A method of inhibiting infection by a tombusvirus and use of tcp1 inhibitors

Abstract

This invention discloses a method for inhibiting Tamdy virus infection and the use of TCP1 inhibitors. Specifically, this invention discloses a method for inhibiting Tamdy virus (TAMV) infection of host cells, the method comprising the step of reducing or inhibiting the expression level and / or activity of the TCP1 gene or its encoded protein in host cells, wherein the reduction or inhibition of the expression level and / or activity of the TCP1 gene or its encoded protein is achieved by methods selected from the group consisting of: gene mutation, gene knockout, gene interruption, RNA interference technology, gene editing technology, introduction of inhibitors of genes or proteins, or combinations thereof.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for inhibiting Tammd virus infection and the use of TCP1 inhibitors. Background Technology

[0002] Tamdy virus (TAMV) was first isolated from the Asian tick *Hygroscopic tick*, which parasitizes sheep in the Tamdinsky region of Bukhara, Uzbekistan. TAMV was first discovered in a febrile patient in Kyrgyzstan in 1973. Subsequently, a TAMV strain derived from *Hygroscopic tick* was successfully isolated using newborn mice. Serological evidence of exposure to the virus has been detected in camels and humans. Immunodeficient mice infected with high doses of TAMV experienced significant weight loss and died 4–5 days post-infection.

[0003] TAMV belongs to the class Bunyaviridae, order Arenavirales, family Nairoviridae, and genus Ortho-Nairovir. Its genome consists of three segments: L, M, and S, which encode RNA-dependent RNA polymerase (RdRp), envelope glycoprotein precursor (GPC), and nucleocapsid (NP), respectively. GPC is cleaved into mature Gn and Gc forms. Envelope glycoproteins play a crucial role in Bunyavirus infection and cross-species transmission. Crimean-Congo Hemorrhagic Fever Virus (CCHFV), which causes Crimean-Congo Hemorrhagic Fever (CCHF), was isolated in 1969 and is currently the most extensively studied Nairovirus. Since 2022, CCHF outbreaks have been reported in Iraq, Afghanistan, Spain, Pakistan, and other countries, becoming a global focus. The RdRp sequence of TAMV shares ≥ 40% homology with that of CCHFV. It is worth noting that at least 12 new tick-borne viruses with the potential to infect humans have been discovered since 2012.

[0004] Tick-borne viruses such as TAMV are posing an increasingly serious threat to human and animal health worldwide. However, there are still no specific drugs or preventive vaccines for the various clinical diseases caused by tick-borne viruses.

[0005] This application develops a method for inhibiting TAMV infection by studying proteins that interact with TAMV envelope glycoproteins. Summary of the Invention

[0006] On one hand, the present invention provides a method for inhibiting Tamdy virus (TAMV) infection, the method being a method for inhibiting Tamdy virus infection of host cells, the method comprising the step of reducing or inhibiting the expression level and / or activity of the TCP1 (T-Complex Protein 1) gene or its encoded protein in host cells.

[0007] In one embodiment, the method is an in vitro or ex vivo method.

[0008] In one embodiment, the reduction or inhibition of the expression level and / or activity of the TCP1 gene or its encoded protein is achieved by a method selected from the group consisting of: gene mutation, gene knockout, gene interruption, RNA interference, gene editing, introduction of an inhibitor of the gene or protein, or a combination thereof.

[0009] In one embodiment, the inhibitor is selected from the group consisting of gene editing reagents, antisense nucleic acids, antibodies, small molecule compounds, small molecule ligands, or combinations thereof.

[0010] In one embodiment, the gene editing reagent includes a Cas enzyme and a gRNA capable of targeting the TCP1 gene.

[0011] In one embodiment, the Cas enzyme may be Cas9 or Cas12a.

[0012] In one embodiment, the antisense nucleic acid is selected from the group consisting of antisense RNA, antisense DNA, interfering RNA, ribozymes, or combinations thereof.

[0013] In one embodiment, the interfering RNA is selected from the group consisting of siRNA, shRNA, RNAi, miRNA, dsRNA, hpRNA, ihpRNA, or combinations thereof.

[0014] In one embodiment, the interfering RNA is siRNA, and the sequence of the siRNA is GCAAGAUCACUUCUUGUUA (SEQ ID No. 7).

