A mutant of the L protein of Rift Valley fever virus and its application

Abstract

This invention relates to the field of biotechnology, and more particularly to a mutant of the Rift Valley fever virus L protein and its applications. The amino acid sequence of the mutant is based on the amino acid sequence shown in SEQ ID NO:1, with mutations made at position 1189 (aspartic acid) and / or position 1205 (tryptophan). Through cryo-electron microscopy structural analysis and in vitro mismatch experiments, this invention has confirmed that both identified key amino acid sites are involved in regulating the synthetic fidelity of viral polymerase: mutation at N1189 decreases the synthetic fidelity of the polymerase, while mutation at W1205 increases it. This invention not only provides a new target for the development of attenuated vaccines but also provides crucial evidence for elucidating the molecular regulatory mechanisms of RNA virus fidelity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a mutant of the L protein of Rift Valley fever virus and its applications. Background Technology

[0002] The family Phenuiviridae, an important branch of the order Bunyavirales, includes a variety of zoonotic pathogens of public health significance, such as Rift Valley Fever Virus (RVFV, Latin name Phlebovirus riftense), Dabie bandavirus (DBV), and Toscana Virus (TOSV). Diseases caused by these viruses face significant challenges in clinical treatment. Currently, there are no approved specific vaccines or antiviral drugs, and prevention and control mainly rely on supportive and symptomatic treatment, making effective prevention and precise intervention difficult.

[0003] Taking Rift Valley fever virus as an example, as a typical representative of the genus *Salvinia* in the family Leukopiviridae, its segmented negative-sense RNA genome exhibits a high mutation rate during replication, increasing the complexity of virus control. While existing veterinary vaccines are used for animal disease prevention, they have significant limitations: live attenuated vaccines, although providing long-term immune protection, pose a potential risk of abortion in pregnant animals and virulence reversion; inactivated vaccines, while having higher safety, require multiple vaccinations, have a short duration of immunity, and are costly to produce, making large-scale application difficult in resource-limited areas. Currently, there is no approved human Rift Valley fever virus vaccine, and candidate vaccines in the research and development stage (such as live attenuated vaccines, inactivated vaccines, and virus-like particle vaccines) have not yet entered Phase III clinical trials; simultaneously, specific antiviral drugs against Leukopiviridae virus infections remain unavailable, and clinical needs urgently need to be met.

[0004] In terms of vaccine design strategies, the L protein, as the largest protein encoded by Bunyavirus, contains an RNA-dependent RNA polymerase (RdRP) whose activity is closely related to viral virulence. Studies have shown that by regulating the fidelity of RdRP (both excessively high and low fidelity can lead to viral attenuation), rational viral attenuation can be achieved, providing new insights for vaccine design. However, since the molecular mechanism of RdRP fidelity in negative-sense RNA viruses is not yet fully elucidated, how to accurately identify and regulate key amino acid sites affecting fidelity remains a core issue facing current vaccine research and antiviral nucleoside analogue design. Summary of the Invention

[0005] In view of this, the present invention proposes a mutant of the Rift Valley fever virus L protein and its application.

[0006] The technical solution of this invention is implemented as follows:

[0007] In a first aspect, the present invention provides a mutant of the Rift Valley fever virus L protein, wherein the amino acids of the mutant are subjected to at least one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO: 1:

[0008] (A1) The aspartic acid at position 1189 is mutated to another amino acid;

[0009] (A2) Tryptophan at position 1205 is mutated into other amino acids.

[0010] Furthermore, in some specific embodiments, the aspartic acid at position 1189 is mutated to glycine, leucine, valine, or glutamine (i.e., N1189G, N1189L, N1189V, or N1189Q).

[0011] Furthermore, in some other specific embodiments, the 1205th tryptophan is mutated to alanine, histidine, or tyrosine (i.e., W1205A, W1205H, or W1205Y).

[0012] Secondly, the present invention provides a nucleic acid molecule encoding the mutant.

