Construction of enterovirus 71 vp1 protein 75th amino acid mutant strain and its application

Abstract

The application relates to the field of microbial technology, in particular to construction and application of a virus strain with mutation of the 75th amino acid of VP1 protein of enterovirus 71. The amino acid sequence of the mutant of VP1 protein of enterovirus 71 is shown in SEQ ID NO. 1. The mutant of VP1 protein and the corresponding coding nucleic acid molecule can make the proliferation ability of enterovirus 71 decrease, but the virulence in mice increases, and can be used for constructing the mutant virus strain of enterovirus 71, and providing effective biological materials for preparing medicines for preventing and treating enterovirus 71 infection and screening and quality control of the medicines.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the construction and application of a mutant viral strain of enterovirus 71 VP1 protein at amino acid position 75. Background Technology

[0002] Enterovirus 71 (EV71) belongs to the family Picornaviridae, genus Enterovirus. Its genome is a single-stranded positive-sense RNA, approximately 7400 nucleotides in length, encoding a polyprotein containing 2193 amino acids. EV71 primarily infects children under 5 years old, spreading via the fecal-oral route or through droplets. It mainly causes hand, foot, and mouth disease (HFMD), which is clinically difficult to distinguish from HFMD caused by Coxsackievirus A16 infection. However, severe EV71 infection can cause aseptic encephalitis, meningoencephalitis, poliomyelitis-like paralysis, neurogenic myocarditis, and pulmonary edema, leading to severe infection and even death.

[0003] The EV71 genome encodes a polyprotein comprising structural proteins in the P1 region (VP4, VP2, VP3, VP1) and non-structural proteins in the P2 region (2A, 2B, 2C) and P3 region (3A, 3B, 3C, 3D). Among these, the VP1 protein, exposed on the surface of the viral capsid, is the primary neutralizing protein and contains numerous antigenic determinants. The VP1 protein also plays a crucial role in the formation of epitopes that bind to cellular receptors, thus playing a key role in viral adsorption. Furthermore, studies have reported that VP1 plays an important role in the stability of the EV71 viral structure and in immunomodulation. Therefore, the VP1 protein may determine the host tropism and pathogenicity of the EV71 virus.

[0004] The VP1 protein of EV71 consists of 297 amino acids, and some studies have revealed that certain sites within it may be potential sites of viral virulence. McMinn et al., by comparing the VP1 gene sequence of the 1999 Australian EV71 epidemic strain, found that the main difference between strains clinically presenting with hand-foot-and-mouth disease and neurotoxic strains lies in the substitution of amino acid 170 of the VP1 protein, from alanine to valine. This change may lead to alterations in the spatial structure of the VP1 protein, thereby altering the virus's ability to bind to host cells and its virulence. Yorihiro et al. found that amino acid 145 of the VP1 protein can regulate the binding of EV71 virus to the PSGL-1 receptor on the surface of leukocytes. When position 145 is glycine (G) or glutamine (Q), the virus can bind to PSGL-1, but after mutation to negatively charged glutamate (E), it loses the ability to bind to PSGL-1. However, when infected with human SCARB2 transgenic mice and macaques, the VP1-145E mutant strain showed stronger virulence, which may be related to the regulation of the adsorption of different receptors at this site and the sensitivity of antibody neutralization.

[0005] Currently, there are no specific drugs for treating EV71 infection, and clinical treatment is mostly symptomatic and supportive. Therefore, developing protein mutants and mutant viral strains that affect the virulence and proliferation ability of EV71 is of great significance for drug development, screening, and quality control for the prevention or treatment of EV71 infection, as well as for the prevention and treatment of EV71 infection. Summary of the Invention

[0006] This invention provides a mutant of enterovirus 71 VP1 protein, a mutant virus strain, and its applications.

[0007] In previous research, the applicant constructed an infectious cDNA clone of the EV71 virus using reverse genetics, which allows for relatively easy modification of the RNA virus genome through genetic engineering techniques. After years of research, it was hypothesized that a highly conserved transtype site in the EV71 virus VP1 protein might be a potential virulence-determining site, leading to site-directed mutagenesis screening of the VP1 protein. Through continuous screening and verification, this invention discovered that a mutation at amino acid position 75 of the VP1 protein, from threonine (T) to alanine (A), significantly affects the maturation and cleavage of the VPO protein during EV71 packaging, thereby impacting the virus's proliferation ability in cells. However, this mutation at this site, observed after intraperitoneal injection into 2-day-old suckling mice, enhances viral virulence in mice. The development of this mutation site, its corresponding protein mutants, and mutant viral strains lays an important foundation for the development of new anti-EV71 drugs and the acquisition of new EV71 vaccine candidates, providing a basis for elucidating the viral pathogenesis and locating the viral virulence-determining site.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] The present invention provides a mutant of enterovirus 71 VP1 protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0010] Compared with the wild-type enterovirus 71 VP1 protein, the amino acid sequence shown in SEQ ID NO.1 above has a mutation at amino acid position 75, where threonine (T) is mutated to alanine (A).

