-dependent RNA polymerase (RdRP). R-spike CD-COVID-19, a peptide corresponding to the C-terminal domain of SARS-CoV-2 S protein with 9 D-arginines at the N-terminus; R-spike CD, a peptide corresponding to the C-terminal domain of the MERS-CoV S protein with nine D-arginines at the N-terminus; R-CP-1, nine D-arginine-conjugated control peptides,

[0075] FIG. 19 is a diagram showing the expression of S and N proteins and the interaction of HCoV-OC43 S protein with HCoV-OC43 N protein in HCoV-OC43-infected cells at 72 hours, (A) Cell lysates prepared from OC43-infected Vero cells and cell lysates were separated by 4-12% gradient SDS-PAGE and analyzed by Western blotting with the indicated antibodies. (B) The association of S protein with N protein. Cell lysates were prepared from Vero cells and HCoV-OC43-infected Vero cells. Lysates were immunoprecipitated with anti-S Ab and immune complexes were subjected to western blotting with anti-N mAbs with the described antibodies. HC, heavy chain. LC, light chain. S, S protein. N, N protein,

[0076] FIG. 20 is a diagram showing the effect of R-Spike CD-OC43 on the production of HCoV-OC43 S and N proteins, showing a decrease in HCoV-OC43 protein production by R-Spike CD-OC43, and the cell lysate was prepared from Vero cells infected with HCoV-OC43 (presence or absence of R-Spike CD-OC43) 48 hours later. Cell lysates were analyzed by Western blotting using the indicated antibodies.

[0077] FIGS. 21 to 22 are diagrams showing the effects of R-Spike CD-OC43 on the production of HCoV-OC43 S and N proteins. Vero cells were infected with HCoV-OC43 (with or without R-Spike CD-OC43). After incubating the cells for 48 h (FIG. 21), S protein was analyzed by confocal microscopy after staining with anti-S Ab and then Alexa Flour 488-conjugated goat anti-rabbit IgG antibody. (FIG. 22) N protein was analyzed by confocal microscopy after staining with anti-N mAb followed by Alexa Flour 488-conjugated rabbit anti-mouse IgG antibody. Scale bar, 20 μm,

[0078] FIG. 23 is a diagram showing the effect of R-Spike CD-OC43 on HCoV-OC43 virus production, showing the inhibition of HCoV-OC43 production by cell-permeable spike CD-OC43 peptide (R-Spike CD-OC43); HCoV-OC43 (0.1 MOI) was treated with Vero cells in a 5% CO.sub.2 incubator at 37° C. After incubation for 1 hour, the medium was removed and then supplemented with DMEM containing 2% FBS. After incubation for 6 hours, cell penetrating spike CD-OC43 peptide (R-Spike-CD-OC43) and control spike CD-OC43 peptide (Spike-CD-OC43) (2 μM each) were each treated. After 48 hours of HCoV-OC43 infection, the virus produced in the cell culture was quantified by a plaque formation test.

MODE FOR INVENTION

[0079] Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are described with the intention of illustrating the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1: Cell Lines and Viruses

[0080] Vero cells, Vero E6 cells and Calu-3 cells were purchased from the Korean Cell Line Bank (Seoul, Korea). Cells was cultured in Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fisher Scientific, MA, USA) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific), 25 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were incubated at 37° C. in atmospheric conditions of 95% air and 5% CO.sub.2. MERS-CoV/KOR/KNIH/002_05_2015 and SARS-CoV-2 (NCCP No. 43326) were obtained from the Korea Disease Control and Prevention Agency. Virus preparation and cell culture procedures were performed under biosafety level 3 (BSL-3) conditions. HCoV-OC43 (KBPV-VR-8) was obtained from the Korea Bank for Pathogenic Viruses (Korea University). HCoV-OC43 preparation and cell culture procedures were performed under biosafety level 2 (BSL-2) conditions.

Example 2: Peptide Synthesis

[0081] The spike CD of MERS-CoV was analyzed from the MERS-CoV S protein sequence (MERS-CoV/KOR/KNIH/002_05_2015 (GI: 829021049)), and the following peptides were designed to investigate the interaction between S protein and N protein:

TABLE-US-00001 Spike CD-F, (SEQ ID NO: 1) .sup.1318TGCGTNCMGKLKCNRC.sup.1333. Spike CD-M, (SEQ ID NO: 2) .sup.1327KLKCNRCCDRYEEYDL.sup.1343. Spike CD-B, (SEQ ID NO: 3) .sup.1336DRYEEYDLEPHKVHVH.sup.1353. Spike CD-Full, (SEQ ID NO: 4) .sup.1318TGCGTNCMGKLKCNRCCDRYEEYDLEPHKVHVH.sup.1353.

