Drug target of plant bunyavirus as well as RNA replicase protein and application thereof

Abstract

The invention relates to a drug target of a plant bunyavirus as well as an RNA replicase protein and application thereof, the RNA replicase protein of the plant bunyavirus is a core protease responsible for virus genome replication, a recognition region and an enzyme activity center of the RNA replicase protein for the virus genome are important functional domains, and the RNA replicase protein can be used as a receptor site for designing a targeted drug and inhibiting virus replication. The invention provides a key site for interaction of tomato spotted wilt virus RNA replicase protein and viral genome RNA, key site information of an enzyme activity center, a double-target action site of a small molecule compound ribavirin 5 '-triphosphate inhibiting RNA replicase, and an RNA replicase protein mutant. According to the structural information, functional sites and drug targets of the tomato spotted wilf virus RNA replicase provided by the invention, targeted selective antiviral drugs can be developed or drug materials can be screened and prepared, and the tomato spotted wilf virus RNA replicase has efficient and safe application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and particularly relates to a drug target of a plant bunyavirus, its RNA replicase protein, and applications thereof. Background Art

[0002] Tomato spotted wilt virus (TSWV) belongs to the genus Orthotospovirus, family Tospoviridae, order Bunyavirales, and is one of the top ten most harmful plant viruses worldwide. TSWV is a segmented negative-strand RNA virus with a wide host range, infecting more than 1,000 plant species in over 80 families, including important cash crops such as tomatoes, peppers, and tobacco. Infected plants exhibit symptoms such as leaf yellowing and wrinkling, plant dwarfing, and concentric ring spots on fruits, causing huge losses to agricultural production.

[0003] The RNA replicase protein encoded by the virus plays a central role in the replication of the viral genomic RNA and is highly conserved among RNA viruses. Therefore, the viral RNA replicase protein is an ideal antiviral drug target, and its structural biology research has always been a focus and difficulty in the field of virology. For a long time, there has been a lack of structural information on the full-length replicase protein of plant RNA viruses, and the ideal drug target sites are unclear. Currently, virtual screening and design of targeted drugs based on the three-dimensional structure of target proteins and artificial intelligence have developed rapidly. Identifying key functional sites based on the three-dimensional structure information of high-resolution drug target proteins is particularly important for further providing ideal drug targets. Summary of the Invention

[0004] Technical problems to be solved: The present invention provides a drug target of a plant bunyavirus, its RNA replicase protein, and applications thereof. Through the high-resolution three-dimensional structure information of the tomato spotted wilt virus RNA replicase protein, key active sites are further mined based on the structural information, and it is confirmed that Y455, K467, T468, T653, K656, D775, R823, P824, Q825, R826, L827, R831, T895, K896, L897, K1008, V1014, H1015, T1018, K1191, E1432 are the key functional sites of the tomato spotted wilt virus RNA replicase protein. Using these as targets to screen inhibitors, it is found that the small molecule compound ribavirin triphosphate can effectively inhibit the tomato spotted wilt virus.

[0005] Technical solution: The tomato spotted wilt virus RNA replicase protein has an amino acid sequence as shown in SEQ ID NO:1.

[0006] Use of the above-mentioned tomato spotted wilt virus RNA replicase protein in screening anti-plant bunyavirus drugs.

[0007] Use of the above-mentioned tomato spotted wilt virus RNA replicase protein in the preparation of a kit for screening anti-plant bunyavirus drugs.

[0008] Use of the above-mentioned tomato spotted wilt virus RNA replicase protein, with its Y455, K467, T468, T653, K656, D775, R823, P824, Q825, R826, L827, R831, T895, K896, L897, K1008, V1014, H1015, T1018, K1191, E1432 sites as drug targets, in the screening of anti-plant bunyavirus drugs.

[0009] Use of ribavirin triphosphate in the preparation of anti-plant bunyavirus drugs by inhibiting the above-mentioned tomato spotted wilt virus RNA replicase protein.