[0011] The aforementioned VP1 protein mutants significantly reduced the proliferation ability of EV71 and significantly enhanced its virulence.

[0012] The present invention provides a nucleic acid molecule that encodes the above-described enterovirus 71 VP1 protein mutant.

[0013] Based on the amino acid sequence and codon rules of the VP1 protein mutant provided above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the VP1 protein mutant. Due to the degeneracy of the codon, the nucleotide sequence of this nucleic acid molecule is not unique, but all nucleic acid molecules capable of encoding the above-mentioned VP1 protein mutant are within the protection scope of this invention.

[0014] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is based on the VP1 protein encoding gene of the EV71 wild-type virus strain, with the codon corresponding to the 75th amino acid mutated to the alanine codon.

[0015] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

[0016] This invention provides a biomaterial comprising the nucleic acid molecules described above or expressing the enterovirus 71 VP1 protein mutant described above;

[0017] The biomaterial is an expression cassette, vector, or host cell.

[0018] The expression cassette may be a recombinant nucleic acid molecule obtained by operatively linking the nucleic acid molecule with transcriptional and / or translational regulatory elements.

[0019] The vectors include, but are not limited to, plasmid vectors, viral vectors, transposons, etc.

[0020] The host cell may be a microbial cell or an animal cell, wherein the microorganism includes bacteria (e.g., Escherichia coli), fungi (e.g., yeast), or viruses (e.g., enterovirus 71). The animal cell may be an animal cell commonly used for culturing viruses (e.g., Vero cells).

[0021] The present invention provides a mutant enterovirus type 71 virus strain, wherein the mutant enterovirus type 71 virus strain comprises the above-described nucleic acid molecule or expresses the above-described enterovirus type 71 VP1 protein mutant.

[0022] The aforementioned enterovirus 71 mutant strain can be any enterovirus 71 strain as the starting strain, and the starting strain can be modified to include the nucleic acid molecules described above or express the enterovirus 71 VP1 protein mutant described above.

[0023] In some embodiments of the present invention, the mutant virus strain is obtained by mutating the genome of a wild-type enterovirus 71 strain so that the 75th amino acid (preferably threonine) of its VP1 protein is mutated to alanine.

[0024] In some embodiments of the present invention, the mutant viral strain is obtained by mutating nucleotide position 2660 of the viral genome of the wild-type enterovirus 71 strain HP (Zhang YX, Huang YM, Li QJ, et al. A highly conserved amino acid in VP1 regulates maturation of enterovirus 71[J]. PLoS Pathogens, 2017, 13(9):e1006625.DOI:10.1371 / journal.ppat.1006625.) from A to G.

[0025] This invention provides the application of the above-described enterovirus 71 VP1 protein mutant, the nucleic acid molecule, or the biological material in constructing enterovirus 71 mutant virus strains.

[0026] Preferably, the enterovirus 71 mutant strain has enhanced virulence and / or reduced replication capacity.

[0027] The present invention provides the application of the above-described enterovirus 71 VP1 protein mutant, the nucleic acid molecule, or the biological material in reducing the proliferation ability of enterovirus 71 and / or enhancing the virulence of enterovirus 71.

[0028] The present invention provides the use of the above-described enterovirus 71 VP1 protein mutant, or the nucleic acid molecule, or the biological material, or the enterovirus 71 strain in the preparation of a medicament for the prevention and / or treatment of enterovirus 71 infection.

[0029] Preferably, the drug includes therapeutic drugs (such as antibodies), vaccines, etc.

[0030] The present invention provides the use of the above-described enterovirus 71 VP1 protein mutant, or the nucleic acid molecule, or the biological material, or the enterovirus 71 strain in the preparation of a product for detecting the presence or level of enterovirus 71 in a sample.

[0031] The present invention provides the use of the above-described enterovirus 71 VP1 protein mutant, or the nucleic acid molecule, or the biological material, or the enterovirus 71 strain in the screening and / or quality control of drugs for the prevention and / or treatment of enterovirus 71 infection.

[0032] Preferably, the drug includes therapeutic drugs (such as antibodies), vaccines, etc.

