Nucleocapsid protein mutant, gene, recombinant expression vector, mutant virus strain and application

Abstract

The invention provides a nucleocapsid protein mutant, a gene, a recombinant expression vector, a mutant virus strain and application, and belongs to the technical field of biological medicine. On the basis of an SARS-CoV-2 virus-like particle (VLP) pseudotyping technology, it is found that N protein alanine mutation occurs, a hydrophobic pocket of N protein CTD is destroyed by L353A, H356A, I357A, D358A and Y360A, and the nucleic acid binding affinity is reduced; it is also found that F403A, F407A and M411A lead to increase of N protein monomers, decrease of tetramers and increase of abnormally high poly N protein, and replication of SARS-CoV-2 wild living viruses and variants is inhibited. Meanwhile, the chiral D-type polypeptide is designed for the CTD region, and it is found that the chiral D-type polypeptide can effectively inhibit assembling of the VLP pseudovirus.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nucleocapsid protein mutant, gene, recombinant expression vector, mutant virus strain, and its applications. Background Technology

[0002] Since the end of 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes novel coronavirus infection (COVID-19), has spread globally. Despite numerous efforts by researchers to develop vaccines and antiviral drugs, effective control of the virus has not yet been achieved. Currently, research on blocking the spread of COVID-19 mainly focuses on the viral spike protein (S protein), primarily because its receptor binding domain (RBD region) is a key region for binding to the human angiotensin-converting enzyme 2 (ACE2) receptor, directly mediating viral invasion of host cells, and it is also the main antigen that induces the body to produce neutralizing antibodies. However, due to the extremely high mutation rate of the S protein, the developed effective neutralizing antibodies easily and rapidly lose their neutralizing activity. Therefore, finding new therapeutic targets for SARS-CoV-2 and developing broad-spectrum antiviral vaccines and drugs have become urgent research needs.

[0003] Currently, the development of broad-spectrum antiviral vaccines and drugs mainly focuses on two approaches: the first is targeting viral proteins, primarily viral enzymes, to interrupt the virus's life cycle by interfering with their function; the second is targeting host proteins involved in the viral life cycle, such as the ACE2 receptor, which has potential for broad-spectrum control of coronaviruses that interact with the ACE2 receptor. However, the ACE2 receptor plays a crucial physiological role in regulating respiration and heartbeat in the human body; therefore, the safety of drugs targeting the ACE2 receptor still requires significant research and resolution.

[0004] Virus-targeting inhibitors aim to disrupt each stage of the SARS-CoV-2 life cycle, so virus-encoded proteins that perform core functions in each stage are naturally considered the main drug targets. Based on the stages of the viral life cycle, the current research status of each target and its corresponding inhibitor is as follows: In the viral entry stage, the spike protein (S protein) is key to mediating viral adsorption and membrane fusion. Inhibitors targeting it are mainly divided into two categories: one is inhibitors that block the interaction between the S protein and the ACE2 receptor, such as ensovibep, FSR16m, and FSR22; the other is peptide inhibitors that target the α-helix region of the heptapeptide repeat of the S protein, such as EK1. In the protein processing stage, papain-like proteases can cleave polyproteins pp1a and pp1ab into various non-structural proteins, such as nsp1 to nsp3. To date, more than 30 papain-like protease inhibitors have been developed. 3C-like proteases can cleave pp1a and pp1ab polyproteins, releasing viral proteins nsp4 to nsp16. Therefore, protease inhibitors targeting 3C-like proteases, such as nimaterevir, ensitavir, and SIM0417, have been used to treat SARS-CoV-2 infection. In the RNA synthesis stage, the core... The target is RNA-dependent RNA polymerase (RdRp), also known as nsp12. This enzyme is a highly conserved enzyme that plays a crucial role in viral RNA replication and transcription, catalyzing template DNA unwinding, RNA synthesis, RNA proofreading, and RNA end capping. RdRp inhibitors developed based on this target can block RNA synthesis by targeting key components of the replication-transcription complex. Representative drugs include Remdesivir, Molnupivir, Favipiravir, and Bemnifosbuvir. In the viral assembly stage, the nucleocapsid protein (N protein) is a key protein, but there is limited research on antiviral drug targets for viral nucleocapsid proteins. The main reason is that the N protein has three flexible regions, which makes the binding of antiviral drugs targeting its N-terminal domain (NTD) and C-terminal domain (CTD) unstable. Therefore, current research has shifted to the direction of "mediating abnormal hyperpolymerization of the N protein" to achieve antiviral effects by disrupting its normal assembly process.

[0005] Replication-defective SARS-CoV-2 virus-like particles (pseudoviruses) contain four structural proteins of the SARS-CoV-2 virus (spike protein, membrane glycoprotein, envelope protein, and nucleocapsid phosphoprotein). They can self-assemble into infectious virus-like particles (VLPs) similar to real viruses. Internally, these VLPs bind to the N protein via an ORF1ab RNA segment, enabling the encapsulation and delivery of exogenous genes. This tool allows for in-depth research into the impact of N protein mutations on viral assembly, thereby screening for mutation sites that may lead to abnormal N protein assembly and providing experimental support for the development of novel antiviral drug targets targeting the N protein. Summary of the Invention

[0006] The purpose of this invention is to provide a nucleocapsid protein mutant, gene, recombinant expression vector, mutant viral strain, and application. Based on SARS-CoV-2 virus-like particle (VLP) pseudotype technology, this invention discovered that alanine mutations in the N protein, L353A, H356A, I357A, D358A, and Y360A, disrupt the hydrophobic pocket of the N protein CTD, reducing nucleic acid binding affinity. It was also found that F403A, F407A, and M411A lead to an increase in N protein monomers, a decrease in tetramers, and an increase in abnormally high-polymerized N protein, inhibiting the replication of SARS-CoV-2 wild-type live virus and mutant strains.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a nucleocapsid protein mutant, which is obtained by amino acid mutation of the nucleocapsid protein, including site mutations in the CTD region or N3 region; The mutation sites in the CTD region include one or more of L353A, H356A, I357A, D358A, and Y360A; The mutation sites in the N3 region include one or more of F403A, L407A, and M411A.