[0010] An anti-plant bunyavirus drug, the active ingredient of which includes ribavirin triphosphate.

[0011] Use of the above-mentioned tomato spotted wilt virus RNA replicase protein in the preparation of a reagent for diagnosing plant bunyavirus infection.

[0012] A kit for diagnosing plant bunyavirus infection, the kit comprising an antibody or nucleic acid probe capable of specifically recognizing the above-mentioned tomato spotted wilt virus RNA replicase protein.

[0013] A method for improving the anti-bunyavirus infection ability of plants, the method comprising introducing a specific fragment or variant of the above-mentioned tomato spotted wilt virus RNA replicase protein into plant cells to induce plants to produce resistance against bunyaviruses.

[0014] Use of the above-mentioned tomato spotted wilt virus RNA replicase protein in the cultivation of transgenic plant varieties resistant to bunyavirus infection.

[0015] Beneficial effects: The beneficial effects of the present invention are as follows: A tomato spotted wilt virus RNA replicase protein with a specific amino acid sequence is provided, which not only provides an accurate target for the screening of anti-plant bunyavirus drugs, but also promotes the development of a kit for screening anti-plant bunyavirus drugs, greatly improving the efficiency and accuracy of drug screening; at the same time, multiple drug target sites on this replicase protein are identified, providing a more specific direction for drug design; in addition, through the inhibitory effect of compounds such as ribavirin triphosphate, the practical application potential of this protein in the preparation of anti-plant bunyavirus drugs is demonstrated, providing strong support for the development of new antiviral drugs; moreover, the invention also expands the application of the tomato spotted wilt virus RNA replicase protein in diagnostic reagents, plant genetic engineering, and the cultivation of transgenic plant varieties resistant to bunyavirus infection, opening up new ways for the prevention and control of plant diseases, and having significant social and economic benefits and application prospects. Description of the Drawings

[0016] Figure 1 It is the three-dimensional structural information of the tomato spotted wilt virus RNA replicase protein.

[0017] Figure 2 It is the key functional sites of the tomato spotted wilt virus RNA replicase protein.

[0018] Figure 3 It is the newly identified conserved motif I located in the enzyme active center of the tomato spotted wilt virus RNA replicase protein.

[0019] Figure 4 It is the dual-targeting sites of ribavirin 5'-triphosphate inhibiting the tomato spotted wilt virus RNA replicase protein.

[0020] Figure 5 It is the control effect of ribavirin 5'-triphosphate on tomato spotted wilt virus diseases. Detailed Implementation Modes

[0021] Example 1: Analyzing the three-dimensional structural information of the tomato spotted wilt virus RNA replicase protein by cryo-electron microscopy technology

[0022] 1) Express and purify to obtain the tomato spotted wilt virus RNA replicase protein, and the specific operation process is as follows:

[0023] The tomato spotted wilt virus RNA replicase gene was cloned into the pFastBac expression plasmid with an N-terminal fusion of the 10×His-msyB tag. Recombinant baculovirus was prepared using SF9 insect cells (11496015, Invitrogen) and protein expression was carried out in High Five cells (B85502, Invitrogen) via the Bac-to-Bac expression system. At 60 hours post-infection of the recombinant baculovirus, the culture was centrifuged to pellet the cells, which were then resuspended in pre-chilled lysis buffer (50 mM Tris [pH 8.0], 200 mM NaCl, 10 mM imidazole) and lysed by sonication. After centrifugation at 38,000 rpm for 1 hour at 4 °C, the supernatant was loaded onto a nickel affinity column (GE Healthcare, USA) pre-equilibrated with lysis buffer. The resin was washed three times with 120 ml of lysis buffer and the bound protein was eluted with elution buffer containing imidazole (50 mM Tris [pH 8.0], 200 mM NaCl, 300 mM imidazole), and then captured onto a 5-ml Q column (GE Healthcare, USA). The protein bound to the column was eluted with a linear NaCl concentration gradient, and the elution fractions that peaked at a NaCl concentration of 380 mM were pooled. Subsequently, this fraction was loaded onto a Superose 6-10 / 300GL column (GE Healthcare, USA) and eluted with 50 mM Tris [pH 8.0], 200 mM NaCl and 1 mM Tris[2-carboxyethyl]phosphine [TCEP] buffer. The samples in the single peak were concentrated, snap-frozen and stored in a -80 °C ultra-low temperature freezer.