[0013] Thirdly, the present invention provides a recombinant vector, expression cassette, or host cell containing the nucleic acid molecule.

[0014] Fourthly, the present invention provides the application of the mutant in the preparation of attenuated virus vaccines.

[0015] Fifthly, this invention provides the application of the mutant in the study of viral fidelity regulation mechanisms.

[0016] In a sixth aspect, the present invention provides a method for regulating the synthetic fidelity of Rift Valley fever virus RdRP, the method comprising at least one of the following (B1) and (B2):

[0017] (B1) By mutating the aspartic acid at position 1189 of the Rift Valley fever virus L protein to another amino acid, the synthesis fidelity of RdRP was reduced.

[0018] (B2) By mutating tryptophan at position 1205 of the Rift Valley fever virus L protein to other amino acids, the synthesis fidelity of RdRP is improved.

[0019] The amino acid sequence of the Rift Valley fever virus L protein is shown in SEQ ID NO: 1.

[0020] Furthermore, the aspartic acid at position 1189 of the Rift Valley fever virus L protein is mutated to glycine, leucine, valine, or glutamine.

[0021] Furthermore, the tryptophan at position 1205 of the Rift Valley fever virus L protein is mutated to alanine, histidine, or tyrosine.

[0022] The beneficial effects of the present invention include at least the following:

[0023] Based on cryo-electron microscopy analysis of the Rift Valley fever virus L protein-nucleic acid complex, this invention reveals that the N1189 site is located near the polymerase active site and interacts with the -1 position of the product chain; the W1205 site is located near the -3 position of the product chain, both sites being situated within the product chain channel. Mutation of the Rift Valley fever virus L protein showed that mutations in the N1189 site decrease the polymerase's synthetic fidelity, while mutations in the W1205 site increase it. When these two sites are combined and mutated, the low-fidelity phenotype caused by the N1189 site can be partially reversed by the W1205 mutation; however, compared to the wild type, the combined mutant still exhibits a high-fidelity effect.

[0024] Therefore, this invention proposes to develop an attenuated vaccine for Rift Valley fever virus based on this principle. Both excessively high and low fidelity can weaken viral virulence, demonstrating good versatility. In addition to serving as a target for attenuated vaccines, the fidelity regulatory site also increases drug sensitivity, making it suitable for developing small molecule inhibitors targeting this site. Furthermore, this invention provides crucial evidence for elucidating the molecular regulatory mechanism of RdRP fidelity. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying 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.

[0026] Figure 1 The image shows the cryo-electron microscopy structure of the Rift Valley Fever Virus (RVFV) L protein-nucleic acid complex. In the image, 1A shows the overall structure of the complex, and 1B shows a detailed view of a local part of the complex, showing the template strand, the product strand, and the corresponding positions of the key residues N1189 and W1205.

[0027] Figure 2 A schematic diagram of the partial sequence of the template (T20), primers (P3), and products (P9, P10) used in the de novo RNA synthesis experiment to detect the fidelity of RVFV L protein.

[0028] Figure 3Polyacrylamide gel electrophoresis (PAGE) analysis results of mismatch experiments at different reaction time points for RVFV wild-type (WT) L protein and its representative mutants;

[0029] Figure 4 PAGE analysis results of mismatch experiments at 60 min for RVFV wild-type L protein and its representative mutants;

[0030] Figure 5 The mismatch rate at different reaction time points in the mismatch experiment of wild-type L protein of RVFV and its representative mutants;

[0031] Figure 6 The results of PAGE analysis in the P10 mismatch type identification test;

[0032] Figure 7 The statistical results of the mismatch rate in the P10 mismatch type identification test;

[0033] Figure 8 PAGE analysis results of the experiment showing the effect of the W1205 site mutation on the fidelity of RVFV L protein;

[0034] Figure 9 The results of PAGE analysis at different reaction time points in the mismatch experiment for the RVFV L protein against different mutation combinations shown in Table 2;

[0035] Figure 10 and Figure 11 The images show structural comparisons of the RVFV wild-type L protein-nucleic acid complex and the mutant complex; the left image shows the overall superimposed structure, and the right image is a magnified view of the -1 site. Figure 10 The comparison target was the N1189G single-point mutant. Figure 11 It is a double-point mutant of N1189G-W1205A.