[0008] The present invention also provides a gene encoding the nucleocapsid protein mutant.

[0009] The present invention also provides a recombinant expression vector comprising the aforementioned gene.

[0010] The present invention also provides the application of the nucleocapsid protein mutant in the construction of a mutant viral strain of severe acute respiratory syndrome coronavirus 2.

[0011] The present invention also provides a mutant viral strain of severe acute respiratory syndrome coronavirus 2, comprising expressing the nucleocapsid protein mutant or the gene described herein.

[0012] The present invention also provides the use of the nucleocapsid protein mutant or the mutant virus strain in the preparation of a severe acute respiratory syndrome coronavirus 2 vaccine.

[0013] The present invention also provides the use of the nucleocapsid protein mutant or the mutant virus strain in screening drugs against severe acute respiratory syndrome coronavirus 2.

[0014] This invention also provides the application of nucleocapsid protein amino acid sites as targets in screening drugs against Severe Acute Respiratory Syndrome Coronavirus 2, wherein the nucleocapsid protein amino acid sites include one or more of the following in the CTD region: leucine at position 353, histidine at position 356, isoleucine at position 357, aspartic acid at position 358, and tyrosine at position 360; or It includes one or more of the following in the N3 region: phenylalanine at position 403, leucine at position 407, and methionine at position 411.

[0015] The present invention also provides a method for reducing the pathogenicity of severe acute respiratory syndrome coronavirus 2, wherein site mutations are performed on the amino acid sequence of the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2, wherein the site mutations include site mutations in the CTD region or the N3 region; The mutation sites in the CTD region include one or more of L353A, H356A, I357A, D358A, and Y360A; The mutation sites in the N3 region include one or more of F403A, L407A, and M411A.

[0016] The present invention also provides a chiral polypeptide, the amino acid sequence of which is shown in SEQ ID No. 1; the chiral polypeptide has the effect of inhibiting the assembly of severe acute respiratory syndrome coronavirus 2.

[0017] The beneficial effects of this invention compared to the prior art are as follows: This invention, based on SARS-CoV-2 virus-like particle (VLP) pseudotype technology, discovered that alanine mutations in the N protein, specifically L353A, H356A, I357A, D358A, and Y360A, disrupt the hydrophobic pocket of the N protein's CTD, reducing nucleic acid binding affinity. This invention designed a chiral D-type polypeptide (d(V)d(I)d(L)d(L)d(N)d(K)d(H)d(I)d(D)d(A)d(Y) (SEQ ID No. 3)) targeting this region and added a transect peptide (YGRKKRRQRRR (SEQ ID No. 4)). Inhibition experiments on VLP pseudovirus packaging showed that it effectively inhibited VLP pseudovirus assembly, with an IC50 concentration of [missing value]. 50 The concentration was 0.016 µM, and its KD was found to be 4.35 × 10⁻⁶ using SPR experiments on N protein and peptide. -5 .

[0018] Furthermore, this invention also found that F403A, F407A, and M411A lead to an increase in N protein monomers, a decrease in tetramers, and an increase in abnormally high-polymerized N protein, thereby causing abnormal viral assembly function. Moreover, this abnormally high-polymerized N protein can inhibit the replication of SARS-CoV-2 wild-type live virus and mutant strains. This invention may provide new drug targets for drug design targeting the SARS-CoV-2 N protein. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Figure showing the effect of truncated N protein regions on VLP pseudovirus assembly. Figure 2 Figure showing the effect of alanine mutations in a key region of the N protein on the infectivity of VLP pseudoviruses; Figure 3 The image shows the Western blot results after alanine mutations at 8 sites in the N protein. Figure 4 Figure showing the thermal stability and structural analysis results of five N-CTD mutant proteins; Figure 5 The figure shows the molecular dynamics simulation results of five mutations in N-CTD; Figure 6 The graph shows the nucleic acid load results of five mutant VLP pseudoviruses in N-CTD. Figure 7The graph shows the changes in the thermal stability of proteins with three alanine mutations in N-N3. Figure 8 The graph shows the changes in monomeric, abnormally high polymeric, and tetrameric states of the three mutated alanine proteins of N-N3. Figure 9 The graph shows the changes in nucleic acid content of VLP pseudoviruses after three alanine mutations in N-N3. Figure 10 Figure 1 shows the results of a nucleic acid competition experiment between wild-type N protein and abnormally high-polymerized N protein during the packaging of VLP pseudoviruses. Figure 11 The graph shows the inhibitory effect of the abnormally high-polymerized N protein on live SARS-CoV-2 virus. Figure 12 This is a diagram showing the inhibitory effect of peptides on VLP pseudovirus packaging. Figure 13 This is a dose-response graph of peptide-D15 concentration versus inhibition rate; Figure 14 The binding and dissociation curves of different concentrations of peptide-D15 with N protein are shown. Figure 15 The graph shows the response values ​​of different concentrations of peptide-D15. Figure 16 This is a comparison chart of the KD values ​​of peptide-D15 and L15. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] This invention provides a nucleocapsid protein mutant, which is obtained by amino acid mutation of the nucleocapsid protein, including site mutations in the CTD region or N3 region; The mutation sites in the CTD region include one or more of L353A, H356A, I357A, D358A, and Y360A; The mutation sites in the N3 region include one or more of F403A, L407A, and M411A.

[0027] In this invention, the nucleocapsid protein is designated MN908947 in the NCBI database.

[0028] The present invention also provides a gene encoding the nucleocapsid protein mutant.

[0029]

[0030] The present invention also provides a recombinant expression vector comprising the aforementioned gene.

[0031] In this invention, the recombinant expression vector further includes an initial expression vector.

[0032] The present invention also provides the application of the nucleocapsid protein mutant in the construction of a mutant viral strain of severe acute respiratory syndrome coronavirus 2.

[0033] The present invention also provides a mutant viral strain of severe acute respiratory syndrome coronavirus 2, comprising expressing the nucleocapsid protein mutant or the gene described herein.