[0024] 2) Preparation and data collection of cryo-EM samples, and the specific operation procedures are as follows:

[0025] The purified tomato spotted wilt virus RNA replicase protein was concentrated to 1.2 mg / mL for sample preparation. The protein was incubated with (i) 5' vRNA (1 - 17 nts, 5'-pAGAGCAAUCAGGUACAA-3'OH) and 3' vRNA (1 - 17 nts, 5'-pUGUACCUGAUGCUCU-3'OH), (ii) 5' vRNA (1 - 10 nts, 5'-pAGAGCAAUCA-3'OH), (iii) ribavirin, or (iv) ribavirin 5'-triphosphate at a 1:1.5 molar ratio for 5 minutes on ice. A glow discharge was performed on a gold grid (Cflat R1.2 / 1.3, 300 mesh) covered with a carbon film using a Solarus 950 plasma cleaner (Gatan) for 30 seconds. 3 μl of the protein sample solution was dropped onto the grid, and cryo-EM samples were prepared using a Vitrobot Mark IV (FEI) device. The tomato spotted wilt virus RNA replicase protein and its complexes with different substrates were imaged using a Titan Krios microscope (Thermo Fisher Scientific) equipped with a K2 detector (Gatan, Pleasanton, CA) at 300 kV, and micrographs were collected using SerialEM automated data collection software. Images (32 frames, every 0.2 seconds, total dose ) were recorded at a final pixel size of , with the defocus between -1.5 and -2.0 μm.

[0026] 3) Image processing, the specific operation process is as follows:

[0027] Almost the same image processing strategy was adopted for all datasets. First, the raw data was processed with MotionCor2, and then aligned and averaged into a motion-corrected sum image. Then, Gctf was used to determine the defocus value of each micrograph. Higher-quality micrographs were selected for further processing. Particles were picked and extracted for 2D calibration, and well-defined particles were selected from them to reconstruct an initial model. The initial model was used as a reference for 3D classification. After refinement and post-processing, according to the gold standard Fourier shell correlation (threshold = 0.143), the overall resolutions of the apo state of the tomato spotted wilt virus RNA replicase protein, the replicase protein - 3'-5' vRNA, the replicase protein - 5' vRNA, the replicase protein - ribavirin, and the replicase protein - ribavirin triphosphate complex reached and The quality of the local resolution was evaluated by ResMap. Structural information of the C-terminal domain (CTD) of the replicase protein was observed in the replicase protein - 5' vRNA dataset. After refinement and post-processing, the overall resolution of the replicase protein containing the CTD reached

[0028] 4) Establishment and improvement of the model, and the specific operation process is as follows:

[0029] The initial structural model was established using the AutoBuild program in Phenix and further manually corrected through the real-space refinement program in COOT. The AlphaFold model containing the CTD region was used to guide the model construction of the CTD part in COOT (in these regions, although there is map density, its quality is too low to build an atomic model structure).

[0030] Example 2: Identification of key functional sites of the tomato spotted wilt virus RNA replicase protein

[0031] The key amino acid sites for the replicase protein to recognize the key elements (nucleotides 1-10 at the 5' end) of the viral genome were obtained through analysis with Pymol and Chimera X software, including: Y455, K467, T468, T653, K656, D775, R823, P824, Q825, R826, L827, R831, T895, K896, L897, K1008, V1014, H1015, T1018, K1191, E1432. By introducing the above amino acid sites into the tomato spotted wilt virus reverse genetic permission system for site-directed mutagenesis one by one, it was found that the above sites are very important for rescuing the virus, indicating that the identified sites can be used as ideal drug targets.