[0036] Additional notes: In the attached figure, P3, P10, P12, P13, P22, etc. represent RNA fragments of different lengths, where the numbers are nucleotide lengths (e.g., P10 is a 10 nt RNA product). Detailed Implementation

[0037] 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. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0038]

[0039] Example 1

[0040] 1. Induction and preparation of Rift Valley fever virus L protein

[0041] Based on the wild-type sequence of the L protein from Rift Valley fever virus strain ZH-501 (GenBank accession number ABD51510.1), to meet the experimental requirements for subsequent structural analysis, we introduced three specific site-directed mutations into the protein, following the methods described in PMID: 34787453 and PMID: 41166549. The D103A mutation aimed to increase the soluble expression level of the protein; the D111A and E125A mutations aimed to inactivate its endonuclease active sites, thereby preventing degradation of the bound nucleic acid substrate during sample preparation and maintaining the structural and functional integrity of the L protein-nucleic acid complex. The resulting L protein, with its amino acid sequence shown in SEQ ID NO: 1, contains the three mutations D103A, D111A, and E125A. This protein was used as a negative control in subsequent polymerase function experiments, also known as the background control (WT group). The three mutation sites were confirmed to not participate in the regulation of polymerase fidelity and are functionally inert sites.

[0042] Based on the amino acid sequence shown in SEQ ID NO:1, the coding sequence was codon-optimized, and the result was inserted into the pPICZB expression vector (which carries a C-terminal 6×histidine tag) using a seamless assembly cloning method to construct the recombinant plasmid pPICZB-RVFV-L-D103A_D111A_E125A. 30 μg of this plasmid was digested with PmeI restriction endonuclease at 37°C for 3–4 h. 10 μL of pre-chilled 3 M sodium acetate (pH 5.2) and 250 μL of anhydrous ethanol were added to the digestion product, mixed well, and incubated at -80°C overnight to precipitate. The precipitate was collected after centrifugation at 4°C, washed twice with 75% ethanol, and dissolved in 20 μL of sterile water to obtain linearized DNA.

[0043] Take 40 μL of SMD1163 competent cells, mix with 10 μL of linearized DNA, and transfer to a pre-chilled electroporation cuvette for electroporation. Then transfer the electroporation buffer to a test tube containing 500 μL of antibiotic-free YPD medium and incubate at 30°C for 3-5 h.

[0044] Spread 10 μL of the revived bacterial culture onto a YPD solid plate containing Zeocin antibiotic and incubate at 30°C until single colonies appear. Pick a single colony and inoculate it into 10 mL of YPD liquid medium containing 100 μg / mL Zeocin. Incubate at 30°C and 220 rpm with shaking for about 20 h to obtain the seed culture. Inoculate the seed culture at 1% into 1 L of BMGY medium and incubate at 30°C and 220 rpm for about 24 h. Centrifuge at 4000 rpm for 15 min to collect the cells, resuspend them in BMMY medium, and transfer them to 1 L of BMMY medium for induction expression. Induce culture at 27°C and 220 rpm for 48 h, adding methanol once during the induction period. Centrifuge to collect the precipitate, and resuspend each 1 L of precipitate in 30 mL of lysis buffer (50 mM Tris pH 8.5, 20 mM imidazole, 1 mM benzidine hydrochloride, 500 mM NaCl, 5% v / v glycerol) for later use.

[0045] 2. Protein purification

[0046] The high-pressure crushed bacterial culture was centrifuged at 34, 220×g for 1 h, and the supernatant was collected for further purification.