[0082] All peptides were synthesized by an automatic peptide synthesizer (Anygen Co., LTD. Gwangju). In order to penetrate the peptides into cells, each peptide is conjugated with nine D-arginines on the N-terminus (R-Spike CD) and/or with biotin on the C-terminus (R-Spike CD-Biotin, Spike CD-Full-Biotin, Spike CD-F-Biotin, Spike CD-M-Biotin, Spike CD-B-Biotin)) and 9 D-Arginine-conjugated control peptides R-CP-1 (NH2-d-RRRRRRRRRR-AQARRKNYGQLDIFP-COOH; (SEQ ID NO: 5)) was used as a control cell penetrating peptide (D. Raina, et al. Cancer Res. 69, 5133-5141 (2009)).

[0083] The spike CD of SARS-CoV-2, a coronavirus infection-19 coronavirus, was synthesized from the SARS-CoV-2 S protein sequence (QHD43416) and the spike CD from SARS-CoV, a severe acute respiratory syndrome coronavirus, with the SARS-CoV S protein sequence (NP_828851.1), and in order to penetrate the peptide into cells, the following peptide conjugated with the spike CD peptide of SARS-CoV-2 with 9 D-arginines on the N-terminus (R-spike CD-COVID-19) was designed: SARS-CoV-2 is the name of the virus that causes COVID-19 (disease name), and when naming the peptide, SARS-CoV-2 is too long and named COVID-19.

TABLE-US-00002 Spike CD-COVID-19, (SEQ ID NO: 6) .sup.1234LCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT.sup.1273 Spike CD-SARS-CoV (SEQ ID NO: 7) .sup.1216LCCMTSCCSCLKGACSCGSCCKFDEDDSEPVLKGVKLHYT.sup.1255
Spike CD-SARS-CoV has one different amino acid sequence from Spike CD-COVID-19, so a separate experiment was not performed by synthesizing Spike CD-SARS-CoV.
The spike CD of human coronavirus OC43 (HCoV-OC43) was analyzed from the HCoV-OC43 S protein sequence (YP_009555241.1), and to penetrate the peptide into cells, the following peptide conjugated with the spike CD peptide of HCoV-OC43 with 9 D-arginines (R-spike CD-OC43) was designed.

TABLE-US-00003 Spike CD-OC43’ (SEQ ID NO: 8) .sup.1320CCTGCGTSCFKKCGGCCDDYTGYQELVIKTSHDD.sup.1353

Example 3: Antibody

[0084] A monoclonal antibody (492-1G10E4E2 clone) against MERS-CoV S protein (anti-S mAb) (B. K. Park, S. Maharjan, S. I. Lee, J. Kim,J-Y. Bae, M.-S. Park, H.-J. Kwon, BMB Rep 52, 397-402 (2019)) and a monoclonal antibod(M158-2D6F11 clone) against MERS-CoV M protein (anti-M mAb) (B. K. Park, S. I. Lee, J.-Y. Bae, M.-S. Park, Y. Lee, H.-J. Kwon, Int J Pept Res Ther, 1-8 (2018)) was prepared, as described in D. Kim, S. Kwon, J. W. Rhee, K. D. Kim, Y.-E. Kim, C.-S. Park, M. J. Choi, J.-G. Suh, D.-S. Kim, Y. Lee, BMC Immunol. 12, 29 (2011), each peptide epitope formulated into a CpG-DNA-liposome complex was used to prepare from hybridoma cells established after immunization of BALB/c mice.

[0085] Spike-492 (.sup.492TKPLKYSYINKCSRLLSDDRTEVPQ.sup.516; (SEQ ID NO: 9)) and MERS-M158 (.sup.158CDYDRLPNEVTVAKPNVLIALKMVK.sup.182; (SEQ ID NO: 10)) were used as B cell epitope sequences for the S protein (Spike glycoprotein universal sequence (GI: 510785803)) and M protein of MERS-CoV, respectively. Rabbit anti-MERS N protein antibody (anti-N Ab) was purchased from Sino Biological (Cat. No. 40068-RP02, Vienna, Austria) and anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, Mo., USA).

[0086] Mouse anti-HCoV-OC43 N protein antibody (anti-N mAb) was purchased from LSBio (Cat No. LS-C79764, Seattle, USA), and rabbit anti-HCoV-OC43 S protein antibody (anti-S Ab) was purchased from LSBio (Cat No. LS-C371066).

