Oligopeptide derivative based on novel coronavirus spike protein SIM site and application thereof

Abstract

The invention discloses an oligopeptide derivative based on a novel coronavirus spike protein SIM site and application of the oligopeptide derivative. The oligopeptide derivative disclosed by the invention is an oligopeptide derivative which comprises cpSIM1, cpSIM1 ', cpSIM2, cpSIM3 and cpSIM4, and the oligopeptide derivative is composed of a cell-penetrating peptide and an oligopeptide; the oligopeptides are oligopeptides obtained by modifying related sites of SIM1, SIM1 ', SIM2, SIM3 and SIM4 on the basis of SARS-CoV-2Spike protein polypeptide SUMO; the cell-penetrating peptide is connected to the N terminal of the oligopeptide. The four short peptide derivatives of the cpSIM1, the cpSIM1 ', the cpSIM3 and the cpSIM4 do not influence the cell activity, have no cytotoxicity, and can promote the expression of the SARS-CoV-2Spike protein on the cell surface and enhance the immunogenicity. The cpSIM2 has high specificity for resisting SARS-CoV-2 infection, the effectiveness of the cpSIM2 is verified in in-vivo and in-vitro models, and the cpSIM2 can be used for preparing drugs for resisting different SARS-CoV-2 mutant strain infection and has important application value for targeted therapy of SARS-CoV-2.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a short peptide derivative based on the SIM site of the spike protein of the novel coronavirus and its use. Background Art

[0002] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the pathogen of coronavirus disease 2019 (COVID-19), has become one of the most widespread and destructive viruses on record and continues to pose a threat to public health security due to its rapid evolution. SARS-CoV-2 is an enveloped single-stranded positive-sense RNA virus with a genome of approximately 29.9 kb, having multiple open reading frames and encoding four structural proteins: spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), 16 non-structural proteins, and 11 accessory proteins. The Spike protein plays a key role in host cell entry and consists of two subunits, S1 and S2. The S1 subunit encodes the receptor binding domain (RBD) responsible for binding to the ACE2 receptor, while the S2 subunit encodes the transmembrane portion of the Spike protein responsible for the fusion of the viral membrane with the cell membrane. The junction between S1 and S2 contains a polyarginine (polybasic) cleavage site that can be recognized by the cellular protease furin, and cleavage at this site is crucial for Spike protein-mediated cell fusion and virus entry into human lung cells. In infected cells, the Spike protein undergoes cleavage by furin during its maturation through the Golgi apparatus and the trans-Golgi network, forming non-covalently linked S1 and S2 subunits, enabling S1 to interact more effectively with ACE2 and promoting the exposure of S2 and its subsequent cleavage and conformational rearrangement. In addition to assembling into virus particles, when expressed on the cell surface, the Spike protein can also mediate intercellular transmission of the virus. Therefore, the intracellular trafficking and cleavage of the Spike protein determine the mode of virus infection. Similar to other viral surface proteins, the Spike protein undergoes various post-translational modifications (PTMs), such as glycosylation, which affect its cleavage and incorporation into virus particles. However, the small ubiquitin-like modifier (SUMO) modification of viral surface proteins and its functional significance have not been reported.

[0003] SUMOylation is a dynamic and reversible post-translational protein modification that plays an important role in the cellular response to various stimuli. The human genome mainly encodes three subtypes of SUMO molecules: SUMO1, SUMO2, and SUMO3. Since SUMO2 and SUMO3 have 98% homology, they are usually collectively referred to as SUMO2 / 3. SUMO1 and SUMO2 / 3 modifications exhibit different physiological functions, and host and viral proteins are more prone to SUMO2 / 3 modification under stress conditions. Previous studies have shown that target proteins can undergo different types of SUMO modifications at different lysine (K) residues and can bind to other proteins through SUMO-interacting motifs (SIMs), providing an additional interaction platform for the recruitment of downstream effector proteins. Increasing evidence indicates that the SUMO modification system plays a key role in the host response to viral infections. Many viral proteins not only serve as substrates for SUMO modification but also regulate the host SUMO pathway. Recent studies have highlighted the link between SUMOylation and SARS-CoV-2 infection. For example, SUMOylation of the SARS-CoV-2 N protein mediated by TRIM28 enhances the oligomerization, RNA-binding, and liquid-liquid phase separation abilities of the N protein. In addition, SUMO3 modification of ACE2 is thought to contribute to SARS-CoV-2 infection. Despite these findings, a comprehensive understanding of SUMO-modified proteins associated with SARS-CoV-2 infection remains to be established. The rapid spread and continuous evolution of SARS-CoV-2, especially the emerging variants such as BA.2.86 / JN.1 and KP.2, KP.3, etc., pose increasing challenges to existing vaccines and treatment methods. There is an urgent need for new treatment strategies and drugs to address these challenges. Therefore, in-depth study of the SUMO modification of SARS-CoV-2 and its role in viral infection is of great significance for the development of new broad-spectrum antiviral drugs and vaccines. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a short peptide derivative based on the SIM sites of the spike protein of the novel coronavirus and its uses. The short peptide derivatives based on 5 different SIM sites (SIM1, SIM1’, SIM2, SIM3, and SIM4) of the SARS-CoV-2 Spike protein in the present invention are polypeptide compounds that can penetrate cell membranes and enter cells and are stable in vivo, and respectively have high specificity and effectiveness against SARS-CoV-2 infection, or have the potential function of promoting the cell surface expression of the SARS-CoV-2 Spike protein and enhancing its immunogenicity.