[0047] 2.1 Nickel ion affinity chromatography

[0048] Use lysis buffer and elution buffer (50 mM Tris pH 8.5, 500 mM imidazole, 1 mM benzidine hydrochloride, 500 mM NaCl, 5% v / v glycerol). First, transfer the supernatant sample after centrifugation to a nickel column at a flow rate of 1 mL / min. Then wash with lysis buffer for 5-10 column volumes, followed by washing with lysis buffer containing 94% v / v lysis buffer and 6% elution buffer for 5 column volumes. Finally, elute with lysis buffer containing 84% v / v lysis buffer and 16% elution buffer and collect the corresponding protein elution peaks.

[0049] 2.2 Heparin affinity chromatography

[0050] Low-salt buffer (20 mM Tris pH 8.5, 2 mM dithiothreitol) and high-salt buffer (1 M NaCl, 20 mM Tris pH 8.5, 2 mM dithiothreitol) were used. The nickel column eluent was mixed with an equal volume of low-salt buffer and loaded onto a heparin column at a flow rate of 1 mL / min. The column was then washed for 5 column volumes with 75% low-salt buffer and 25% high-salt buffer to remove unbound proteins. The NaCl concentration was then gradually increased using linear elution to elute and collect the target protein peak.

[0051] 2.3 Gel Filtration Chromatography

[0052] A Superose 6 10 / 300 GL column and GF buffer (20 mM Tris pH 8.5, 500 mM NaCl) were used. The L protein purified by the heparin column was concentrated to about 0.5 mL and passed through a gel filtration chromatography column at a flow rate of 0.3 mL / min. The corresponding protein elution peak was collected, and TCEP (tris(2-carboxyethyl)phosphine) was added to a final concentration of 5 mM.

[0053] 2.4 Protein Concentration and Quantification

[0054] The RVFV L protein, purified in three steps, was concentrated to approximately 28 mg / mL. After diluting 100-fold with 6 M guanidine hydrochloride, its absorbance at 280 nm and 320 nm was measured using a spectrophotometer. The absorbance coefficient of the RVFV L protein was 1.081, and its mass concentration was (A... 280 -A 320 The molar concentration is calculated by multiplying the dilution factor by the absorbance coefficient and then calculating the molar concentration based on the molecular weight of 237913.79 Da.

[0055] 3. Obtaining and preparing RVFV L protein-nucleic acid complexes using cryo-electron microscopy

[0056] 3.1 In vitro assembly of the complex

[0057] First, a functional L protein-nucleic acid complex was assembled in vitro. This complex consisted of: RVFV L protein, promoter RNA (5' vRNA, sequence shown in Table 1), and template RNA (T20, sequence shown in Table 1) with the trinucleotide primer pGGA (P3). The specific assembly steps were as follows: Template RNA T20 and primer P3 were mixed at a molar ratio of 1:5 and incubated at 45°C for 3 min, then slowly cooled to room temperature. Subsequently, in a 20 μL reaction system, 6 μM of 5' vRNA, 4 μM of T20 / P3 double strands, 6 μM of RVFV L protein, 300 μM of ATP, 300 μM of UTP, 50 mM Tris-HCl (pH 7.0), 2 mM MgCl2, and 5 mM TCEP were added to a final concentration. The reaction system was incubated at 20°C for 30 min. After the reaction, the precipitate was removed by centrifugation, and the supernatant was used for subsequent cryo-electron microscopy sample preparation.

[0058] 3.2 Cryo-electron microscopy sample preparation, data collection, and structural analysis

[0059] The three-dimensional structure of the above-mentioned complex was determined using cryo-electron microscopy. Sample preparation was performed using a Vitrobot Mk IV (Thermo Fisher Scientific) cryo-electron microscope. First, a Quantifoil R1.2 / 1.3 copper mesh (200 mesh) was subjected to glow discharge. The instrument parameters were set as follows: adsorption time 2 seconds, waiting time 10 seconds, and adsorption force 8. 3.5 μL of the clarified complex sample was dropped onto the hydrophilic surface of the treated copper mesh. After removing excess liquid by adsorption with filter paper, the mesh was quickly immersed in liquid ethane pre-cooled to below -180°C with liquid nitrogen.