Example 4: MERS-CoV Infection and Co-Immunoprecipitation Method

[0087] Vero cells were cultured for 12 hours at a density of 6×10.sup.5 cells/10 cm dish. MERS-CoV (0.1 MOI) was inoculated into Vero cells in serum-free medium, and then incubated at 37° C. in a 5% CO.sub.2 incubator for 1 hour. After incubation, the supernatant was removed and each dish was replenished with DMEM medium containing 25 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin. 3 days post infection, MERS-CoV-infected Vero cells were lysed in cell lysis buffer (10 mM HEPES, 150 mM NaCl, 5 mM EDTA, 100 mM NaF, 2 mM Na.sub.3VO.sub.4, protease inhibitor cocktail and 1% NP-40) at 4° C. for 30 min. Cell lysates were centrifuged to remove cell debris, and cell lysates were incubated with anti-S mAb or anti-M mAb at 4° C. for 3 hours.

[0088] Protein A beads (CaptivAtm PriMAB 52% (v/v) slurry, REPLIGEN, Waltham, Mass., USA) were added, followed by centrifugation to collect immune complexes Immune complexes were separated by 4-12% gradient SDS-PAGE (Bottom 4-12% Bis-Tris Plus gel, Thermo Fisher Scientific) and stained with Coomassie Brilliant Blue G-250.

Example 5: MERS-CoV S Protein Binding Protein Analysis

[0089] After immunoprecipitation with anti-S mAb in MERS-CoV-infected cell lysates, the immune complexes were separated by 4-12% gradient SDS-PAGE, followed by excision of the protein band of interest. Protein bands were analyzed by Proteinworks Co (Seoul, Korea).

[0090] The protein band in the gel was digested with trypsin and the resulting peptide was analyzed using a Poros reversed phase R2 column (PerSeptive Biosystems, Framingham, Mass., USA). The isolated peptides were investigated using the electrospray ionization time of a flight mass spectrometer/mass spectrometer (4700 MALDI-TOF/TOF, Applied Biosystems, Thermo Fisher Scientific). Peptide sequences were analyzed using the database of the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov).

Example 6: Western Blotting and Immunoprecipitation

[0091] Uninfected Vero cell lysates and MERS-CoV-infected cell lysates were prepared with cell lysis buffer (20 mM Tris HCl pH 8.0.5 mM EDTA, 150 mM NaCl, 100 mM NaF, 2 mM Na.sub.3VO.sub.4, 1% NP-40) and after centrifugation at 14,000 rpm for 20 mM at 4° C., separation was performed on a 4-12% Bis-Tris gradient gel (Thermo Fisher Scientific).

[0092] The isolated protein was transferred to a nitrocellulose membrane and then the membrane was incubated with anti-S mAb, anti-M mAb, anti-N Ab or anti-β actin antibody overnight at 4° C. After incubating the membrane with horseradish peroxidase-conjugated secondary antibody, the immunoreactive band was reacted with enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific).

[0093] To perform the binding properties of each MERS-CoV protein, co-immunoprecipitation analysis was performed with each MERS-CoV protein as described above. Coimmunoprecipitated proteins were identified by Western blotting using anti-S mAbs, anti-M mAbs or anti-N Abs.

Example 7: Analysis of the Interaction Between MERS-CoV Spike CD Peptide and N Protein

[0094] MERS-CoV-infected cell lysates was incubated with one of the following biotinylated peptides at 4° C. for 2 hours,

[0095] Spike CD-whole-biotin,

[0096] Spike CD-F-Biotin,

[0097] Spike CD-M-biotin and

[0098] Spike CD-B-Biotin

[0099] After incubation, after addition of streptavidin-agarose (Thermo Fisher Scientific), immune complexes were collected by centrifugation.

[0100] Immune complexes were separated on 10% SDS-PAGE and then analyzed by Western blotting using anti-N Ab. The band density was analyzed by the program of Quantity One (Bio-Rad, Hercules, Calif., USA).

[0101] To determine the major regions of Spike-CD involved in the interaction with the N protein, MERS-CoV infected cell lysates were incubated with the respective peptides of Spike CD-F, Spike CD-M and Spike CD-B at 4° C. After incubation for 1 hour, Spike CD-Biotin was added to each sample and then incubated at 4° C. for 2 hours. The interaction of the N protein with Spike CD-biotin was determined by immunoprecipitation with streptavidin-agarose as described above.