[0033] This invention provides the application of the above-described enterovirus 71 VP1 protein mutant, the nucleic acid molecule, the biological material, or the enterovirus 71 strain in the preparation of animal models of enterovirus 71 infection.

[0034] The present invention provides a method for reducing the proliferation capacity of enterovirus 71 and / or enhancing the virulence of enterovirus 71, the method comprising: mutating the genome of enterovirus 71 such that the 75th amino acid (preferably threonine) encoding the VP1 protein is mutated to alanine.

[0035] The beneficial effects of the present invention include at least the following: the enterovirus 71 VP1 protein mutant and its corresponding encoding nucleic acid molecule provided by the present invention can reduce the proliferation ability of enterovirus 71 and enhance its virulence. It can be used to construct enterovirus 71 mutant virus strains, and provides effective biomaterials for the preparation of drugs for the prevention and treatment of enterovirus 71 infection, as well as for drug screening and quality control. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1This is the sequencing verification result of the mutation site in Example 1 of the present invention.

[0038] Figure 2 The results of immunoblotting in Example 4 of this invention are shown, where Ctrl represents the control, EV71-HP represents the wild-type HP strain, and EV71-T75A represents the HP-T75A mutant strain.

[0039] Figure 3 The results of the HP-T75A mutant strain proliferation phenotype detection on Vero cells in Example 5 of this invention are shown. In this example, Ctrl represents the control, FY represents the wild-type HP strain, and T75A represents the HP-T75A mutant strain.

[0040] Figure 4 The above figure shows the results of virus infectivity titer detection in Example 5 of the present invention. The upper figure shows the results when the inoculation amount MOI=10, and the lower figure shows the results when the inoculation amount MOI=1. EV71-HP represents the wild strain HP, and T75A represents the HP-T75A mutant strain.

[0041] Figure 5 The results of protein component and RNA detection of virus particles with different sedimentation coefficients separated and purified by density gradient centrifugation in Example 6 of the present invention are shown. In this example, EV71-HP represents the wild strain HP and T75A represents the HP-T75A mutant strain.

[0042] Figure 6 The results of the virulence test of the HP-T75A mutant strain in BalB / c suckling mice in Example 7 of this invention are shown. In this example, A represents the degree of hind limb paralysis in the suckling mice, B represents the weight change and mortality rate, and C represents the tissue distribution of the virus as determined by qPCR. MOCK represents the control, EV71-HP represents the wild-type HP strain, and T75A represents the HP-T75A mutant strain. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0044] Example 1: Construction of an infectious clone of enterovirus 71 (HP) strain VP1 mutant at amino acid position 75 (HP-T75A) cDNA

[0045] Using an infectious clone plasmid of the wild-type virus strain (EV71-HP strain) as a template, the 75th amino acid position of the VP1 protein was mutated from a threonine residue to an alanine residue (i.e., the 2660th nucleotide position of the viral genome was mutated from A to G) via site-directed mutagenesis. First, a plasmid with the mutated site and nick was amplified using primers 75-F (5'-GTGTTCTTAACTCGC ACAGTGCAGCTGAGACCACTCTTGAT-3') and 75-R (5'-ATCAAGAGTGGTCTCAGCTGCACTGTGCGA GTTAAGAACAC-3'). After PCR, the methylated original plasmid template was digested with the restriction endonuclease DpnⅠ. The processed PCR product was transformed into *E. coli* DH5α competent cells, where the nicked mutant plasmid was repaired and amplified. Finally, sequencing confirmed the successful introduction of the mutation site. Figure 1 ), thus obtaining an infectious clone of FY-T75AcDNA.

[0046] Example 2: Rescuing EV71 HP-T75A mutant virus through infectious cloning

[0047] In vitro transcription: The 3' end of the FY-T75A cDNA infectious clone was digested with restriction endonuclease HindIII to obtain a linearized DNA template. After purification using a gel extraction purification kit (CW2301M, purchased from CWBIO), the full-length viral genomic RNA was transcribed in vitro using the T7 RNA polymerase in vitro transcription kit MEGAscript High Yield Transcription Kit (Call No. #ON-040, purchased from HONGENE BIOTECH). The RNA was then purified with lithium chloride.

[0048] Cell transfection: Vero cells were cultured in DMEM medium containing 10% FBS. After seeding in six-well plates for 24 hours, the medium was replaced with serum-free DMEM. Transfection was performed using RNAiMAX transfection reagent (catalog number: 13778150, purchased from Thermo Fisher Scientific), with 2 μg of RNA transfected per well. Cytopathic effect (CPE) was observed daily after transfection. Virus-infected cells and culture supernatant were harvested for identification and passage after 7 days of cell rounding and detachment (more than 75%).