[0005] The first object of the present invention is to provide short peptide derivatives of the SIM site of the novel coronavirus spike protein. The short peptide derivatives include cpSIM1, cpSIM1', cpSIM2, cpSIM3 and cpSIM4. The short peptide derivatives are composed of a cell-penetrating peptide and a short peptide. The short peptide is a short peptide based on the SIM1, SIM1', SIM2, SIM3 and SIM4 sites related to SUMO modification of the SARS-CoV-2 Spike protein polypeptide. The cell-penetrating peptide is connected to the N-terminus of the short peptide.

[0006] In some embodiments of the present invention, the amino acid sequences of the short peptides at the SIM1, SIM1', SIM2, SIM3 and SIM4 sites are shown as SEQ ID No.1-4 in sequence.

[0007] The chemical structural formulas of the short peptides at the SIM1, SIM1', SIM2, SIM3 and SIM4 sites are as follows:

[0008] Chemical structural formula of the short peptide at the SIM1 site:

[0009]

[0010] Chemical structural formula of the short peptide at the SIM1' site:

[0011]

[0012] Chemical structural formula of the short peptide at the SIM2 site:

[0013]

[0014] Chemical structural formula of the short peptide at the SIM3 site:

[0015]

[0016] Chemical structural formula of the short peptide at the SIM4 site:

[0017]

[0018] In some embodiments of the present invention, the amino acid sequence of the cell-penetrating peptide is shown as SEQ ID No.5.

[0019] In some embodiments of the present invention, the amino acid sequences of the cpSIM1, cpSIM1', cpSIM2, cpSIM3 and cpSIM4 are shown as SEQ ID No.6-9 in sequence. The short peptide derivatives are prepared by solid-phase synthesis.

[0020] The second object of the present invention is to provide a composition comprising the short peptide derivatives of the SIM site of the novel coronavirus spike protein.

[0021] The third object of the present invention is to provide an antiviral drug, comprising a short peptide derivative of the SIM site of the novel coronavirus spike protein or the composition. Further, the short peptide derivative of the SIM site of the novel coronavirus spike protein or the composition comprises cpSIM2.

[0022] In some embodiments of the present invention, the virus comprises the novel coronavirus SARS-CoV-2 or SARS-CoV.

[0023] Further, the short peptide derivative (such as cpSIM2) can inhibit the increase in the viral copy number of SARS-CoV-2.

[0024] The fourth object of the present invention is to provide a viral vaccine adjuvant, comprising a short peptide derivative of the SIM site of the novel coronavirus spike protein or the composition. Further, the short peptide derivative of the SIM site of the novel coronavirus spike protein or the composition comprises cpSIM1, cpSIM1', cpSIM3 or cpSIM4.

[0025] In some embodiments of the present invention, the virus comprises the novel coronavirus SARS-CoV-2 or SARS-CoV.

[0026] In some embodiments of the present invention, the short peptide derivative promotes the expression of the SARS-CoV-2 Spike protein on the cell surface and enhances the immunogenicity.

[0027] In the present invention, the infection of SARS-CoV-2 triggers a comprehensive reconstruction of the host SUMO modification network, significantly affecting the localization of SUMO1 and SUMO2 / 3. The SUMO2 modification of the SARS-CoV-2 Spike protein inhibits its cleavage, thereby preventing virus-mediated intercellular transmission. In the present invention, through a series of experiments, the sites of SUMO modification and interaction of the SARS-CoV-2 Spike protein were detected, and the polypeptides related to the SIM1, SIM1', SIM2, SIM3 and SIM4 sites were screened out. This peptide segment can competitively inhibit the interaction between the Spike protein and other host proteins, promote or inhibit the expression of the Spike protein, thereby playing a role in preventing or resisting SARS-CoV-2 virus infection.

[0028] The above technical solutions of the present invention have the following advantages compared with the prior art:

[0029] The present invention designs a variety of polypeptide compounds cpSIM1, cpSIM1’, cpSIM2, cpSIM3, and cpSIM4 derived from the SIM sites of the SARS-CoV-2 Spike protein. The peptide sequence and length are determined through the protein sequence of Spike and specific SUMO interaction sites. The effects of different polypeptide compounds on the infection and replication of different SARS-CoV-2 mutants are compared through techniques such as reverse transcription-quantitative PCR, immunoblotting, and immunofluorescence analysis. Using the hACE2 transgenic mouse model, the potential preventive effect of cpSIM1 / 1’ / 3 / 4 against SARS-CoV-2 infection and the therapeutic effect of cpSIM2 against SARS-CoV-2 infection are further verified, providing new strategies for the prevention and treatment of SARS-CoV-2 and its mutants.

[0030] The present invention provides five short peptide derivatives based on the SIM sites of the SARS-CoV-2 Spike protein, namely cpSIM1, cpSIM1’, cpSIM2, cpSIM3, and cpSIM4. Among them, the four short peptide derivatives cpSIM1, cpSIM1’, cpSIM3, and cpSIM4 do not affect cell viability and have no cytotoxicity, and can promote the surface expression of the SARS-CoV-2 Spike protein and enhance immunogenicity. cpSIM2 has high specificity and effectiveness against SARS-CoV-2 infection, which has been verified in both in vitro and in vivo models, laying a foundation for the treatment of SARS-CoV-2 infection. Brief Description of the Drawings

[0031] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, where

[0032] Figure 1 SUMO2 modification of the SARS-CoV-2 Spike protein in Example 1 of the present invention results in perinuclear punctate localization of SUMO2; A) SUMO modification of the SARS-CoV-2 Spike protein; B) SUMO modification of the exogenous CoV-2 Spike protein; C) SUMO2 gene knockout affects the perinuclear punctate localization of the Spike protein; D) SUMO2 KO increases the cleavage and modified forms of the Spike protein.