Example 8: Cell Permeation of MERS-CoV Spike CD Peptides

[0102] Vero cells (5×10.sup.4) were seeded on cover glasses in 12 well plates. One day later, the cells were incubated with R-Spike CD-Biotin in a 5% CO.sub.2 incubator at 37° C. for 30 min.

[0103] After fixing the cells with 4% paraformaldehyde, the cells were permeabilized with PBST containing 1% BSA. Alexa flour-488-attached streptavidin (Jackson ImmunoResearch Laboratories Inc.) was added and incubated for 1 hour, then the samples were washed with PBST. Nuclei were stained by addition of Hoechst 33258 (Thermo Fisher Scientific). The slides were analyzed by Carl Zeiss LSM710 (Carl Zeiss Co. Ltd. Oberkochen, Germany).

Example 9: Analysis of MERS-CoV S Protein Expression Using Confocal Microscopy

[0104] Vero cells (5×10.sup.4) were cultured overnight on cover glass in 12 well plates and infected with MERS-CoV (0.1 MOI) with PBS or R-Spike CD in serum-free medium.

[0105] After 48 h, cells were fixed and then permeabilized with PBST containing 1% BSA. Permeabilized cells were incubated with anti-S mAb for 2 h. Cells were washed with PBST containing 1% BSA and then incubated with Alexa Flour 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) for 1 hour. Nuclei were stained with Hoechst 33258 and then slides were investigated with Carl Zeiss LSM710.

Example 10: Plaque Formation Assay

[0106] Vero cells (6×10.sup.5 cells/well) were plated on 6-well plates (Corning, N.Y., USA) and incubated for 12 hours. MERS-CoV (200 pfu) was mixed with R-Spike-CD or R-CP-1 serially diluted 2-fold in PBS.

[0107] A mixture of MERS-CoV and peptide was added to Vero cells and incubated for 1 hour. After incubation, the supernatant was removed, and the plate was replenished with 3 ml of DMEM/F12 medium (Thermo Fisher Scientific) containing 0.6% Oxoid agar. Cells were stained with crystal violet after 4 days, plaque counts were counted and compared to control samples treated with virus only.

Example 11: Confirmation of SARS-CoV-2 Spike CD Peptide Inhibition of SARS-CoV-2 Production

[0108] Vero cells (2×10.sup.5 cells/well) were plated on 24-well plates (Corning, N.Y., USA) and incubated for 12 hours. Cells were washed with PBS and then infected with SARS-CoV-2 (0.1 MOI) in a 5% CO.sub.2 incubator at 37° C. for 1 hour.

[0109] After incubation, the supernatant was removed, and the plates were replenished with DMEM containing 2% FBS. After incubation for 6 h, 5 μM of the cell-penetrating spike CD-COVID-19 peptide (R-Spike CD-COVID19), the control cell-penetrating peptide (R-CP-1) and the cell-penetrating MERS-CoV spike CD peptide (R-Spike CD (MERS), was treated respectively. After incubation for 17 hours, viral RNA was isolated from the cell culture medium, and cDNA was prepared. The produced virus was quantified by performing real-time RCR using primers for SARS-CoV-2 RNA-dependent RNA polymerase (RdRP).

Example 12: Real-Time RT-PCR

[0110] RNA isolation from virus in cell culture was performed using QIAamp Viral RNA Mini Kit (Catalog No. 52904, Qiagen, Hilden, Germany), and cDNA was synthesized using Reverse Transcription System kit (Catalog No. A3500, Promega, Madison, Wis., USA).

[0111] To quantify SARS-CoV-2 in cell culture, the following primers for the RNA-dependent RNA polymerase (RdRP) gene of SARS-CoV-2 were synthesized. [Reference, Jeong-Min Kim et al., Identification of Coronavirus Isolated from a Patient in Korea with COVID-19. Osong Public Health Res Perspect. 2020 February; 11(1): 3-7];

TABLE-US-00004 Forward primer, (SEQ ID NO: 11) 5-GTGAAATGGTCATGTGTGGCGG′ Reverse primer, (SEQ ID NO: 12) 5′-CAAATGTTAAAAACACTATTAGCATA-3′, TaqMan ® Probe, (SEQ ID NO: 13) 5′-FAM-CAGGTGGAACCTCATCAGGAGATGC-TAMRA-3′

[0112] Primers and TaqMan® Probe sequences were synthesized by Genotech (Daejeon, South Korea). 10 μL of GoTaq®Probe qPCR Master Mix (Promega, Madison, Wis., USA) was added to 10 μL of forward and reverse primers (125 nM each) and TaqMan®Probe (250 nM) mixture, and 1 μL of cDNA solution was added. The mixture was heated at 95° C. for 5 minutes, followed by 45 cycles of PCR at 95° C. for 15 sec and 60° C. for 1 minute each. The copy number of the RdRP gene was calculated by obtaining a standard curve from the cDNA of the RdRP gene.