[0049] Example 3: RT-PCR identification of the VP1 sequence of the rescued mutant virus strain HP-T75A

[0050] Genomic RNA was extracted from the mutant virus using Trizol LS as a template. The viral genome sequence (containing the coding sequence of the VP1 protein) from positions 2366 to 3401 was amplified by RT-PCR using universal primers EV71-qPCR-F (5'-GCAGCCCAAAAGAACTTCACTATG AAA-3') and EV71-qPCR-R (5'-GAGTGGCAAGATGTCGGTTG-3'). Sequencing analysis identified the rescued mutant virus strain, showing that the mutant virus rescued from the HP-T75A infectious clone contained mutation sites consistent with its infectious clone.

[0051] Example 4: Detection of HP-T75A mutant virus protein expression by Western blotting

[0052] 24 h after Vero cells were seeded into six-well plates, each well was transfected with 2 μg of viral genomic RNA. 48 h after transfection, cells were collected with 1 mL of pre-chilled PBS, centrifuged at 800 rpm for 10 min, the PBS was discarded, and 1×SDS loading buffer was added. The plates were then incubated at 100°C for 30 min before electrophoresis. The expression of viral structural proteins VP1, VP2, and VP0 was detected by Western blotting using specific antibodies. Primary antibodies were anti-EV71 VP1 mouse monoclonal antibody (MAB1255-M08, purchased from Abnova) and anti-EV71 VP0 / 2 mouse monoclonal antibody (MAB979, purchased from Millipore). Secondary antibody was horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG secondary antibody (ZB-2305, purchased from Zhongshan Jinqiao Company). Western blot results showed that specific viral protein expression was detected in cells transfected with both the HP strain and its mutant strain HP-T75A, and the expression levels of VP1, VP2, and VPO proteins in the mutant strain HP-T75A were significantly lower than those in the wild-type strain. Figure 2 ).

[0053] Example 5: Proliferation phenotype of HP-T75A mutant in Vero cells

[0054] Vero cells were transfected with 2 μg of viral genomic RNA in each well of a six-well plate. The progression of cell pathogenesis (CPE) was observed daily. When 90%-100% of the cells showed pathogenesis, the supernatant was collected, thus rescuing the corresponding virus. Cell CPE was recorded under a microscope. Figure 3After transfection of cells with HP strain genomic RNA, viral proliferation and the progression of cytopathic effects (CPE) were significantly faster than those of HP-T75A strain. One day after transfection, both strains produced sporadic cytopathic effects. However, three days after transfection, HP strain produced over 80% CPE, while HP-T75A strain showed no significant progression in CPE. It wasn't until seven days after transfection that HP-T75A strain produced over 80% CPE. Figure 3 This indicates that mutations at the 75th amino acid site of the VP1 protein can significantly affect the viral replication phenotype.

[0055] The infectivity titer of the virus was determined by microtiter. Vero cells were seeded in 96-well plates, with 1 × 10⁶ cells per well. 4 Vero cells were collected. On the second day, after the cells had grown into a monolayer, the virus solution was serially diluted 10-fold with DMEM medium, and 100 μL of the diluted virus solution was inoculated into each well. The CPE progression of cells in each well was observed and recorded daily until day 5 post-inoculation. The TCID of the virus was calculated using the Reed-Muench formula. 50 In addition, HP-T75A mutant and wild-type virus strains were inoculated into Vero cells at high infection doses (MOI = 10) and low infection doses (MOI = 0.1), respectively, and cultured at 37°C. Samples were collected at 0h, 2h, 4h, 8h, 12h, 24h, 36h, 48h, and 72h post-infection with the high infection dose, and at 1d, 2d, and 3d post-infection with the low infection dose. The viral titer in the supernatant was determined by microtiter, and the proliferation kinetics curves of the corresponding viral strains were plotted. The results showed that after inoculation into Vero cells at MOI = 10, the viral titers of both viruses gradually increased with increasing culture time, but the viral titer of the HP-T75A mutant strain remained lower than that of the wild-type virus within 72h post-infection. When both viruses were inoculated into Vero cells at MOI = 1, the viral titer of the HP-T75A mutant strain was significantly lower than that of the wild-type strain, only reaching approximately the same level at 72h post-infection. Figure 4 ).

[0056] Example 6: The VPO precursor protein of the HP-T75A mutant strain has a defect during maturation cleavage.