[0033] Figure 2Identification of SUMO sites and SIM motifs of SARS-CoV-2 Spike protein in Example 2 of the present invention; A) Schematic diagram of GPS-SUMO predicting the conservation analysis of SUMO sites and SIM motifs of Spike protein; B) Three-dimensional spatial distribution of SUMO sites and SIM motifs of Spike protein; C) Effect of SUMO site and SIM motif mutations on Spike protein cleavage; D) Effect of SUMO site and SIM motif mutations on SUMOylation of Spike protein; E) Schematic diagram of SUMO modification types occurring at SUMOylation sites and SIM motifs of Spike protein.

[0034] Figure 3 HPLC detection of the purity of different Spike peptides synthesized in Example 3 of the present invention; among them, A) cpSIM1; B) cpSIM1'; C) cpSIM2; D) cpSIM3; E) cpSIM4.

[0035] Figure 4 Effect of different Spike peptides of the present invention on cell fusion and replication of SARS-CoV-2 in Example 4; A) Schematic diagram of different Spike peptides; B) Effect of different Spike peptides on cell morphology after CoV-2 infection; C) Effect of different Spike peptides on CoV-2 replication; D) Effect of different Spike peptides on the expression level of Spike protein in cells after CoV-2 infection; D) cpSIM2 inhibits the replication of CoV-2 VOC strain in A549-hACE2 and Calu3 cell lines, *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0036] Figure 5 cpSIM2 can effectively inhibit the replication of SARS-CoV-2 in mice in Example 5 of the present invention; A) Schematic diagram of CoV-2-infected mice and cpSIM2 treatment; B) Detection of viral load in mouse lung tissue by reverse transcription-quantitative PCR targeting the CoV-2 N gene. ****p<0.0001, indicating a significant statistical difference; C) Lung tissues were stained with hematoxylin and eosin (H&E) and immunohistochemically stained with an antibody against CoV-2 N protein to evaluate the degree of pathological damage and viral load in the lung tissue. The following figure is a representative enlarged image. D) Quantitative results of the percentage of N-positive cells in the lung tissue. Detailed implementation manners

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0038] Example 1. SUMO2 modification of the SARS-CoV-2 Spike protein inhibits Spike protein cleavage

[0039] 1. SUMO2 modification of the SARS-CoV-2 Spike protein, which undergoes SUMO modification, results in perinuclear punctate localization of SUMO2

[0040] To verify whether the SARS-CoV-2 Spike protein undergoes SUMO modification, A549-hACE2 cells infected with the SARS-CoV-2 original strain SH01 were harvested, and denaturing immunoprecipitation was performed using endogenous SUMO1 and SUMO2 / 3 antibodies, followed by immunoblotting with a Spike antibody. The results showed that Spike could be captured by SUMO1 and SUMO2 / 3 antibodies, indicating that Spike undergoes SUMO modification ( Figure 1 A). In addition, the N protein, which is known to undergo SUMO modification, was also detected. The results showed that the N protein was effectively immunoprecipitated by SUMO1 and SUMO2 / 3 antibodies, indicating the reliability of the detection method ( Figure 1 A).

[0041] Viral infection is a complex process involving multiple viral proteins. To exclude the interference of other viral proteins, SARS-CoV-2 Spike was co-expressed with HA (a small fragment of the influenza virus hemagglutinin protein, Zhang Y, Dong Z, Gu F, et al. Degradation of TRIM32 is induced by RTA for Kaposi's sarcoma-associated herpesvirus lytic replication. J Virol. 2024 Jun 13;98(6):e0000524. doi: 10.1128 / jvi.00005-24. PubMed PMID: 38717113; PubMed Central PMCID: PMCPMC11237441. eng.)-tagged SUMO1, SUMO2, and SUMO3 in 293T cells, and Spike was detected by co-immunoprecipitation. The results showed that Spike interacted with SUMO1, SUMO2, and SUMO3, and overexpression of SUMO increased the expression level of Spike ( Figure 1 B).

[0042] 2. SUMO2 modification of the SARS-CoV-2 Spike protein results in perinuclear punctate localization of SUMO2

[0043] To study the effect of SUMO modification on the function of SARS-CoV-2 Spike protein, A549-hACE2 SUMO1, SUMO2, and SUMO3 knockout cell lines were constructed using the CRISPR-Cas9 system. These cell lines were infected with the original SARS-CoV-2 strain SH01, and the effects of different SUMO molecules on the expression and localization of Spike protein were further detected by immunofluorescence and immunoblotting. The results are as Figure 1 shown in C. SARS-CoV-2 infection led to a perinuclear punctate distribution of SUMO2 / 3, and Spike colocalized with SUMO2 / 3 in the perinuclear region. Compared with A549-hACE2 wild-type cells, SUMO2 knockout blocked the perinuclear punctate distribution of SUMO2 / 3 and led to a decrease in the abundance of Spike protein ( Figure 1 C), indicating that SUMO2 modification of SARS-CoV-2 Spike protein results in perinuclear punctate localization of SUMO2.

[0044] 3. SUMO2 modification can inhibit the cleavage of SARS-CoV-2 Spike protein

[0045] Since the Spike protein needs to be cleaved to form its active conformation for virus transmission, the expression of SARS-CoV-2 Spike protein in wild-type A549-hACE2 cells and cells knocked out of different SUMO molecules after infection was evaluated by immunoblotting. The results showed that SUMO1 knockout led to an increase in the trimeric form of Spike protein and a decrease in the cleavage products, while SUMO2 knockout led to an increase in the cleavage form of Spike and a decrease in the trimeric form ( Figure 1 D). These results indicate that SUMO2 modification can inhibit the cleavage of Spike protein.