[0034] The present invention also provides the use of the nucleocapsid protein mutant or the mutant virus strain in the preparation of a severe acute respiratory syndrome coronavirus 2 vaccine.

[0035] The present invention also provides the use of the nucleocapsid protein mutant or the mutant virus strain in screening drugs against severe acute respiratory syndrome coronavirus 2.

[0036] This invention also provides the application of nucleocapsid protein amino acid sites as targets in screening drugs against Severe Acute Respiratory Syndrome Coronavirus 2, wherein the nucleocapsid protein amino acid sites include one or more of the following in the CTD region: leucine at position 353, histidine at position 356, isoleucine at position 357, aspartic acid at position 358, and tyrosine at position 360; or It includes one or more of the following in the N3 region: phenylalanine at position 403, leucine at position 407, and methionine at position 411.

[0037] In this invention, the leucine at position 353, histidine at position 356, isoleucine at position 357, aspartic acid at position 358, tyrosine at position 360, phenylalanine at position 403, leucine at position 407, and methionine at position 411 are preferably all mutated to alanine.

[0038] The present invention also provides a method for reducing the pathogenicity of severe acute respiratory syndrome coronavirus 2, wherein site mutations are performed on the amino acid sequence of the nucleocapsid protein of severe acute respiratory syndrome coronavirus 2, wherein the site mutations include site mutations in the CTD region or the N3 region; The mutation sites in the CTD region include one or more of L353A, H356A, I357A, D358A, and Y360A; The mutation sites in the N3 region include one or more of F403A, L407A, and M411A.

[0039] The present invention also provides a chiral polypeptide, the amino acid sequence of which is shown in SEQ ID No. 1; the chiral polypeptide has the effect of inhibiting the assembly of severe acute respiratory syndrome coronavirus 2.

[0040] In this invention, the amino acid sequence of the chiral polypeptide is YGRKKRRQRRRVILLNKHIDAY (SEQ ID No. 1), and the chiral polypeptide preferably includes D-type and L-type, with the chiral polypeptide preferably being D-type.

[0041] Example 1: The effect of truncated N protein key regions on VLP pseudovirus assembly To investigate the effects of different domains of the N protein on the release and assembly of SARS-CoV-2 VLP pseudoviruses, specific consecutive amino acid deletions were performed on the N protein gene sequence, and the remaining amino acid sequence of the N protein was synthesized and cloned into the PCDNA3.1 expression vector. This study aimed to investigate the impact of deleting truncated amino acid regions on the packaging of VLP pseudoviruses.

[0042] Four expression plasmids for S protein, M&E protein, N protein, and packaging signal (synthesized and cloned by General Biol and ligated into the pcDNA3.1(+) expression vector; the sequences of all four plasmids were derived from Wuhan-Hu-1 (RefSeq:NC_045512.2)) were co-transfected into 293T cells. After 48 hours, the virus was harvested, yielding nine SARS-CoV-2 VLP pseudoviruses with truncated N protein variants: N-del (50-90 aa), N-del (90-130 aa), N-del (130-170 aa), N-del (170-210 aa), N-del (210-250 aa), N-del (240-290 aa), N-del (290-340 aa), N-del (340-390 aa), and N-del (391-419 aa). The detection method for luminescent signals of SARS-CoV-2 VLP pseudoviruses is as follows: The intensity of the luminescent signal represents the strength and presence of viral infectivity. Add 100 μL of culture medium to the 60 wells in the center of a 96-well plate (excluding the 36 wells at the edge, which are sealed with the culture medium). Add 50 μL of the prepared VLP pseudovirus solution to the 6 wells B2-G2. Starting from wells B2-G2, serially dilute the solution 3-fold to wells B3-G3 and subsequent wells. Discard the 50 μL of mixed solution taken from wells B10-G10. Add 100 μL of 293T cells overexpressing ACE2 and Furin to the 60 wells in the center of the 96-well plate (10,000 cells / well). Seal the edge wells with 260 μL of sterile water. Incubate the diluted 96-well plate in a 5% CO2, 37°C incubator for 18 hours. After incubation, discard 100 μL of the diluted solution from each well and add 100 μL of the culture medium. Bright-Glo™ fluorescence detection reagent, after being placed at room temperature in the dark for 2 minutes, is pipetted 5 times, and 150 μL of the liquid is transferred to a white or black plate. The luminescence value of each well is read using a microplate spectrophotometer. The results are as follows: Figure 1 A and Figure 1 As shown in C.

[0043] In this context, N-del (50-90aa) represents the deletion of 50-90 amino acids from the full-length amino acid sequence of the N protein, and N-del (90-130aa) represents the deletion of 90-130 amino acids from the full-length amino acid sequence of the N protein. The meanings of the other names follow the same pattern.

[0044] The results showed that the truncated N protein variants (N-del340-390aa) and (N-del391-419aa) resulted in the loss of infectivity of the SARS-CoV-2 VLP pseudovirus.

[0045] Therefore, every 5 amino acids between 340-390 aa were truncated to construct SARS-CoV-2 VLP pseudoviruses of the N protein truncated variant (N-del(340-344 aa), N-del(345-349 aa), N-del(350-354 aa), N-del(355-359 aa), N-del(360-364 aa), N-del(365-369 aa), N-del(370-374 aa), N-del(375-379 aa), N-del(380-384 aa), N-del(385-389 aa), and N-del(390-394 aa)). Infectivity studies were conducted following the same procedures described above. Results are as follows... Figure 1 B and Figure 1 As shown in D in the diagram.

[0046] The results showed that the N-protein variants truncated from N-del (350-354aa), N-del (355-359aa), and N-del (360-364aa) lost their infectivity.