[0057] When progeny EV71 virus particles are packaged, the precursor proteins VP0, VP1, and VP3 first assemble into a hollow protein capsid. Then, the viral genomic RNA enters the capsid. At the same time, due to the change in protein conformation, the VP0 precursor protein undergoes spontaneous hydrolysis and is cleaved into VP4 and VP2 proteins, thereby producing mature virus particles. Therefore, this cleavage process of VP0 is also called maturation cleavage.

[0058] To investigate whether the T75A mutation affects the maturation and cleavage of EV71, viral particles with different sedimentation coefficients were separated and purified by density gradient centrifugation, and their protein components and RNA were analyzed. The supernatants of EV71-HP and HP-T75A mutant strains were subjected to three freeze-thaw cycles, centrifuged at 3000 rpm for 10 minutes, and the supernatants were then filtered through a 0.22 μm filter to remove cell debris. The collected viral fluid was centrifuged at 37000 rpm for 2.5 h at 4 °C (Beckman SW41Ti) and concentrated using a 15% sucrose buffer. The concentrated viral sample was then centrifuged at 37000 rpm for 70 minutes under a continuous sucrose density gradient of 15%–35% (Beckman SW41Ti) to separate viral particles with different sedimentation coefficients. After centrifugation, samples were taken from the top in 0.5 mL portions. RNA was extracted from half of each sample using Trizol LS, and the copy number of viral genomic RNA was quantified by RT-qPCR. The other half was concentrated by ultrafiltration and then analyzed for viral protein composition using Western blotting. The experimental results showed that ( Figure 5 The RNA-containing viral particles showed two peaks in samples 8–11 (135S) and 13–15 (160S), corresponding to EV71 A particles and mature viral particles, respectively. Notably, in the 160S mature particles, the HP-T75A strain had significantly more VPO precursor protein than the EV71-HP strain, indicating that the mutation at this site can affect the maturation and cleavage of the VPO protein. Furthermore, the HP-T75A strain's 135S viral particles contained significantly less protein than the EV71-HP strain, suggesting that the uncoating process of the HP-T75A strain's viral particles was also affected.

[0059] Example 7: Enhanced virulence of the HP-T75A mutant in BalB / c suckling mice

[0060] 10 animals were injected intraperitoneally (100 μL / animal). 7 lgTCID 50 HP-T75A mutant strain was injected into 2-day-old BALB / c suckling mice (5 mice / group) at a concentration of / mL, with the wild-type strain as a control. The mice were observed for 16 consecutive days, and the degree of hind limb paralysis was recorded daily. Figure 6 A) Weight change and mortality ( Figure 6 (B) After the observation, the spleen, stomach and muscle tissue of the mice were dissected, and the supernatant was ground and used for qPCR to determine the tissue distribution of the virus. Figure 6 The results showed that mice infected with the HP-T75A mutant strain experienced a more severe decrease in body weight and a higher mortality rate than the wild-type strain, indicating that the mutation at amino acid position 75 of the VP1 protein can enhance the virulence of EV71 in mice.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mutant of enterovirus 71 VP1 protein, characterized in that, The amino acid sequence of the VP1 protein mutant is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the enterovirus 71 VP1 protein mutant as described in claim 1.

3. A biomaterial, characterized in that, The biomaterial comprises the nucleic acid molecule of claim 2 or expresses the enterovirus 71 VP1 protein mutant of claim 1; The biomaterial is an expression cassette, vector, or host cell.

4. A mutant strain of enterovirus 71, characterized in that, The enterovirus 71 mutant strain comprises the nucleic acid molecule of claim 2 or expresses the enterovirus 71 VP1 protein mutant of claim 1.

5. The enterovirus 71 mutant strain according to claim 4, characterized in that, The mutant virus strain was obtained by mutating the genome of the wild-type enterovirus 71 strain so that the 75th amino acid of its VP1 protein was mutated to alanine.

6. The use of the enterovirus 71 VP1 protein mutant of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in constructing enterovirus 71 mutant virus strains.

7. The use of the enterovirus 71 VP1 protein mutant of claim 1, the nucleic acid molecule of claim 2, or the biomaterial of claim 3 in reducing the proliferation capacity of enterovirus 71 and / or enhancing the virulence of enterovirus 71; the use is for purposes other than disease diagnosis and treatment.

8. A method for reducing the replication capacity and / or enhancing the virulence of enterovirus 71 for purposes other than disease diagnosis and treatment, characterized in that, The method includes: mutating the genome of enterovirus 71 so that the 75th amino acid encoding the VP1 protein is mutated to alanine; Enterovirus 71 is a wild-type HP strain.

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