[0046] The experimental steps involved in this example are as follows:

[0047] 1. SARS-CoV-2 cell infection model:

[0048] 1) Prepare cells with good growth and pre-culture them at 37°C and 5% CO2 for 24 hours according to experimental needs to reach a density of 90%.

[0049] 2) Dilute the SARS-CoV-2 virus stock solution with DMEM medium containing 2% FBS to the required multiplicity of infection (MOI), discard the original culture medium, add the virus solution, add 100 μl of virus dilution to each well, and place it in the cell culture incubator.

[0050] 3) After incubating in the cell culture incubator for 24 hours, collect cell and supernatant samples for subsequent detection as needed.

[0051] 2. Preparation of cell sample lysis:

[0052] 1) Collect an appropriate amount of cells, centrifuge at 2500 rpm at room temperature for 3 min, and discard the culture medium.

[0053] 2) Wash twice with pre-cooled PBS, centrifuge at 2500 rpm at room temperature for 3 min, and discard the PBS.

[0054] 3) For cells transfected for 48 hours or infected for 24 hours, discard the supernatant, wash twice with PBS, add trypsin and digest for 5 min, add serum-containing culture medium to neutralize trypsin, pipette the cells repeatedly, transfer the cell suspension to a new centrifuge tube, count the cells, and take a sample with approximately 3×10 7 viable cells in total.

[0055] 4) Denaturation step (a proprietary step for denaturing immunoprecipitation): Add 180 μL of protein lysis buffer RIPA (1% NP-40, 2 mM EDTA, 150 mM NaCl, 50 mM Tris (pH 7.6), 1 mM PMSF, 1 mM NEM, 1 μg / mL aprotinin, 1 μg / mL leupeptin, and 1 μg / mL pepstatin), and 20 μL of 20% SDS solution. Pipette the cell sample to mix well, and incubate in a 100°C water bath for 10 min.

[0056] 5) Cool to room temperature, add 1.8 mL of RIPA protein lysis buffer containing protease inhibitor, and lyse on ice for 30 min, mixing by shaking every 5 min.

[0057] 6) Centrifuge the lysed cells at 14500 rpm for 5 min in a 4°C centrifuge.

[0058] 7) Transfer the supernatant after centrifugation to a new EP tube, which is the whole cell lysate. Measure the protein concentration according to the Coomassie Brilliant Blue G-250 method.

[0059] 8) Aliquot the protein samples according to the loading amount, add 6x protein loading buffer, and boil in a 100°C metal bath for 5 min until the protein is denatured.

[0060] 9) Store the protein samples at -80°C for future use or directly perform immunoprecipitation experiments.

[0061] 2. Immunoprecipitation:

[0062] 1) Pretreatment of Protein A / G: 30 μL each of Protein A / G, wash with TBS, centrifuge at 1000 g for 30 s at 4°C, and repeat 3 times.

[0063] 2) Protein supernatant after the previous step of cell lysis:

[0064] a) IP group: Add an appropriate amount of negative control (Control IgG) and an appropriate amount of washed Protein A / G pre-mixed agarose beads, incubate at 4°C with sufficient rotation for 30 min -1 ·h to eliminate non-specific background.

[0065] b) Input group: Take 2.5%-5% of the whole cell lysate as the Input group, add 6x protein loading, boil at 100°C for 10 min, and store at -20°C for later use.

[0066] 3) Antibody incubation:

[0067] a) After IgG incubation, centrifuge at 4°C, 1000g for 30 s, and let it stand at room temperature for 10 min to allow the IgG antibody and the bound protein to precipitate slowly. Keep both the supernatant and the precipitate after centrifugation. The precipitate is used as the IgG negative control group.

[0068] b) Supernatant: Transfer the supernatant to a new EP tube, add the appropriate volume of the primary antibody according to the antibody instruction manual, and incubate at 4°C with slow rotation overnight (8-12 h) to facilitate the full binding of the antibody and the target protein.

[0069] c) Precipitate (IgG negative control group): Resuspend with 1 mL of TBS, centrifuge at 4°C, 1000g for 30 s, and repeat this three times; resuspend with 20 μL of TBS, then add 6×protein loading and boil the protein for 10 min, and store at -20°C.

[0070] 4) Sample protein denaturation:

[0071] a) The next day, add 30 μL of Protein A / G pre-mixed agarose beads to the supernatant, and continue to incubate at 4°C with slow rotation for 3 h to allow the supernatant to fully bind to the beads.

[0072] b) After 3 h, centrifuge at 4°C, 1000g for 30 s, and discard the supernatant. Wash the precipitate three times with 1 mL of pre-cooled TBS, each time centrifuge at 4°C, 1000g for 30 s. Resuspend with 20 μL of TBS, add 4 μL of 6×protein Loading and boil the protein, which is used as the IP group and stored at -20°C.

[0073] 5) Protein immunoblot analysis: Detect the Input group, IgG group, and IP group together.

[0074] 9. Denaturing immunoprecipitation:

[0075] 1) Preparation of ordinary cell protein: The same as the previous immunoprecipitation operation steps.

[0076] 2) Protein A / G pretreatment: The same as the previous immunoprecipitation operation steps.

[0077] 3) Obtaining whole cell lysates:

[0078] a) For each sample, add 200 μL of protein lysis buffer RIPA (containing the required protease inhibitors PMSF, NEM, aprotinin, leupeptin, and pepstin) and a mixed solution containing 2% SDS according to the cell amount, and mix the cell samples by shaking.

[0079] b) Place the shaken and mixed samples in a 100 °C water bath for 10 min to heat-denature the proteins.