[0047] Example 2: The effect of alanine mutation in the key region of the N protein on the infectivity of VLP pseudoviruses Fourteen alanine mutations were performed at 14 sites in the V350-P364 region of the CTD. The effect of alanine mutations in the key N protein region on the infectivity of VLP pseudoviruses was investigated according to the method described in Example 1. Simultaneously, the titer of SARS-CoV-2 VLP pseudoviruses was detected using the method for detecting luminescent signals described in Example 1. The viral titer was calculated with a cutoff value of 10 times that of the cell control, and the VLP pseudovirus titer was calculated using the Reed-Muench method. The results are as follows: Figure 2 As shown in A in the diagram.

[0048] The results showed that five mutations in the CTD region of the N protein affected viral assembly: L353A, H356A, I357A, D358A, and Y360A, which reduced viral titers by 176-fold, 53-fold, 101-fold, 58-fold, and 149-fold, respectively.

[0049] Based on the above results, alanine mutations were performed at 25 sites in the T391-Q418 region of the N3 protein to investigate the impact of alanine mutations in key N protein regions on the infectivity of VLP pseudoviruses. The results are as follows: Figure 2 As shown in B in the diagram.

[0050] The results showed that three mutations in the N3 region of the N protein affected viral assembly: F403A, L407A, and M411A, which reduced viral titers by 380-fold, 660-fold, and 396-fold, respectively.

[0051] Example 3 Western Blot (WB) assay The effects of the eight mutation sites obtained in Example 2 on protein expression were verified by Western blotting experiments, as follows: The S protein, M&E protein, eight N proteins containing alanine mutations, and four expression plasmids for packaging signals were synthesized and cloned by General Biol and ligated into the pcDNA3.1(+) expression vector to obtain plasmids containing the eight N proteins with alanine mutations. These plasmids, along with wild-type controls (N proteins), were transiently transfected into 293T cells. Cells were collected 24 hours after transfection, lysis buffer was added, and the cells were centrifuged. The supernatant was washed with 5× protein loading buffer (Coolaber, catalog number: SL1170), boiled at 100°C for 10 min, and then subjected to 8% loading buffer followed by electrophoresis on an 80V polyacrylamide gel for 20 min. Then, 8% separation gel was added, and electrophoresis was performed on a 120V polyacrylamide gel for 1.5 h. The electrophoresed proteins were transferred to a PVDF membrane at 250 mA for 2 h. The membrane was then used to extract the proteins using a 1:1000 diluted mouse novel coronavirus (2019-nCoV) nucleocapsid antibody and a mouse monoclonal antibody (Sino...). Biological antibody (catalog number: 40588-MM123) was used as the primary antibody to incubate PVDF membranes for detecting the expression of the N protein variant; and the membrane was developed using 1:2000 diluted goat anti-mouse IgG HRP (purchased from Abbkine, catalog number: A21010). A transfer control was performed using 1:1000 diluted anti-GAPDH mouse monoclonal antibody (purchased from Abbkine, catalog number: ABL1010). The intensity of the target protein was quantified using the EasySee Western blot Kit (TransGen Biotech, catalog number: DW101). Results are as follows. Figure 3 As shown.

[0052] Western blot results showed that the eight N proteins after the alanine mutation could still be expressed normally.

[0053] Example 4: Thermal stability and structural analysis of five sites in the CTD region of the N protein. To investigate how five sites in the CTD region of the N protein affect the assembly of SARS-CoV-2 VLP pseudoviruses, a truncated CTD (248-364) of the wild-type N protein (NCBI ID MN908947) was used as a control group. Proteins N-CTD-WT, N-CTD-L353A, N-CTD-H356A, N-CTD-357A, N-CTD-D358A, and N-CTD-Y360A were expressed. The thermal stability of this truncated variant was determined using thermofluorescence. The specific procedures are as follows: Based on the full-length N protein of wild-type SARS-CoV-2 (GenBank: MN908947), a plasmid was constructed (according to the method in Example 1) to generate three protein mutants: F403A, L407A, and M411A. The Lys248-Pro364 amino acid residues of the N-CTD were amplified by PCR. The product was further digested with NdeI and XhoI enzymes and ligated into a modified pET28a plasmid (purchased from addgene) containing an N-terminal hexahistine tag and a TEV protease cleavage site. Using the recombinant plasmid as a template, the N-CTD genes of the L353A, H356A, I357A, D358A, and Y360A mutants were constructed. The PCR amplification reaction system consisted of: 15 μL 2×PrimeSTAR (Premix), 1 μL F primer (10 μM), 1 μL R primer (10 μM), 0.5 μL plasmid template, and 13 μL ddH2O. The PCR amplification program consisted of 35 cycles, performed in two stages: 20 cycles followed by 15 cycles to ensure amplification accuracy. The pre-denaturation process was performed at 98℃ for 5 min, followed by denaturation for 15 s. The annealing temperature was set in a gradient of 55℃ with a 2℃ fluctuation, and the extension was performed at 72℃. After 20 cycles, a second denaturation, annealing, and extension reaction was performed for 15 cycles. The amplified products were stored at 4℃, with an extension rate of 1 kb / min. The primers are shown in Table 1. Table 1 PCR amplification primers

[0054] All plasmids were transformed into Escherichia coli BL21(DE3) strain and cultured overnight at 37°C in 10 ml LB medium. Colonies resistant to kanamycin (50 μg / ml) were inoculated into 1 L LB medium and cultured at 37°C and 220 rpm until the OD600 reached 0.6. IPTG was added to a concentration of 0.5 mM, and the cells were induced at 16°C and 220 rpm for 18 h. The cells were collected by centrifugation at 3800 rpm for 20 min and resuspended in 40 mL Lysis Buffer (25 mM Tris-HCl, 100 mM NaCl, 2 mM DTT, 10 mM imidazole, 1 mM PMSF, pH 6.8). The cells were then sonicated at 300 W for 30 min (3 s on, 6 s off). Centrifuge at 15000 rpm for 20 min, separate the supernatant precipitate, and prepare 20 μL samples. Equilibrate the nickel column with Buffer A beforehand, repeatedly attach the supernatant to the column for 2 h, and wash with a gradient of Buffer A containing 20 mM and 50 mM imidazole, using the first 20 μL of eluent for each wash. Elute with Buffer A containing 300 mM imidazole, using the first 20 μL of eluent for each wash. Electrophoresis confirms that all purification steps are correct. Concentrate the eluent to a final volume of 500 μL at 3700 rpm, centrifuge at 13300 rpm, transfer the supernatant to a new centrifuge tube, and further separate and purify according to molecular weight differences using size-exclusion chromatography (SEC). The mobile phase is Buffer A, the flow rate is 0.5 mL / min, and a 1 mL loading loop is used. Samples are taken based on the UV280 and UV260 peak profiles, prepared, and electrophoresed to confirm protein distribution. The target peak is concentrated, the concentration is measured, and then aliquoted and frozen.