Example 13: HCoV-OC43 Infection and S Protein Binding Protein Analysis

[0113] Vero cells (6×10.sup.5 cells/well) were cultured in 6-well plates for 12 h. HCoV-OC43 (0.1 MOI) was inoculated into Vero cells in PBS and incubated for 1 hour at 37° C. in 5% CO.sub.2 incubator. After incubation, the supernatant was removed and each dish was supplemented with DMEM medium containing 2% FBS, 25 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin.

[0114] 3 days post infection, HCoV-OC43-infected Vero cells were lysed in cell lysis buffer (10 mM HEPES, 150 mM NaCl, 5 mM EDTA, 100 mM NaF, 2 mM Na.sub.3VO.sub.4, protease inhibitor cocktail and 1% NP-40) at 4° C. for 30 mM. Uninfected Vero cell lysates and HCoV-OC43-infected cell lysates were prepared with cell lysis buffer (20 mM Tris HCl pH 8.0.5 mM EDTA, 150 mM NaCl, 100 mM NaF, 2 mM Na.sub.3VO.sub.4, 1% NP-40) and after centrifugation at 14,000 rpm for 20 min at 4° C., separation was performed on a 4-12% Bis-Tris gradient gel (Thermo Fisher Scientific).

[0115] The isolated protein was transferred to a nitrocellulose membrane and then the membrane was incubated with anti-S Ab, anti-N mAb or anti-β actin antibody overnight at 4° C. After incubation of the membrane with horseradish peroxidase-conjugated secondary antibody, the immunoreactive band was reacted with enhanced chemiluminescence (ECL) reagent (Thermo Fisher Scientific).

[0116] After immunoprecipitation with anti-S Ab from HCoV-OC43-infected cell lysates, the immune complexes were separated by 4-12% gradient SDS-PAGE, and then co-immunoprecipitated proteins were identified by western blotting using anti-N mAbs.

Example 14: Analysis of HCoV-OC43 S Protein and N Protein Expression Using Confocal Microscopy

[0117] Vero cells (5×10.sup.4) were cultured overnight on cover glasses in 12 well plates, infected with HCoV-OC43 (0.1 MOI) in PBS for 1 hour, and cultured in DMEM medium containing 2% FBS. After 6 hours, R-Spike CD-OC43 was treated.

[0118] After 48 h, cells were fixed and then permeabilized with PBST containing 1% BSA. Permeabilized cells were incubated with anti-S Ab or anti-N mAb for 2 h. Cells were washed with PBST containing 1% BSA and then incubated with Alexa Flour 488-conjugated goat anti-mouse IgG antibody (Thermo Fisher Scientific) or goat anti-rabbit IgG antibody (Thermo Fisher Scientific) for 1 hour. Nuclei were stained with Hoechst 33258 and then slides were examined with Carl Zeiss LSM710.

Example 15: Confirmation of Inhibition of HCoV-OC43 Production by HCoV-OC43 Spike CD Peptide

[0119] Vero cells (2×10.sup.5 cells/well) were plated on 24-well plates (Corning, N.Y., USA) and incubated for 12 hours. Cells were washed with PBS and then infected with HCoV-OC43 (0.1 MOI) in a 5% CO.sub.2 incubator at 37° C. for 1 hour. After incubation, the supernatant was removed, and the plates were supplemented with DMEM medium containing 2% FBS. After incubation for 6 hours, the cell-permeable spike CD-OC43 peptide (R-Spike CD-OC43) and the spike CD peptide of HCoV-OC43 (Spike CD-OC43) (2 μM each) were respectively treated. After 42 hours of incubation, the virus in the cell culture medium was identified through plaque formation assay.

[0120] Vero cells (6×10.sup.5 cells/well) were plated on 6-well plates (Corning, N.Y., USA) and incubated for 12 hours. after adding the cell-permeable spike CD-OC43 peptide (R-Spike CD-OC43) and the spike CD peptide of HCoV-OC43 (Spike CD-OC43) (2 μM each) to Vero cells incubated for 1 hour. After incubation, the supernatant was removed, and the plate was replenished with 3 ml of DMEM/F12 medium (Thermo Fisher Scientific) containing 0.6% Oxoid agar. Cells were stained with crystal violet after 5 days, plaque counts were counted and compared to control samples treated with virus only.