[0060] Frozen samples were acquired using a 300 kV JEOL CryoARM 300 electron microscope equipped with a K3 Summit direct electron detector (Gatan). Images were automatically acquired at 50,000x magnification using SerialEM software, corresponding to a pixel size of 0.475 Å. The underfocus value was set to vary from -0.5 μm to -2.5 μm, and the total cumulative electron dose was 40 eE. - / Å 2 A total of approximately 4,000-6,000 microscopic images were collected.

[0061] The acquired raw images (.mrc format) were imported into CryoSPARC software suite for data processing. After steps including film motion correction, contrast transfer function (CTF) estimation, grain selection, 2D classification, initial 3D model construction, and non-uniform refinement, a high-resolution 3D electron density map was finally obtained. This density map was initially fitted to a homologous model in UCSF Chimera, followed by manual adjustments and optimizations in Coot. Finally, Phenix software was used for multiple rounds of real-time spatial refinement to ensure that all validation metrics of the model met the standards for cryo-electron microscopy structure resolution, thus obtaining the final PDB file.

[0062] 4. Results

[0063] The cryo-electron microscopy structure of the RVFV L protein-nucleic acid complex is shown below. Figure 1 As shown, analysis of the upstream channels of the RVFV product chain reveals that residue N1189 is located near the polymerase active site, at the top of the motif E loop, and interacts with the -1 position of the product chain. Residue W1205 is located on the helix adjacent to motif E, near the -3 position of the product chain, and the amino acid side chain of residue W1205 has a hydrophobic interaction with the -3 position of the product chain.

[0064] Example 2

[0065] 1. Preparation of Rift Valley Fever Virus L Protein Mutant

[0066] Based on the analysis results of Example 1, the following mutations were designed targeting the N1189 and W1205 residues:

[0067]

[0068] We also selected K964, which is spatially close to N1189, and W995, which is located in the active site, as well as R1204 and A1208, which are close to W1205; the following mutations were designed for these residues as controls:

[0069]

[0070] The mutant of RVFV L protein was obtained using the QuickChange method (Vandeyar, MA, Weiner, MP, Hutton, CJ & Batt, CA A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene 65, 129-133 (1988).).

[0071] 2. Fidelity was determined using a de novo RNA synthesis experiment.

[0072] 2.1 Methods

[0073] The fidelity of RVFV L protein and its mutants was determined using a de novo RNA synthesis experiment. A schematic diagram of the assay is shown below. Figure 2 , Figure 2 The mismatch experimental system shown includes the sequences of a template (T20), primers (P3), products (P9, P10), and NTPs (ATP and UTPs). This system, through the design of mismatched primer-template complexes and the provision of restriction NTPs, compares the differences in the recognition and processing of terminal mismatches by polymerases (i.e., L proteins). The specific method steps are as follows:

[0074] The main reaction system setup for the de novo RNA synthesis experiment is exactly the same as the "3.1 In vitro assembly of the complex" step in Example 1. First, the reaction system was assembled according to the established plan and the reaction was started. Unlike the single-point detection in Example 1, this experiment used a stop reaction solution (95% [v / v] formamide, 20 mM EDTA [pH 8.0], 0.02% [w / v] bromophenol blue, 0.02% [w / v] xylene blue) to stop the reaction after sampling at different time points. Then, the samples were heated at 100℃ for 1 min, followed by polyacrylamide gel electrophoresis and staining with Stains-All (purchased from Sigma-Aldrich). The treatment method was the same as that in the published literature (Wu, J., Lu, G., Zhang, B. & Gong, P. Perturbation in the conserved methyltransferase-polymerase interface of flavivirus NS5 differentially affects polymerase initiation and elongation. Journal of Virology 89, 249-261, doi:10.1128 / JVI.02085-14). Consistent with (2015).