[0055] For the full-length N protein and its mutants, harvested cells were resuspended in buffer A (25 mM Tris-HCl, 300 mM NaCl, 5 mM MgCl2, 5% glycerol, 5 mM β-mercaptoethanol, pH 8.0). Ni Sepharose™ FastFlow beads (GE Healthcare) were added to the supernatant. A gradient elution method was used to elute the protein using buffer B (25 mM Tris-HCl, 300 mM NaCl, 5 mM MgCl2, 5% glycerol, 5 mM β-mercaptoethanol, 300 mM nitrosoimidazole, pH 8.0). The concentration and purification of the target protein were performed by centrifugation and gel filtration chromatography (Superdex 200; GE Life Sciences) in gel filtration buffer (5 mM Tris-HCl, 300 mM NaCl, 5 mM MgCl2, 5% glycerol, 1 mM DTT, pH 8.0).

[0056] N-CTD protein and its mutants were purified using the same method described above, with resuspension buffer A (20 mM Tris-HCl, 500 mM NaCl, pH 7.5); elution buffer B (20 mM Tris-HCl, 500 mM NaCl, 300 mM nitrosoimidazole, pH 7.5); and gel filtration buffer (20 mM Tris-HCl, 500 mM NaCl) at pH 7.5. The mutant N-CTD protein was further identified by SDS-PAGE. The full-length protein was validated by Native-PAGE. The purified protein was concentrated and stored at 4°C for subsequent analysis. Results are as follows: Figure 4 As shown in A in the diagram.

[0057] The results show that, compared with the wild-type CTD, the thermal stability of all mutants decreased significantly, from 52℃ in the wild type to 37℃ in L353A, 39℃ in H356A, 39℃ in I357A, 42℃ in D358A, and 39℃ in Y360A.

[0058] Example 5: Molecular dynamics simulation analysis of 5 sites in the CTD region The 3D structure of the SARS-CoV-2 N protein was derived from PDB (6WZO) and predicted by GROMACS-2021. The dynamic system was prepared using an OPLS force field and a TIP3P water model, with the addition of Na... + and Cl - Ions were used to achieve charge neutralization. To ensure energy minimization, a steepest descent algorithm was employed to achieve a maximum force of 1000 kJ / mol. The NVT series was performed using the Nose-Hoover method at 300 K, while the Parinello-Rahman algorithm was used to assemble the NPT. At 1 bar, the assembly of NVT and NPT was used to ensure temperature and pressure equilibrium, respectively. Molecular dynamics simulations were finally run from a random initial velocity with periodic boundary conditions applied until 5 ns. Unbonded and long-range electrostatic interactions were handled using the Verlet cutoff scheme and the particle grid Ewald method, respectively. Short-range electrostatic and van der Waals interactions were calculated with a 12 Å cutoff. The last 3 ns frames of the molecular dynamics simulations were used to create the average structure of the four complexes. The ΔG prediction between RBD and antibody was performed using ROSETTA's InterfaceAnalyzer with Atomic_burial_cutoff, sasa_calculator_probe_radius, and interfaces_cutoff values ​​set to 0.01, 1.4, and 8.0, respectively. The results are as follows: Figure 4 B to G and Figure 5 As shown.

[0059] Further structural analysis revealed that these mutations were distributed in the α5 and η3 domains of the N protein's CTD structure (e.g., Figure 4 (As shown in B). L353 and I357 play a role in stabilizing the dimer; L353 forms a large-area hydrophobic pocket with homosynthetic F286 / F307 / L339 and heterosynthetic M233 / L331. Figure 4 In the C group, I357 forms a hydrophobic pocket with homostic F286 and heterostic I320 / M322. Figure 4 Amino acid mutations at these two sites (E in the chain) can disrupt the hydrophobic pocket between the chains, thus affecting stability. Figure 4 (C / E ratio in the middle). D358 mainly utilizes hydrogen bonds to stabilize the dimer. D358 forms two hydrogen bonds with the homo-chain N285 and the hetero-chain R319 to exert its function. Figure 4 (On F in the middle), mutations at this site disrupt these two hydrogen bonds, thus affecting stability. Figure 4 (F below). H356 and Y360 mainly utilize hydrophobic interactions to stabilize the internal structure of the single-stranded CTD domain of the N protein. H356 forms a hydrophobic pocket with F286 / L291 / Y298, and simultaneously forms three hydrogen bonds with D288 and Y298. Figure 4 The site at point D in the molecular dynamics energy dissociation exhibits numerous interactions, consistent with the finding that this site contributes the most to the energy reduction during energy dissociation (refer to the gmx_mmPBSAj results). Therefore, a mutation at H356 would lead to the disappearance of these interactions. Figure 4 (D below). Similarly, the mutation of Y360 is due to the disruption of the hydrophobic pocket formed by Y360 / R277 / G278 / P279, resulting in reduced stability. Figure 4 The G in the text further reduces the infectivity of VLP pseudoviruses.

[0060] Results of interaction energy decomposition of the CTD structure using molecular dynamics simulation software Gromacs and gmx_MMPBSA ( Figure 5 The results show that, except for 358, all of the above mutations contribute positively to dimer formation and structural stability.