[0015] On the other hand, the present invention also provides the use of TCP1 inhibitors in the preparation of medicaments for inhibiting host infection with Tamud virus, said inhibitors being able to reduce or inhibit the expression level and / or activity of the TCP1 gene or its encoded protein.

[0016] In one embodiment, the inhibitor includes gene editing reagents, antisense nucleic acids, interfering RNA, antibodies, small molecule compounds, small molecule ligands, or other reagents that can cause a decrease in the expression level and / or activity of the TCP1 gene or its encoded protein.

[0017] In this invention, other agents that can reduce the expression level and / or activity of the TCP1 gene or its encoded protein include agents that can cause TCP1 gene mutation, gene knockout, or gene interruption; for example, the art can reduce or inhibit the expression level and / or activity of the TCP1 gene or its encoded protein by homologous recombination to mutate or knock out the TCP1 gene.

[0018] In this invention, "reduction or inhibition" means that the expression or activity of the TCP1 gene or its encoded protein is reduced by at least 10%, at least 20%, at least 40%, at least 60%, or at least 80% compared to the TCP1 level in wild-type host cells.

[0019] In one implementation, reducing or inhibiting the expression level and / or activity of the TCP1 gene or its encoded protein can suppress the replication capacity or release efficiency of TAMV in host cells.

[0020] In one implementation, the host is an animal, such as a human, an Asian glass-eyed tick, or a tiny fan-headed tick.

[0021] In one embodiment, the host is human, and the amino acid sequence of TCP1 has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with SEQ ID No. 1. Preferably, the amino acid sequence of TCP1 is as shown in SEQ ID No. 1.

[0022] In one embodiment, the host is *Hypericum asiaticum*, and the amino acid sequence of TCP1 has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with SEQ ID No. 3. Preferably, the amino acid sequence of TCP1 is as shown in SEQ ID No. 3.

[0023] In one embodiment, the host is *Ixodes microphylla*, and the amino acid sequence of TCP1 has at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity with SEQ ID No. 5. Preferably, the amino acid sequence of TCP1 is as shown in SEQ ID No. 5. Attached Figure Description

[0024] Figure 1 CO-IP screening of TAMV interaction proteins in HEK293T cells; (A) Western Blot results of plasmids successfully expressing Flag tag in 293T cells, and (B) CO-IP silver staining to obtain interaction proteins.

[0025] Figure 2 . Validation of the interaction between human protein hTCP1 and TAMV viral protein; (A) Co-IP results of endogenous hTCP1 and TAMV protein, (BE) bidirectional Co-IP validation of exogenous Myc-hTCP1 and Flag-Gn / Gc.

[0026] Figure 3 The effect of knocking down hTCP1 expression on TAMV infection was verified; among them, (A) hTCP1 knockdown efficiency verification, (B) TAMV viral load detection in cell supernatant, (C) relative expression level of TAMV mRNA in cells, and (D) TAMV-NP protein level detection in cells.

[0027] Figure 4 . Validation of the interaction between hTCP1 and three Bunyavirus glycoproteins: (A) Validation of the interaction between CCHFV-Gn, Gc and TCP1, (B) Validation of the interaction between YEZV-Gn, Gc and TCP1, and (C) Validation of the interaction between SFTSV-Gn and TCP1.

[0028] Figure 5 Verification of co-precipitation of two ticks' TCP1 with TAMV Gn and Gc: (A, B) HyTCP1 with TAMV-Gn, (C, D) HyTCP1 with TAMV-Gc, (E, F) RmTCP1 with TAMV-Gn, and (G, H) RmTCP1 with TAMV-Gc. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for implementing the present invention, except as specifically mentioned below, are all common knowledge and general knowledge in the art, and the present invention does not have any particular limitations.

[0030] Example 1, Experimental Method

[0031] 1. Cell resuscitation

[0032] (1) Remove the frozen cells at -80℃ and quickly place them in a 37℃ water bath to thaw them completely;

[0033] (2) Centrifuge the cell suspension tube at 1500 rpm for 5 minutes;

[0034] (3) Discard the supernatant, resuspend the cell pellet with 1 ml of 10% culture medium and add it to the T25 culture flask, add the culture medium to 5 ml, and incubate in a 37℃ incubator containing 5% CO2.