[0075] Catalyzed by RVFV L protein or its mutants, primer P3 can extend by 7 nucleotides to generate product P9 with a length of 9 nucleotides. In addition to P9, there is also a mismatch product (P10) with a mismatch at the 10th nucleotide position. The experiment was repeated 3 times, and the gel electrophoresis results were analyzed by grayscale quantification using ImageJ software. The mismatch rate of RVFV L protein was calculated by the following formula: mismatch rate = [P10] / ([P9] + [P10]).

[0076] 2.2 Results

[0077] In mismatch experiments, the products of wild-type L protein and its different mutants at different reaction time points were analyzed by polyacrylamide gel electrophoresis, and the results are as follows: Figure 3 and Figure 4 As shown.

[0078] The results of mismatch rates at different reaction times in the mismatch experiment for wild-type L protein and its different mutants are as follows: Figure 5 As shown.

[0079] Figure 3 and Figure 4The results showed that the single-point mutants (W995H, W995F, K964R, K964M, R1204K, R1204A, A1208N, A1208S, A1208T, and A1208G) constructed targeting the four sites W995, K964, R1204, and A1208 were completely consistent with the WT group, and no mismatch products were detected in this experimental system. However, mismatch products were consistently detected in the N1189G, N1189L, and N1189V mutants. Therefore, mutations at the W995, K964, R1204, and A1208 sites have no significant regulatory effect on polymerase fidelity, while mutations at the N1189 site are involved in the regulation of polymerase fidelity.

[0080] Figure 5 The results showed that the mismatch rate of the W1205A mutant was not significantly different from that of the WT mutant, while the mismatch rates of the N1189G, N1189L, and N1189V mutants were all higher than those of the WT mutant to varying degrees. However, the mismatch rate of the W1205A-N1189G double-point mutant returned to a level not significantly different from that of the WT mutant. These results indicate that mutations at both the N1189 and W1205 sites are involved in the regulation of polymerase fidelity.

[0081] 3. Identification of P10 mismatch types

[0082] By using single-factor variables, the concentrations of ATP or UTP were varied to observe their differential effects on the yields of correctly paired and mismatched products, thereby identifying the type of base required for mismatch. The specific steps are as follows:

[0083] Two different NTP concentration combinations were used to conduct mismatch incorporation experiments: In the first group, the reaction system had a fixed UTP concentration of 300 μM, combined with ATP at gradient concentrations of 50 μM, 100 μM, 300 μM, and 500 μM; in the second group, the reaction system had a fixed ATP concentration of 300 μM, combined with UTP at gradient concentrations of 50 μM, 100 μM, 300 μM, and 500 μM. Samples were taken for electrophoresis at 60 min and 120 min after the polymerase (RVFV L protein N1189G mutant) reaction in both groups. The experiments were repeated three times. ImageJ software was used to perform grayscale quantitative analysis of the gel electrophoresis results to calculate the mismatch rate. The results are shown below. Figure 6 , Figure 7 As shown.

[0084] Statistical results showed that changing the ATP concentration had no significant effect on the mismatch rate (P > 0.05), while changing the UTP concentration significantly altered the mismatch rate (P < 0.05). Therefore, the mismatch product originates from the mismatch incorporation of UTP, i.e., the mismatch type of P10 is G:U mismatch.