[0032] Example 3: Discovery of new enzymatic activity sites of the tomato spotted wilt virus RNA replicase protein

[0033] Interaction site analysis was performed using Pymol and Chimera X software, and a new key site K1008 in the enzymatic active center of the tomato spotted wilt virus RNA replicase was discovered. Further, multiple sequence alignment analysis of amino acid sequences and three-dimensional structure spatial alignment analysis were performed through ESPript 3.0 / ENDscript, and it was found that the K1008 site is highly conserved in bunyavirus RNA replicases. By introducing the above amino acid sites into the tomato spotted wilt virus reverse genetic permission system for site-directed mutagenesis one by one, it was found that the above sites are very important for rescuing the virus, indicating that the identified sites can be used as ideal drug targets.

[0034] Example 4: Revealing the dual drug target sites of the tomato spotted wilt virus RNA replicase protein

[0035] Using the region where the above-mentioned site is located as the target, molecular docking of nucleoside small molecule compounds was carried out using AutoDock Vina software, and it was found that ribavirin 5'-triphosphate had a good binding effect. Further, by analyzing the cryo-electron microscopy structure of the replicase protein and ribavirin triphosphate complex, it was found that ribavirin had two binding sites on the replicase protein: 1) the entry channel of the 5' vRNA hook-like structure binding cavity; 2) the polymerase active center. The key susceptibility sites were: M777, T779, Y908, K1191, K1370, Q1455, N1538, K1540, K1541.

[0036] Example 5: Discovery of the inhibitory effect of ribavirin 5'-triphosphate on Tomato spotted wilt virus

[0037] One hour after rubbing and inoculating Tomato spotted wilt virus on Nicotiana benthamiana leaves, ribavirin 5'-triphosphate (100 μg / ml) was evenly applied to the inoculation site using a writing brush. After 14 days, no typical symptoms of virus infection were observed on the tobacco, and no viral proteins were detected in the systemic leaves of the tobacco. In contrast, the tobacco that was not treated with ribavirin 5'-triphosphate after virus inoculation showed typical symptoms after virus infection 14 days later, and viral proteins were detected in the systemic leaves of the tobacco. These data indicate that ribavirin 5'-triphosphate can effectively inhibit the infection of Tomato spotted wilt virus.

[0038] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Tomato spotted wilt virus RNA replicase protein, characterized in that The amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the tomato spotted wilt virus RNA replicase protein according to claim 1 in screening anti-plant bunyavirus drugs.

3. Use of the tomato spotted wilt virus RNA replicase protein according to claim 1 in preparing a kit for screening anti-plant bunyavirus drugs.

4. The tomato spotted wilt virus RNA replicase protein according to claim 1, wherein Y455, K467, ​​T468, T653, K656, D775, R823, P824, Q825, R826, L827, R831, T895, K896, L897, K1008, V1014, H1015, T1018, K1191, and E1432 sites thereof are used as drug targets in screening for drugs against plant bunyaviruses.

5. Use of ribavirin triphosphate in the preparation of anti-plant bunyavirus drugs by inhibiting the RNA replicase protein of tomato spotted wilt virus according to claim 1.

6. An anti-plant bunyavirus drug, characterized in that: The active ingredient includes ribavirin triphosphate.

7. Use of the tomato spotted wilt virus RNA replicase protein according to claim 1 in the preparation of a reagent for diagnosing plant bunyavirus infection.

8. A kit for diagnosing plant bunyavirus infection, characterized in that: The kit comprises an antibody or a nucleic acid probe capable of specifically recognizing the RNA replicase protein of the tomato spotted wilt virus according to claim 1.

9. A method for improving the resistance of plants to Bunyavirus infection, characterized in that: The method comprises introducing the specific fragment or variant of the RNA replicase protein of tomato spotted wilt virus according to claim 1 into plant cells to induce the plant to produce resistance to Bunyavirus.

10. Use of the tomato spotted wilt virus RNA replicase protein according to claim 1 in cultivating transgenic plant varieties resistant to bunyavirus infection.

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