[0061] Example 6: Changes in viral nucleic acid load at 5 sites in the CTD region To investigate the mechanism by which the mutated N protein leads to a decrease in viral titer, viral RNA was extracted from 140 mL of purified pseudovirus using the QIAamp Viral RNA Mini Kit (QIAGEN, Cat#52906). The extracted RNA was then used as a template for reverse transcription, which was performed using the SuperScript III First-Strand Synthesis System RT-PCR Kit (Invitrogen, Cat#18080-051). Real-time PCR for viral quantification was performed using TB Green Premix Ex TaqII (TaKaRa, Cat#RR820A) according to the manufacturer's instructions. The PS9 gene from the SARS-CoV-2 VLP pseudovirus was inserted into the vector pCDNA3.1(+) as a plasmid standard for calculating viral copy number. Primers PS9-F: AGTGATATAGTACGACCCT, PS9-R: TCTTTTATAGCCACGGAAC. Results are as follows: Figure 6 As shown.

[0062] The nucleic acid extraction method is as follows: Add an appropriate amount of binding buffer (usually containing ethanol) to the lysed sample, mix thoroughly, and transfer the sample to a centrifuge column. The silica membrane inside the column selectively adsorbs nucleic acids under high salt and ethanol conditions. Centrifuge at 8000g for 2 minutes to allow the nucleic acids to bind to the silica membrane. Add washing buffer (usually containing ethanol) to wash the silica membrane containing the adsorbed nucleic acids, removing residual proteins, salts, and other impurities. Wash twice, centrifuging at the same speed and time after each wash. Add an appropriate amount of elution buffer (such as nuclease-free water or TE buffer) to the washed centrifuge column, with a volume of 100 μL. After incubating the centrifuge column at room temperature for 1 minute, centrifuge at 3000g for 2 minutes to elute the nucleic acids from the silica membrane. Collect the eluent, which contains the extracted viral nucleic acid.

[0063] The reverse transcription method is as follows: purified RNA, reverse transcriptase, primers, deoxyribonucleoside triphosphates (dNTPs), reaction buffer, and other components are mixed to prepare a reverse transcription reaction system. The reaction system is placed in a constant temperature device, and the reaction is carried out according to the optimal reaction temperature and time of reverse transcriptase. Under the action of reverse transcriptase, cDNA complementary to RNA is synthesized using RNA as a template according to the base complementary pairing principle.

[0064] The method for quantitative real-time PCR is as follows: Prepare template nucleic acid cDNA, a pair of primers, dNTPs (deoxyribonucleoside triphosphates), Taq DNA polymerase, and buffer. First, add the buffer, dNTPs, primers, and template nucleic acid, and finally add the Taq enzyme. Gently mix to avoid generating air bubbles. The sample addition process should be performed on ice to prevent premature inactivation of the Taq enzyme. Incubate at 94℃ for 5 min to completely denature the template DNA and unwind the double strands. This includes three steps: denaturation at 94℃ for 20 s; annealing at 50℃ for 20 s; and extension at 72℃ for 1 min per 1 kb length. Repeat for 40 cycles, incubating at 72℃ for 10 min each time, to ensure complete extension of all products. Use the PS9 plasmid as a positive control.

[0065] Quantitative results showed that the viral RNA load of the mutant strains was reduced compared with the wild-type strain (SARS-CoV-2 VLP pseudovirus), suggesting that these point mutations affected the interaction between the N protein and nucleic acid. Among them, the viral RNA load of the L353A, H356A, and I357A mutant VLP pseudoviruses was reduced by 2.0-4.6 times.

[0066] Example 7: Changes in the thermal stability of three alanine mutant proteins in the N3 region To investigate how the three alanine mutations in the N3 region affect the assembly of SARS-CoV-2 VLP pseudoviruses, and since the N3 domain of the N protein has not yet been resolved, a full-length N protein was constructed, and wild-type and mutant N proteins were expressed. The thermostability of the full-length proteins of the wild-type and the three mutants was then determined using thermofluor. Protein expression and purification were performed according to the method described in Example 4. Details not described herein are the same as in Example 4.

[0067] The protein thermostability assay was performed using an MX3005 qPCR instrument (Agilent, Santa Clara, USA) to detect exposed hydrophobic residues. The total reaction volume was 20 μL, pH set to 8.0, containing 20 μg of the target protein (i.e., N protein and its mutation, N-CTD and its mutation). The system was labeled with a 1000-fold concentration of SYPRO Orange fluorescent probe (Invitrogen, Carlsbad, USA), and the temperature was gradually increased from 25°C to 99°C, with fluorescence signals recorded three times for each 1°C increase to obtain accurate results. In this experiment, the SYPRO Orange fluorescent probe was used to detect the protein denaturation process. As the temperature increased, hydrophobic residues were exposed, and the fluorescence signal intensified, allowing monitoring of the protein's folding state and stability. This method provides important quantitative data for studying the impact of mutations on protein conformation and stability. Results are as follows: Figure 7 As shown.

[0068] The results showed that the Tm value remained unchanged at 49℃, indicating that the mutation at this site did not cause a change in the thermal stability of the N protein.

[0069] Example 8: Tetramer, monomer, and anomalous polymerization changes of proteins after alanine mutations at three sites in the N3 region. Previous studies have reported (Qiaozhen Y, VMAW, Steve S, et al. Architecture and self-assembly of the SARS-CoV-2 nucleocapsid protein.[J]. Protein science: a publication of the Protein Society, 2020, 29(9):1890-1901.DOI:10.1002 / pro.3909.) that the N3 domain may mediate the formation of oligomers such as N protein tetramers. Therefore, the expression and purification protein (prepared according to the method in Example 4) was used to identify the aggregation state of wild-type N protein and mutant N protein by non-denaturing polyacrylamide gel electrophoresis (native-page) according to the method in Example 4, and the different bands were identified by peptide fingerprinting. Operations not described herein were the same as in Example 4. The results are as follows: Figure 8 As shown.

[0070] The results showed that these three mutations did indeed lead to a decrease in the proportion of tetramers and a corresponding increase in the proportion of monomers, thereby reducing the generation of SARS-CoV-2 VLP pseudoviruses. Figure 8 The F403A, F407A, and M411A mutations led to a 2.2-fold, 2.9-fold, and 1.8-fold increase in N protein monomer content, respectively; furthermore, these mutations reduced N protein tetramer formation by 18%, 19%, and 8%, respectively; these mutations also led to an increase in aberrant polymers between dimers and tetramers, by 20%, 58%, and 16%, respectively. Figure 8 (B in the middle).