[0080] c) After 10 min, remove the samples and wait for them to cool to room temperature. Be sure not to place them directly on ice.

[0081] d) After the samples return to room temperature, add 1.8 mL of RIPA lysis buffer containing protease inhibitors (containing the required protease inhibitors PMSF, NEM, aprotinin, leupeptin, and pepstin), and lyse the samples on ice at 4 °C for 30 min, shaking and mixing every 5 min.

[0082] 4) The remaining operation methods are the same as those for immunoprecipitation.

[0083] 3. Steps of Western blot experiment:

[0084] 1) SDS-PAGE electrophoresis: Add the above denatured protein samples to an SDS-PAGE gel with an appropriate concentration for gel electrophoresis separation.

[0085] 2) Transfer: Soak a 0.45 μm NC membrane in 1X NC membrane transfer equilibration buffer, and use the standard program of the Kingsley eBlot TM L1 rapid wet transfer instrument to transfer the membrane for about 10 min.

[0086] 3) Blocking: Place the NC membrane in 5% non-fat milk powder (prepared with PBS) and incubate at room temperature for 1 h.

[0087] 4) Primary antibody incubation: Wash 3 times with TBST at room temperature for 5 min each time, add the primary antibody diluted in an appropriate ratio, and incubate overnight at 4 °C.

[0088] 5) Secondary antibody incubation: Wash 3 times with TBST at room temperature for 5 min each time, and use the corresponding fluorescent secondary antibody 800 of the appropriate species.

[0089] 6) Incubate in the dark at room temperature for 1 h; Wash 3 times with TBST at room temperature for 5 min each time.

[0090] 7) Development: Develop using an Odyssey dual-color imager.

[0091] 4. Immunofluorescence

[0092] 1) Add 4% paraformaldehyde to the cell slides and fix for 20 min, then wash three times with PBS.

[0093] 2) Dilute the primary antibody to the recommended ratio in Blocking Buffer, vortex to mix well, and centrifuge at 14,000 rpm for 2 min. Pipette 50 μL of the primary antibody dilution onto the sealing film, gently place the front side of the slide on the droplet, and incubate at room temperature for 2 h.

[0094] 3) Transfer the slide with the front side up to the well plate and wash three times with Blocking Buffer.

[0095] 4) Dilute the secondary antibody to the recommended ratio in Blocking Buffer, vortex to mix well, and centrifuge at 14,000 rpm for 2 min. Pipette 50 μL of the secondary antibody dilution onto the sealing film, gently place the front side of the slide on the droplet, and incubate at room temperature in the dark for 1 h.

[0096] 5) Transfer the slide with the front side up to the well plate and wash three times with Blocking Buffer.

[0097] 6) Dilute DAPI to 1:1000 in PBS, vortex to mix well, centrifuge at 4000 rpm for 2 min, add 1 mL / well of the DAPI dilution, incubate in the dark for 3 min, wash three times with PBS, and air-dry the slides in order in the dark.

[0098] 7) Drop 20 μL of resin onto the glass slide, gently cover the slide with the front side down on the glass slide, and apply the mounting medium around the slide to seal it.

[0099] 5. Recombinant plasmid construction:

[0100] 1) Primer design: Query the target sequence on NCBI and design primers. The primer sequences are shown in the following table.

[0101]

[0102]

[0103]

[0104] 2) PCR amplification of the target fragment: Obtain the reverse transcription product; Obtain the existing plasmid using Max DNA Polymerase (R045A, Takara),

[0105] Preparation of PCR Amplification System

[0106]

[0107] PCR Amplification Reaction Program

[0108]

[0109] 3) Enzyme Digestion:

[0110] Preparation of Enzyme Digestion System

[0111]

[0112] Enzyme Digestion Reaction Program

[0113] Temperature Time 37 °C water bath 2h

[0114] 4) Ligation and Sequencing: Use the Beyotime seamless cloning kit (D7010M) for ligation. Specific precautions can be referred to the product manual.

[0115] 6. Construction of Short hairpin RNA (ShRNA) Plasmid

[0116] 1. Primer Design: Design the target sequence of the gene of interest according to the following method. The following primers are shown in the table below.

[0117] (1) Consult relevant literature on the gene of interest

[0118] (2) Website query: https: / / zlab.bio / guide-design-resources

[0119]

[0120] 2. Synthesis of Primer Sequences:

[0121] (1) Prepare a 20 μL system for synthesizing primer sequences

[0122] Preparation of Oligos Synthesis System

[0123]

[0124] (2) Annealing program: In the PCR instrument, set the gradient cooling program: 95 °C for 30 s, 72 °C for 2 min, 37 °C for 2 min, 25 °C for 2 min.

[0125] (3) Dilute the above annealed fragment with ddH2O at a ratio of 1:200.

[0126] 3. Enzyme Digestion: Linearize the LentiCRISPR vector with the restriction endonuclease BsmBI at 55 °C for 3 h.

[0127] Preparation of Restriction Enzyme Reaction System

[0128]

[0129] 4. Ligation

[0130] Use Beyotime Ultra-Fast T4 DNA Ligase (D7009M). For specific precautions, please refer to the product manual.

[0131] 5. Transformation: Use competent cells Stbl3.

[0132] 6. Lentivirus Packaging and Purification:

[0133] 1) Prepare cells: The passage number of HEK293T cells revived in each batch should not exceed 20 passages. The virus packaging can start from the third passage after revival.

[0134] 2) Prepare plasmids: The total amount of plasmids is 40 μg (14 μg of target plasmid, 5 μg of VSVG, 21 μg of Plet-CMV-GFP-Hygro).