[0035] 2. Cell passage

[0036] (1) Remove the cells from the incubator and discard the old culture medium;

[0037] (2) Add 1 ml of PBS solution and gently shake, discard the waste liquid, and repeat the operation once;

[0038] (3) Add 1 ml of trypsin to the culture flask, cover the cells, and then put it into the incubator for digestion. The time can be adjusted according to the different cell types. Observe the degree of cell digestion under a microscope and stop digestion when appropriate.

[0039] (4) Add 1 ml of 10% complete culture medium to stop digestion;

[0040] (5) According to the experimental needs, dispense the cell suspension into new cell culture flasks and add culture medium to 5 ml;

[0041] (6) Mark the passage time and number of passages on the bottle wall, transfer the cell bottle to the incubator for culture, gently shake the bottle body using the cross-shaking method, and observe under a microscope whether the cells in the bottle are shaken evenly.

[0042] 3. Cell cryopreservation

[0043] (1) Remove the cells from the incubator and discard the supernatant;

[0044] (2) Same cell passage steps (2)-(4);

[0045] (3) After adding complete culture medium to stop digestion, mix well by pipetting and transfer the cell suspension to a centrifuge tube, centrifuge at 1500 rpm for 5 min;

[0046] (4) Prepare cell cryopreservation solution in the dark with a serum to DMSO ratio of 9:1. Mix the components by inverting. After centrifugation, discard the supernatant. Gently pipette the cryopreservation solution and cell pellet together and transfer it to cryovials. Place the cryovials in a programmed cooling box and store them in a -80°C freezer.

[0047] II. Plasmid transformation, culture, and extraction

[0048] All plasmids used were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0049] (1) Centrifuge 4 μg of lyophilized powder plasmid at 10,000 rpm for 5 min in a biosafety cabinet, add 40 μL of sterile enzyme-free water to dilute; shake to mix, and centrifuge briefly for 10 s;

[0050] (2) Take out a tube of competent cells (DH5α) from the -80℃ freezer and insert it into ice to thaw;

[0051] (3) Add 50 μL of competent cells and 2 μL of plasmid to a 1.5 EP tube, tighten the cap, gently stir the centrifuge tube to mix, and let stand on ice for 30 min; Note: Do not use force or pipette tip to mix during the process.

[0052] (4) Preheat the water bath to 42°C, place the EP tube from (3) in the 42°C water bath for 90 seconds, and then quickly place it on ice for 2 minutes.

[0053] (5) Add 700 μL of antibiotic-free liquid LB medium to the 1.5 EP tube in (4) and shake at 37°C for 1 h;

[0054] (6) Centrifuge at 5000 rpm for 5 min and discard the supernatant. The remaining bacterial culture of about 150 μL is mixed with the pipette tip and aspirated onto an LB agar plate containing ampicillin (Amp). Use a disposable sterile spreader to draw lines in sections and incubate overnight at 37°C for 12-16 h.

[0055] (7) The next day, a single colony grows on the plate. If the culture is not shaken for a short time, place the plate in a refrigerator at 4°C.

[0056] (8) Picking bacteria: Use a sterile 10μL pipette tip to pick a single colony from the plate into a 2ml LB medium with resistance, and place it in a constant temperature shaker at 37℃ and 220rpm for about 6 hours.

[0057] (9) Observe the liquid LB medium change from clear to turbid, take 20ul of bacterial solution from the shaking tube and add it to a shaking bottle containing 15ml of resistant LB medium;

[0058] (10) Perform plasmid extraction and detect plasmid concentration according to the instructions of Kangwei Century Plasmid Extraction Kit.

[0059] III. Agarose Gel Electrophoresis

[0060] (1) Prepare all necessary reagents and consumables, including conical flasks, agarose gel casting plate holders, combs, agarose powder, and 1×TAE solution;

[0061] (2) Weigh 0.3g of agar powder and add it to the conical flask, then add 30mL of 1×TAE solution and mix well;

[0062] (3) Heat in a microwave oven, wearing gloves, take out the conical flask every 4-5 seconds, shake gently to mix, and stop heating immediately when the liquid changes from cloudy to clear;

[0063] (4) After the conical flask has cooled down, add 3 μL of nucleic acid dye to the conical flask and gently shake the conical flask to mix evenly;

[0064] (5) Slowly pour the mixture into the agarose gel casting plate holder with the comb inserted, avoiding the formation of air bubbles;

[0065] (6) Let it solidify at room temperature, then pull out the comb in parallel.