[0071] Example 9: Nucleic acid content of VLP pseudovirus after alanine mutation at three sites in the N3 region. To investigate the mechanism by which the N3-mutated N protein leads to a decrease in viral titer, nucleic acid was extracted from SARS-CoV-2 VLP pseudoviruses and reverse transcribed according to the method described in Example 6, followed by qPCR quantification. The WT (wild-type), F403A, F407A, and M411A mutated N proteins packaged 2.13 pg, 3.8 pg, 5.12 pg, and 4.38 pg of viral nucleic acid, respectively. The results are as follows... Figure 9 As shown.

[0072] Quantitative results showed that the nucleic acid content of the mutant strains was increased compared to the wild-type strain, suggesting that these point mutations increased the interaction between the N protein and nucleic acids. This indicates that the abnormally high polymerization of the mutant N protein dimers and tetramers has a higher nucleic acid binding capacity than that of the normal tetramer. Due to their stronger nucleic acid binding capacity, and the fact that the abnormally high polymerization of the N protein causes the assembled virus to lose its infectivity, this resulted in a significant decrease in the titer of SARS-CoV-2 VLP pseudoviruses.

[0073] Example 10: Competition experiment on nucleic acids during the VLP pseudovirus packaging process of wild-type N protein and anomalously high-polymer N protein. To further verify at the biological level that the abnormally highly polymerized N protein has a stronger binding affinity for nucleic acids, a nucleic acid competition experiment was conducted during the VLP pseudovirus packaging process of normal N protein and abnormally highly polymerized N protein. Control groups were set up with doses of 5 µg and 10 µg of wild-type N protein, and experimental groups were set up with doses of 5 µg wild-type N protein and 5 µg mutant N protein. The results are as follows: Figure 10 As shown.

[0074] The preparation method for VLP pseudovirus is as follows: One day in advance, 293T cells grown to 90%-100% confluency are passaged at a ratio of 1:2.5 and seeded in T75 or T150 cell flasks for plasmid transfection; transfection is performed at 75% cell density, as the transfection reagent lipo3000 is toxic and causes significant damage to cells; two EP tubes are prepared, and 750 μL of opti-MEM is added to each tube. In tube 1, 1.5 μg of Spike plasmid, 7.5 μg of M&E plasmid, 15 μg of N protein plasmid, and PS9 (PS966) are added. Pack 15 μg of the signal plasmid, then add 30 μL of P3000; add 30 μL of Lipo3000 to the Opti-MEM in tube 2, and mix the two tubes thoroughly; (Note that Opti-MEM is more likely to form liposomes if kept at room temperature), pour the contents of tube 2 into tube 1, and incubate at room temperature for 20 min; pouring the plasmid-free contents of tube 2 into tube 1 is to reduce plasmid waste and loss during transfer; add the mixed liposomes to the cell culture flask and mix gently (you can aspirate about 30% of the culture medium to increase the plasmid concentration and improve the excretion efficiency); 5% After culturing in a CO2, 37℃ incubator for 12 hours, the original DMEM was aspirated, and 15 mL of DMEM complete medium was added. The culture was continued for 36 hours. The supernatant DMEM was aspirated, centrifuged at 4000 rpm for 10 min, filtered through a 0.45 μm filter, and frozen at -20℃. The VLP pseudovirus solution was concentrated by ultrafiltration at 4000 rpm to prepare for subsequent ultrafiltration and quantification of pseudovirus (100 KD can remove some empty protein).

[0075] The results showed that the F403A, F407A, and M411A mutants reduced the VLP pseudovirus titer by 376-fold, 473-fold, and 215-fold, respectively. This indicates that when wild-type and mutant N protein particles are added simultaneously to package VLP pseudoviruses, the mutant N protein binds more strongly to nucleic acids than the wild-type N protein. As a result, the nucleic acids carrying the reporter gene are all packaged into the viral particles of the abnormally high-polymerized N protein, while the wild-type N protein packages less nucleic acid. The VLP pseudovirus composed of the abnormally high-polymerized N protein loses its infectivity, thus leading to a decrease in VLP titer.

[0076] Example 11 Inhibition of live SARS-CoV-2 virus by abnormally polymerized N proteins generated from F403A, L407A, and M411A mutations. The inhibitory effect of aberrant high-polymerized N proteins generated by F403A, L407A, and M411A mutations on live virus was evaluated. Using wild-type N protein plasmids as a control, the plasmids of the mutant N protein were transfected into 293T-ACE2 cells. Live virus infection experiments were performed 24 h post-transfection, and viral particles were collected from the supernatant 48 h post-infection. Viral titers were determined through infection experiments. Results are as follows: Figure 11 As shown in A in the diagram.

[0077] The experimental method was as follows: 293T-ACE2 cells were pre-seeded in six-well plates 24 hours in advance. When the cells reached 80-90% confluency, they were transfected with mutant N protein plasmids (N-F403A, N-L407A, and N-M411A) and wild-type N protein plasmids (N-WT), respectively. After 48 hours, 0.05 MOI of live novel coronavirus virus (Wuhan-Hu-1, RefSeq:NC_045512.2) was added. After 96 hours, the supernatant virus was collected, and 50 µl of the collected supernatant virus solution was used to infect 293T-ACE2 cells. Cytopathic effects were observed after 48 hours, and the viral titer was calculated.

[0078] The results showed that the F403A, L407A and M411A mutations led to a 23.7-fold, 9.0-fold and 6.9-fold reduction in live virus titers, respectively.

[0079] To investigate the broad-spectrum characteristics of the anomalously polymerized N protein against SARS-CoV-2, a triple-mutant inhibitory assay was performed on wild-type, Delta (preserved in the P3 laboratory of the Institute of Medical Biology), and XBB.1.5 SARS-CoV-2 variant (preserved in the P3 laboratory of the Institute of Medical Biology). The results are as follows: Figure 11 As shown in B and C.