[0135] 3) Prepare for transfection: Use the product (Polyethylenimine Linear (PEI) MW40000 (rapid lysis)) or Lipofectamine TM 3000 for transfection operation.

[0136] 4) Collect supernatants: Collect the cell supernatants at 24 h and 48 h after medium change into 50 mL centrifuge tubes respectively.

[0137] 5) Concentrate supernatants: Centrifuge the cell supernatants at 2000 rpm for 5 min and filter them using a 0.45 μm filter.

[0138] 6) Centrifuge at 10000 g at 4 °C for 18 h, with an acceleration of 5 and a deceleration of 0.

[0139] 7) Store the virus: Take out the virus tube from the centrifuge, carefully aspirate the supernatant, leaving about 1.5 mL of supernatant. Vigorously pipette to mix well, aliquot into 3 tubes, and store at -80 °C. Label the virus name, date, and batch number.

[0140] 7. Construction of Knockout Cell Lines:

[0141] 1) Determine the optimal antibiotic screening concentration: Before lentivirus infection, first determine the optimal killing drug screening concentration for the target cells required.

[0142] 2) Plate cells: For adherent cells, one day in advance, seed 500 μL of 0.5×10 6 cells to be infected into a 24-well plate for transfection the next day.

[0143] 3) Prepare the virus: Take out the virus stored in the -80 °C refrigerator in advance and place it on ice to wait for melting.

[0144] 4) Virus infection: Take out the cells plated the previous day and add the prepared virus drop by drop.

[0145] 5) Supplement the culture medium: After 24 h and 48 h, replenish the liquid in time and divide into wells.

[0146] 6) Cell screening: Start cell screening 72 h after virus infection, replace the culture medium containing 1 μg / mL puromycin, and screen in the cell incubator for 48 h.

[0147] 7) Monoclonal screening: Digest, count the screened cells, take 80 - 100 cells and dilute them in 10 mL of culture medium, spread the cell suspension into a 96-well plate, 100 μL / well. Culture for about 2 weeks. When the monoclonal cells grow to about 50% of the wells, they can be digested and then transferred to a 24-well plate for amplification culture.

[0148] 8) Cell identification: When the cells picked into the 24-well plate grow to confluence, digest them. Transfer half of the cells to a 6-well plate and use the other half to extract DNA for PCR identification. Confirm the knocked-out cells by PCR identification, expand the culture, preserve them, and perform WB identification at the same time.

[0149] Example 2 Identification of SUMOylation Specific Sites and SIM Motifs in SARS-CoV-2 Spike

[0150] To identify specific SUMOylation sites on the SARS-CoV-2 Spike protein, the Spike sequence was scanned using the GPS-SUMO tool (http: / / sumo.biocuckoo.cn / advanced.php), and multiple SUMO modification sites and potential SUMO-interacting motifs (SIMs) were found. These motifs match the consensus sequence valine (V) / isoleucine (I) / leucine (L)-X-V / I / L-V / I / L motif. Among these motifs, there are two adjacent SIM motifs, named SIM1 and SIM1' respectively. By comparing the conservation of these sites between SARS-CoV and SARS-CoV-2 variant of concern (VOC), it was found that the SUMO modification sites and SIMs of the Spike protein are highly conserved among SARS-CoV-2 variants, even the sites located in the N-terminal domain (NTD) and receptor-binding domain (RBD) of the highly variable S1 subunit ( Figure 2A). In contrast, the conservation of these sites between SARS-CoV and SARS-CoV-2 is not consistent: in SARS-CoV, sites 462 and 933 are not lysine (K) residues, and the linker sequences of SIM1 and SIM1' are also different from those in SARS-CoV-2. This indicates that the SUMOylation sites and SIMs on the Spike protein play important roles in the evolution of SARS-CoV-2. Further projecting these sites onto the three-dimensional structure of the extracellular domain of the Spike protein, including its closed, open, and post-fusion conformations. The results show that all identifiable SUMOylation sites are located on the surface of the three-dimensional structure. The opening of the RBD further exposes sites 986 and 462, while SIM1 and SIM1' maintain spatial proximity, and only SIM2 is located inside the RBD domain ( Figure 2 B).

[0151] To explore the functions of these sites, the predicted SUMOylation sites were mutated by replacing lysine (K) with arginine (R) and residues on SIMs with alanine (A), and these mutants were named Spike mSIM1 / 2 / 3 / 4. Then, these mutants were transfected into 293T wild-type (WT) and SUMO knockout cell lines. Immunoblot analysis showed that mutations at sites K129 and K1269 significantly enhanced the cleavage of the Spike protein. In the WT cell group, both K129R and K1269R mutants existed in the cleaved form, while in the SUMO2 knockout cell group, the K1269R mutant partially reverted to the original size (S0). In contrast, all other mutants inhibited the cleavage of the Spike protein and maintained the S0 form ( Figure 2 C). Next, these mutants were co-expressed with HA-tagged SUMO1, SUMO2, and SUMO3 in 293T cells, and their SUMOylation status was examined by co-immunoprecipitation. The results showed that single-site mutations did not completely block the SUMOylation of Spike, but some sites showed a preference for specific SUMOylation. For example, K462R reduced SUMO1 modification, while mSIM1 reduced SUMO1 and SUMO3 modifications ( Figure 2 D). The Spike protein exhibits diverse SUMOylation patterns, and the specific modification types at each site are summarized in Figure 2 E. These findings demonstrate that the SUMOylation sites and SIMs on the Spike protein are involved in regulating its cleavage and suggest potential antiviral mechanisms.