[0066] (7) Place the DNA gel in the electrophoresis tank, ensuring the electrophoresis solution covers the DNA gel;

[0067] (8) Sample loading: Mix 5 μL of the extracted plasmid with 1 μL of 6× Loading buffer solution and add it to the sample loading well. Select the appropriate sample loading marker according to the size of the target fragment, and electrophoresis at 120V for 30 min.

[0068] (9) After electrophoresis, remove the DNA gel and expose it in a UV gel irradiator to check for clear DNA bands.

[0069] IV. Plasmid Transfection

[0070] (1) One day before transfection, 293T cells were evenly seeded into a 10cm cell culture dish. After 12 hours of cell culture, the cells were in good growth condition and the density reached 60%-80%. Then, the cells were transfected with plasmids. Three replicate wells were set up for each treatment group.

[0071] (2) The samples required for this experiment are TAMV-Gn, TAMV-Gc, and TAMV-Np plasmids;

[0072] (3) Prepare the transfection dilution solution. Add serum-free DMEM medium and PEI transfection reagent to the centrifuge tube. This is the transfection dilution solution.

[0073] (4) Prepare plasmid DNA dilution solution. Add 500 μL of serum-free DMEM medium and 10 μg of plasmid to centrifuge tubes respectively, mix well and let stand at room temperature for 5 min.

[0074] (5) Take 500 μL of transfection dilution buffer and add it to centrifuge tubes containing each target gene plasmid. Gently pipette to mix and let stand at room temperature for 10 min.

[0075] (6) Remove the plated cells, discard the original culture medium in the dish, add 6 mL of serum-free DMEM culture medium stock solution along the wall, and gently shake the culture dish until the bottom of the cell dish is completely covered by the liquid.

[0076] (7) Add 1 mL of the target gene plasmid and transfection reagent mixture below the liquid surface, and set up a control group without plasmid. Shake the culture dish to mix well.

[0077] (8) Place the culture dish in a cell culture incubator and incubate for 4-6 hours. After incubation, discard the original solution and add 10 mL of 2% DMEM medium.

[0078] (9) After 48 hours of transfection, cell proteins were collected for subsequent experiments; 1 mL of CO-IP lysis buffer was added to the collected cell pellet before total protein extraction.

[0079]

[0080] V. Immunoprecipitation

[0081] (1) Collect cell pellet: Digest the cell sample from a 10cm dish with trypsin and transfer it to a 1.5ml EP tube. Centrifuge at 1500rpm for 5min, discard the supernatant, add 1mL PBS, centrifuge at 1500rpm for 5min, discard the PBS and keep the cell pellet.

[0082] (2) Cell lysis: Prepare Co-IP protein lysis buffer by adding PMSF, Cocktail, and 2mM DTT to Wash Buffer and mixing well; add 1ml of Co-IP lysis buffer to each cell sample and mix by pipetting to lyse the cells; place the EP tube on ice for 30min; use an ultrasonic disruptor to fully disrupt the lysed cells 10-15 times; centrifuge at 4℃ and 12000rpm for 10min, and aspirate the supernatant into a new EP tube to detect the protein concentration;

[0083] (3) Turn on the induction cooker in advance to heat the water to 100°C, and boil the 5× protein loading buffer for 5 minutes;

[0084] (4) Prepare the Input group: Aspirate 50 μL of protein supernatant into a new sterile centrifuge tube, add 12.5 μL of 5× protein loading buffer, mix well, and centrifuge at 13000 rpm for 5 min; after the centrifugation, collect the supernatant and transfer it to a new sterile centrifuge tube. This is the Input group sample.

[0085] (5) Balancing magnetic beads: Add 20 μL of Flag magnetic beads and 1 mL of Wash Buffer lysis buffer to a new sterile centrifuge tube, mix well, and place it on a 360° rotary mixer at 4°C for 5 min. After the mixture is finished, remove it and place it on a magnetic rack. Let it stand at room temperature for 1 min and then discard the supernatant. Add 1 mL of Wash Buffer lysis buffer again and repeat the above balancing magnetic bead process 5 times.

[0086] (6) Preparation of IP group: Add 950 μL of protein supernatant to the equilibrated magnetic beads, place in a 360° rotary mixer at 4℃ and incubate for 6-8 h. After incubation, remove and place on a magnetic rack, let stand at room temperature for 1 min, discard the supernatant, add 1 mL of Wash Buffer lysis buffer, and place in a 360° rotary mixer at 4℃ and invert for 5 min. After inversion, remove and place on a magnetic rack, let stand at room temperature for 1 min, discard the supernatant, add 1 mL of Wash Buffer lysis buffer, and repeat the above steps 5 times.