[0080] The results showed that the abnormal N protein expressed by the triple mutant plasmid effectively reduced the wild-type virus titer by 93%, while also achieving inhibition rates of 80% and 62% against the Delta and XBB.1.5 variants, respectively. Figure 11 (B in the text). However, no inhibitory effect was observed against live RSV virus ( Figure 11 (C in the middle).

[0081] In addition, natural mutation analysis of the coronavirus N protein was performed on the F403, L407, and M411 sites associated with the abnormally high-polymerized N protein (mutation status was statistically analyzed after sequence alignment), and the results are as follows: Figure 11 As shown in D in the diagram.

[0082] These sites were found to be completely conserved in the N protein of Sarbecoviruses, with no known natural mutations. Figure 11 (D in the text). Therefore, these sites may be potential targets for developing broad-spectrum coronavirus drugs.

[0083] Example 12 Surface Plasmon Resonance (SPR) Experiment D-type chiral peptides (d(V)d(I)d(L)d(L)d(N)d(K)d(H)d(I)d(D)d(A)d(Y)) were designed to target the CTD region of the N protein, which affects viral assembly, and a membrane-penetrating peptide (YGRKKRRQRRR) was added to enable peptide D15 (d(Y)d(G)d(R)d(K)d(K)d(R)d(Q)d(R)d(R)d(V)d(I)d(L)d(L)d(N)d(K)d(H)d(I)d(D)d(A)d(Y)) to enter the cell. By adding peptide D15, L-type peptides, and membrane-penetrating peptides during the assembly of VLP pseudoviruses, surface plasmon resonance (SPR) detection was performed. During the testing, control, shuttle peptide (i.e., membrane-penetrating peptide) treatment, peptide-L9 treatment, peptide-L15 treatment, and peptide-D15 treatment groups were set up. Peptide-L9 was an L-type peptide (YGRKKRRQRRRVILLN (SEQ ID No. 15)); peptide-L15 was an L-type peptide (i.e., the chirality of peptide D15 was changed to L-type). Results are as follows... Figures 12 to 16 As shown.

[0084] The experimental method was as follows: different concentrations of peptides were added during the VLP pseudovirus packaging process, namely 10µM, 3.3µM, 1.1µM, 0.36µM, 0.12µM, 0.04µM, 0.01µM, 0.004µM, and 0.0001µM. The packaged VLP pseudoviruses were then collected and titrated (as above) to detect the luminescence value, the inhibition rate at different concentrations, and the IC50 was calculated.

[0085] The results showed that peptide-D15 could inhibit the assembly of VLP pseudoviruses, and its IC50 value was [missing information]. 50 The concentration was 0.016 µM; through SPR experiments of N protein and peptide, its KD was found to be 4.35 × 10⁻⁶. -5 .

[0086] As can be seen from the above embodiments, this invention provides a nucleocapsid protein mutant, gene, recombinant expression vector, mutant viral strain, and applications. Based on SARS-CoV-2 virus-like particle (VLP) pseudotype technology, this invention discovered that alanine mutations in the N protein, specifically L353A, H356A, I357A, D358A, and Y360A, disrupt the hydrophobic pocket of the N protein CTD, reducing nucleic acid binding affinity. Furthermore, it was found that F403A, F407A, and M411A lead to an increase in N protein monomers, a decrease in tetramers, and an increase in abnormally high-polymerized N protein, inhibiting the replication of SARS-CoV-2 wild-type live virus and mutant strains. Simultaneously, this invention designed a peptide-D15 targeting the aforementioned CTD region, which was found to effectively inhibit the assembly of VLP pseudoviruses.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nucleocapsid protein mutant, characterized in that, The nucleocapsid protein mutant is obtained by amino acid mutation of the nucleocapsid protein, including site mutations in the CTD region or N3 region. The mutation sites in the CTD region include one or more of L353A, H356A, I357A, D358A, and Y360A; The mutation sites in the N3 region include one or more of F403A, L407A, and M411A.

2. A gene encoding a mutant of the nucleocapsid protein of claim 1.

3. A recombinant expression vector, characterized in that, Includes the gene described in claim 2.

4. The use of the nucleocapsid protein mutant of claim 1 in the construction of a mutant strain of severe acute respiratory syndrome coronavirus 2.

5. A mutant strain of Severe Acute Respiratory Syndrome Coronavirus 2, characterized in that, This includes expressing the nucleocapsid protein mutant of claim 1 or the gene of claim 2.

6. The use of the nucleocapsid protein mutant of claim 1 or the mutant virus strain of claim 5 in the preparation of a severe acute respiratory syndrome coronavirus 2 vaccine.

7. The use of the nucleocapsid protein mutant of claim 1 or the mutant virus strain of claim 5 in screening for drugs against severe acute respiratory syndrome coronavirus 2.

8. The application of nucleocapsid protein amino acid sites as targets in screening drugs against severe acute respiratory syndrome coronavirus 2, characterized in that, The nucleocapsid protein amino acid sites include one or more of the following in the CTD region: leucine at position 353, histidine at position 356, isoleucine at position 357, aspartic acid at position 358, and tyrosine at position 360; or It includes one or more of the following in the N3 region: phenylalanine at position 403, leucine at position 407, and methionine at position 411.

9. A method for reducing the pathogenicity of severe acute respiratory syndrome coronavirus 2, characterized in that, Site mutations were performed on the amino acid sequence of the severe acute respiratory syndrome coronavirus 2 nucleocapsid protein, including site mutations in the CTD region or the N3 region; The mutation sites in the CTD region include one or more of L353A, H356A, I357A, D358A, and Y360A; The mutation sites in the N3 region include one or more of F403A, L407A, and M411A.

10. A chiral polypeptide, characterized in that, The amino acid sequence of the chiral polypeptide is shown in SEQ ID No. 1; the chiral polypeptide has the effect of inhibiting the assembly of severe acute respiratory syndrome coronavirus 2.

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