[0152] Example 3 Synthesis of Short Peptides Related to SUMO Modification Sites of SARS-CoV-2 Spike

[0153] In this example, based on the short peptides at the SUMO modification-related SIM1, SIM1', SIM2, SIM3, and SIM4 sites of the SARS-CoV-2 Spike protein polypeptide screened in Example 2, corresponding short peptide derivatives cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4 for treating SARS-CoV-2 were designed. The short peptide derivatives consist of the above-mentioned short peptides and a leading transmembrane peptide; the amino acid sequences of the short peptides at the SIM1, SIM1', SIM2, SIM3, and SIM4 sites are SEQ ID No.1: KTQSLLIVNNATN, SEQ ID No.2: NATNVVIKVCEFQ, SEQ ID No.3: QPYRVVVLSFELL, SEQ ID No.4: INASVVNIQKEID, SEQ ID No.5: DSEPVLKGVKLHY in sequence. The leading transmembrane peptide is connected to the N-terminus of the above-mentioned short peptide, and the amino acid sequence of the transmembrane peptide is SEQ ID No.6: YGRKKRRQRRR. The short peptides at the SIM1, SIM1', SIM2, SIM3, and SIM4 sites correspond to the short peptide derivatives cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4 in sequence. Therefore, the amino acid sequences of the short peptide derivatives cpSIM1, cpSIM1', cpSIM2, cpSIM3, and cpSIM4 are SEQ ID No.7: YGRKKRRQRRRKTQSLLIVNNATN, SEQ ID No.8: YGRKKRRQRRRNATNVVIKVCEFQ, SEQ ID No.9: YGRKKRRQRRRQPYRVVVLSFELL, SEQ ID No.10: YGRKKRRQRRRINASVVNIQKEID, SEQ ID No.11: YGRKKRRQRRRDSEPVLKGVKLHY in sequence.

[0154] The above polypeptide sequences were all prepared by solid-phase synthesis method, and the purity of the polypeptide was detected by HPLC as Figure 3 shown in A-H of [Figure] and Table 2A-2H. It can be seen from the chromatogram that the purity of the five polypeptides is greater than 95%.

[0155] Table 2A. HPLC detection related parameters of cpSIM1

[0156]

[0157] Table 2B. HPLC detection related parameters of cpSIM1'

[0158]

[0159] Table 2C. Related parameters for HPLC detection of cpSIM2

[0160]

[0161]

[0162] Table 2D. Related parameters for HPLC detection of cpSIM3

[0163]

[0164] Table 2E. Related parameters for HPLC detection of cpSIM4

[0165]

[0166] Broad-spectrum inhibitory effect of cpSIM2 on SARS-CoV-2 infection and cell fusion in Example 4

[0167] A549-hACE2 cells were infected with SARS-CoV-2 (SH01), and after 1 hour, the above five polypeptide drugs were used for treatment respectively ( Figure 4 A). The results of high-content scanning analysis showed that cpSIM1, cpSIM1’, cpSIM3, and cpSIM4 all increased the SARS-CoV-2 infection rate in a dose-dependent manner. Microscopic photography showed that the treatment with these four drugs promoted the formation of a large number of syncytia and shedding in infected cells, while the uninfected control group did not show this effect, excluding the possibility of drug toxicity. In contrast, cpSIM2 significantly inhibited the infection of SARS-CoV-2 in a dose-dependent manner ( Figure 4 B). Reverse transcription-quantitative PCR results further confirmed that the treatment with cpSIM2 could inhibit the replication and release of intracellular virus in a dose-dependent manner, and 20 μM cpSIM2 could reduce the virus release amount in the supernatant by 100-fold ( Figure 4 C). The results of immunoblotting (IB) detection showed that the treatment with cpSIM2 significantly reduced the expression level of Spike protein ( Figure 4 D). To evaluate whether cpSIM2 has broad-spectrum antiviral potential, A549-hACE2 and Calu3 cells (a human lung cell line that can be naturally infected with SARS-CoV-2) were infected with SARS-CoV-2 Delta and Omicron mutants and treated with cpSIM2. The results showed that the treatment with cpSIM2 could also significantly inhibit the intracellular replication and virus release of SARS-CoV-2 variants, and also showed obvious antiviral effects in Calu3 cells ( Figure 4 E).

[0168] The experimental procedures involved in this example are as follows:

[0169] 1. SARS-CoV-2 Cell Infection and Polypeptide Treatment Model:

[0170] 1) Prepare 100 μL of cell suspension in a 96-well plate and incubate it for 24 hours according to the experimental requirements (under the conditions of 37 °C and 5% CO2).

[0171] 2) Dilute the SARS-CoV-2 virus solution to the desired multiplicity of infection (MOI) using DMEM medium with 2% FBS. Discard the liquid in the culture plate, add 100 μL of virus dilution to each well, and place it in the cell culture incubator for 1 hour.

[0172] 3) Discard the virus solution in the culture plate, wash it three times with PBS, and add PBS or polypeptide drugs at different concentrations.

[0173] 5) After incubating in the cell culture incubator for 18 hours, collect the cell and supernatant samples for subsequent detection as needed.

[0174] 2. Quantification of Virus Copy Number:

[0175] 1) Extract RNA from cells and supernatants using TRIzol reagent (Thermo Fisher). Refer to the instruction manual for detailed methods.