[0085] 4. The effect of W1205 site mutation on RNA polymerase fidelity

[0086] Single-point mutants (W1205Y, W1205H, and W1205A) of the Rift Valley fever virus L protein at the W1205 site were prepared according to the aforementioned method. The elongation ability of the single-point mutants W1205Y, W1205H, W1205A, and the double-point mutant W1205A-N1189G for erroneous substrates was then tested, using the following specific methods:

[0087] A 33 nt RNA template (T33, sequence shown in SEQ ID NO:6) was hybridized with primer pGGA (P3) to form a primer-template complex. At 20°C, using ATP and UTP as substrates, the primer was extended from P3 to P10. After 0.5 h of reaction, unincorporated free NTPs were completely removed by ultrafiltration to obtain the T33 / P10 extension complex. Starting with the T33 / P10 complex, the extension reaction was carried out at 25°C. The reaction system contained only GTP and the incorrect substrate probe 4'-fluorouridine triphosphate (4'-FIU-TP, used to replace the substrate UTP, which is structurally similar to but different from UTP and can be "mistakenly" recognized and incorporated by polymerases. High-fidelity polymerases will reject or slow incorporation due to its structural abnormality, causing the extension to "pause"; while low-fidelity polymerases are more likely to incorporate it and continue the extension). Sampling was performed at time gradients of 0, 2, 5, 10, 30, and 60 min. A parallel control group containing only the correct substrate, GTP and UTP, was set up. All extension reaction products were analyzed by gel electrophoresis (P22 is the full-length extension product).

[0088] The results are as follows Figure 8 As shown, under the conditions of providing GTP and the erroneous substrate 4'-FIU-TP, different polymerase mutants exhibited significant differences: compared with the WT group, the W1205Y, W1205H, and W1205A mutants showed more pronounced chain pausing after incorporation of the erroneous substrate, and their efficiency in extending to the full-length product P22 was also lower; among them, the pausing effect of W1205A was the most significant. This indicates that the mutation at the W1205 site has the effect of improving polymerase fidelity. Meanwhile, the double-site mutant W1205A-N1189G reversed the above-mentioned chain pausing phenomenon, indicating that under the high-fidelity background of W1205A, the introduction of the known low-fidelity site N1189G mutation can offset its effect and restore the ability to extend to the erroneous substrate to a certain extent. However, compared with the wild type, the combined mutant still exhibited a high-fidelity effect.

[0089] 5. The impact of other different combinations of mutations on mismatches

[0090] Table 2. Five two-point mutants used to investigate the co-regulatory effects of N1189 and W1205 sites.

[0091]

[0092] To further investigate the co-regulatory effect of the N1189 and W1205 sites on fidelity, we constructed different combinations of two-point mutants, including N1189G-W1205A, N1189G-W1205Y, N1189G-W1205H, N1189Q-W1205H, and N1189V-W1205A. Mismatch experiments were conducted using the aforementioned method.

[0093] The results are as follows Figure 9 As shown, using wild-type (WT) as a control, when the W1205 site is mutated to a tyrosine residue (Y) with a large side chain, a mismatch extension product of approximately 10 nt can still be detected when combined with the N1189 mutant. However, when the W1205 site is mutated to a small side chain amino acid (alanine A, histidine H) and combined with different N1189 mutants, the corresponding double-point mutants do not produce a visible 10 nt mismatch product. These results indicate that the high-fidelity mutation of W1205 can effectively reverse the mismatch extension effect caused by the N1189 series of low-fidelity mutations.

[0094] Example 3

[0095] Using the aforementioned method, cryo-electron microscopy samples of the representative N1189G mutant and N1189G-W1205A double-point mutant of L protein were prepared, and then each mutant model was compared with the wild-type model in detail by superposition.

[0096] The results are as follows Figure 10 and Figure 11 As shown, the N1189G mutation causes the -1 nucleotide of the product chain to shift upward by about 1.3 angstroms, while the W1205A mutation affects the swing of the entire product chain, thereby restoring the -1 nucleotide shift caused by the N1189G mutation to a position similar to that of the wild type, about 0.3 angstroms away from the wild type.