[0176] 2) Quantification of virus copy number: Perform reverse transcription polymerase chain reaction (RT-PCR) detection on a CFX Connect real-time system (Bio-Rad) instrument using the HiScript II One Step RT-qPCR SYBR Green Kit (Q221-01, Vazyme). The Q-PCR primers are shown in Table 1 below:

[0177] Table 1

[0178]

[0179] Example 5 Inhibition of SARS-CoV-2 Virus Replication in Vivo by Short Peptide cpSIM2

[0180] To further verify the antiviral effect of cpSIM2 in vivo, a study was conducted using a 6 - 8-week-old K18-hACE2 C57BL / 6 transgenic mouse infection model. Briefly, mice were infected intranasally with 1×10 ^4 PFU of SARS-CoV-2 Omicron BA5.2, and PBS, cpControl (cpCtrl), or cpSIM2 (at a dose of 25 mg / kg) were intraperitoneally injected at 2, 24, 48, and 72 hours after infection ( Figure 5A). On the 5th day after infection, all mice were sacrificed, and lung tissues were collected for further analysis. The qPCR results showed that compared with the PBS-treated group and the cpCtrl-treated group, cpSIM2 treatment significantly inhibited the replication of SARS-CoV-2 in the lungs of mice ( Figure 5 B). Hematoxylin and eosin (H&E) staining showed that SARS-CoV-2 infection caused severe lung injury in mice in the PBS-treated group and the cpCtrl-treated group, while cpSIM2 treatment could antagonize lung injury at the lowest level. Figure 5 C). Immunohistochemical staining of the N protein showed that the distribution of the virus in the lungs of cpSIM2-treated mice was significantly reduced. Figure 5 C and Figure 5 D).

[0181] The experimental steps involved in this example are as follows:

[0182] 1. SARS-CoV-2 infection treatment model in K18-hACE2 mice:

[0183] 1) Preparation of mice: Female K18-hACE2-2A-CreERT2 C57BL / 6J mice aged 6 - 8 weeks were purchased, acclimated in a biosafety level 3 (BSL-3) laboratory at Fudan University (Shanghai), China for 7 days, and weighed and recorded one day before inoculation.

[0184] 2) Infection of mice: Each mouse was infected intranasally with 1×10 ^4 PFU of the SARS-CoV-2 Omicron BA.5.2 strain.

[0185] 3) Drug treatment: After infection, 25 mg / kg of PBS, or cpCtrl or cpSIM diluted in PBS was intraperitoneally injected daily, and the body weights of the mice were measured.

[0186] 4) Sacrifice of mice: On the 5th day after infection, the mice were euthanized and sacrificed to collect lung tissues.

[0187] 2. Hematoxylin and eosin staining:

[0188] The lungs of the mice were fixed with 4% formaldehyde. The experiment was performed by Sevier Company. The experimental steps included: paraffin embedding, sectioning, hematoxylin staining, eosin staining, dehydration and mounting. Stained tissue sections were photographed using a WS-10Zhiyue panoramic scanner.

[0189] 3. Immunohistochemical staining and analysis:

[0190] The lung tissue sections of each group of mice were subjected to immunohistochemical staining using the N protein antibody (A18797, Abclonal, dilution ratio 1:200). The experiment was performed by Sevier Company, and the experimental steps included: dewaxing the sections, antigen retrieval, treatment with 3% hydrogen peroxide, serum blocking, incubation with the primary antibody overnight at 4°C, incubation with the secondary antibody at room temperature, color development, hematoxylin nuclear staining, dehydration, permeabilization, mounting, and microscopic examination. The tissue sections were photographed for stained images using a WS-10Zhiyue panoramic scanner, and the immunohistochemical positive rate was calculated using ImageJ software.

[0191] Obviously, the above embodiments are merely examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A short peptide derivative based on the SIM site of the novel coronavirus spike protein, characterized in that: The short peptide derivatives include cpSIM1, cpSIM1', cpSIM2, cpSIM3 and cpSIM4, and the short peptide derivatives are composed of a transmembrane peptide and a short peptide; the short peptide is a short peptide based on SARS-CoV-2 Spike protein polypeptide SUMO modification related SIM1, SIM1', SIM2, SIM3 and SIM4 sites; the transmembrane peptide is connected to the N-terminus of the short peptide.

2. The short peptide derivative based on the SIM site of the novel coronavirus spike protein according to claim 1, characterized in that: The amino acid sequences of the short peptides at the SIM1, SIM1', SIM2, SIM3 and SIM4 sites are shown in SEQ ID No. 1-4 respectively.

3. The short peptide derivative based on the SIM site of the novel coronavirus spike protein according to claim 1, characterized in that: The amino acid sequence of the cell-penetrating peptide is shown in SEQ ID No.

5.

4. The short peptide derivative based on the SIM site of the novel coronavirus spike protein according to claim 1, characterized in that: The amino acid sequences of the cpSIM1, cpSIM1', cpSIM2, cpSIM3 and cpSIM4 are shown in SEQ ID No.6-9 respectively.

5. A composition, characterized in that Including a short peptide derivative based on the SIM site of the novel coronavirus spike protein as described in any one of claims 1 to 4.

6. An antiviral drug, characterized in that: It includes the short peptide derivative based on the SIM site of the novel coronavirus spike protein as described in any one of claims 1 to 4 or the composition as described in claim 5.

7. The antiviral drug according to claim 6, characterized in that The virus includes the novel coronavirus SARS-CoV-2 or SARS-CoV.

8. A viral vaccine adjuvant, characterized in that It includes the short peptide derivative based on the SIM site of the novel coronavirus spike protein as described in any one of claims 1 to 4 or the composition as described in claim 5.

9. The viral vaccine adjuvant according to claim 8, characterized in that The virus includes the novel coronavirus SARS-CoV-2 or SARS-CoV.

10. The viral vaccine adjuvant according to claim 8, characterized in that The short peptide derivative promotes the expression of SARS-CoV-2 Spike protein on the cell surface and enhances immunogenicity.

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