[0097] These results indicate that when the amino acid at the W1205 site is mutated to the small side chain W1205A, combined mutations at these two sites can regulate each other. The low-fidelity mutant phenotype of N1189 can be reversed by the W1205A mutant, which suggests that the effect of W1205A on polymerase fidelity is the opposite of that of the low-fidelity mutant of N1189.

[0098] In summary, the results of this study indicate that in the Rift Valley fever virus L protein, two key functional residues, N1189 and W1205, located in the upstream channel of the product chain, form a regulatory module. N1189 and W1205 residues jointly determine the spatial positioning of key sites in the product chain, directly regulating the fidelity of RdRP synthesis. Mutations in N1189 decrease the polymerase synthesis fidelity, while mutations in W1205 increase it. Furthermore, mutations in W1205 residues can compensate for the positioning deviations caused by N1189 mutations by regulating the overall conformational dynamics of the product chain. This interaction maintains the conformational homeostasis of RdRP, ensuring the inherent fidelity of nucleotide incorporation during viral RNA replication.

[0099] Example 4

[0100] Amino acid sequences of the L proteins from representative viral strains of 23 genera within the Leukoviridae family were obtained from the UniProt database. Multiple sequence alignment was performed using Mega software. Using the N1189 and W1205 sites and their surrounding conserved domains of the Rift Valley fever virus L protein as references, corresponding equivalent sites in the L proteins of the remaining 22 viral strains were identified and recorded through alignment; these were amino acid residues located in the same or highly similar spatial positions and functional domains (Table 3).

[0101]

[0102]

[0103] Given the high conservation of the N1189 and W1205 sites within the Leukoviridae family, and their proven mechanism of action in jointly regulating replication fidelity in RVFV, it is reasonable to infer that this molecular mechanism, in which two key residues dynamically regulate polymerase fidelity through interaction, is a common and conserved inherent regulatory pattern among viruses in this family. Based on this, attenuated vaccines and small-molecule inhibitors targeting these sites can be developed to fill the gaps in vaccines and antiviral drugs for viruses in this family, reversing the widespread situation of "no vaccines, no specific drugs" for these viruses.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mutant of the L protein of Phlebovirus riftense, characterized in that, The amino acid sequence of the mutant is formed by at least one of the following amino acid mutations in the amino acid sequence shown in SEQ ID NO: 1: (A1) The aspartic acid at position 1189 is mutated to another amino acid; (A2) Tryptophan at position 1205 is mutated into other amino acids.

2. The mutant as described in claim 1, characterized in that, The aspartic acid at position 1189 is mutated to glycine, leucine, valine, or glutamine.

3. The mutant as described in claim 1, characterized in that, The 1205th tryptophan is mutated to alanine, histidine, or tyrosine.

4. A nucleic acid molecule encoding the mutant as described in claim 1.

5. A recombinant vector, expression cassette, or host cell containing the nucleic acid molecule as described in claim 4.

6. The use of the mutant as described in claim 1 in the preparation of an attenuated virus vaccine.

7. The application of the mutant as described in claim 1 in the study of viral fidelity regulation mechanisms.

8. A method for regulating the synthetic fidelity of Rift Valley fever virus RdRP, characterized in that, The method includes at least one of the following: (B1) and (B2): (B1) By mutating the aspartic acid at position 1189 of the Rift Valley fever virus L protein to another amino acid, the synthesis fidelity of RdRP was reduced. (B2) By mutating tryptophan at position 1205 of the Rift Valley fever virus L protein to other amino acids, the synthesis fidelity of RdRP is improved. The amino acid sequence of the Rift Valley fever virus L protein is shown in SEQ ID NO:

1.

9. The method as described in claim 8, characterized in that, The aspartic acid at position 1189 of the Rift Valley fever virus L protein is mutated to glycine, leucine, valine, or glutamine.

10. The method as described in claim 8, characterized in that, The 1205th tryptophan in the Rift Valley fever virus L protein is mutated to alanine, histidine, or tyrosine.

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