[0087] (7) Add 50 μL Wash Buffer lysis buffer and 12.5 μL 5× protein loading buffer to the cleaned protein sample, boil at 100℃ for 5 min, and let stand at room temperature. This is the IP experimental group.

[0088] VI. Silver Dye

[0089] (1) Fixation: After electrophoresis, take the gel and put it into about 100ml of fixative. Shake it on a shaker at room temperature for 20 minutes at a speed of 60-70rpm. (Preparation of fixative: add 50ml of ethanol, 10ml of acetic acid and 40ml of ultrapure water in sequence, mix well and you will get 100ml of fixative).

[0090] (2) Washing with 30% ethanol: Discard the fixative, add 100 ml of 30% ethanol, and shake on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm;

[0091] (3) Washing with water: Discard 30% ethanol, add 200ml of ultrapure water, and shake on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm;

[0092] (4) Sensitization: Discard the water, add 100ml of silver staining sensitization solution (1×), and shake on a shaker at room temperature for 2 minutes at a shaking speed of 60-70 rpm. (Preparation of silver staining sensitization solution (1×): Add 1ml of silver staining sensitization solution (100×) to 99ml of ultrapure water, mix well and it is silver staining sensitization solution (1×));

[0093] (5) Wash with water (twice): Discard the original solution, add 200 ml of ultrapure water, and shake on a shaker at room temperature for 1 minute at a speed of 60-70 rpm. (Repeat twice);

[0094] (6) Silver staining: Discard the water, add 100ml of silver solution (1×), and shake on a shaker at room temperature for 10 minutes at a shaking speed of 60-70rpm (Preparation of silver solution (1×): Add 1ml of silver solution (100×) to 99ml of ultrapure water, mix well and it is silver solution (1×)).

[0095] (7) Washing with water: Discard the original solution, add 100ml of ultrapure water, and shake on a shaker at room temperature for 1-1.5 minutes at a shaking speed of 60-70rpm;

[0096] (8) Development: Discard the water, add 100ml of silver staining development solution, and shake on a shaker at room temperature for 3-10 minutes until the desired protein band appears. The shaking speed is 60-70 rpm. (Preparation of silver staining development solution: Add 20ml of basic silver staining development solution (5×) to 80ml of ultrapure water, then add 0.05ml of silver staining development acceleration solution (2000×), mix well and the silver staining development solution is ready).

[0097] (9) Termination: Discard the silver staining developer, add 100ml of silver staining termination solution (1×), and shake on a shaker at room temperature for 10 minutes at a shaking speed of 60-70 rpm. (Preparation of silver staining termination solution (1×): Add 5ml of silver staining termination solution (20X) to 95ml of ultrapure water, mix well and it is silver staining termination solution (1×)).

[0098] (10) Washing with water: Discard the silver staining stop solution, add 100ml of ultrapure water, and shake on a shaker at room temperature for 2-5 minutes at a shaking speed of 60-70rpm;

[0099] (11) Storage: Can be stored in ultrapure water;

[0100] (12) Cutting: Visually inspect the differential stripes and cut them with a blade;

[0101]

[0102] VII. siRNA transfection and cell inoculation (using a 24-well cell plate as an example)

[0103] The small interfering RNA was synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0104] (1) The day before transfection, RD cells were administered at a dose of 1×10 5 Evenly spread cells per well onto a 24-well cell plate;

[0105] (2) After 12 hours of plating, when the cell density reaches about 60%, the plated cells are transfected with siRNA. At the same time, three replicate wells are set up for each treatment group. The medium is replaced with Opti-MEM medium (without antibiotics) 1 hour before transfection.

[0106] (3) Add ddH2O to the siRNA product and adjust the concentration to 10 μM;

[0107] (4) Dilute siRNA: Dilute siRNA with Opti-MEM at a ratio of 1:50 to adjust the concentration to 10 μM; Dilute Lipofectamine: Dilute Lipo reagent with Opti-MEM at a ratio of 3:50;

[0108] (5) Mix the diluted Lipo reagent and diluted siRNA, incubate at room temperature for 5 min, and then add 50 μL of the diluted mixture to each well below the liquid surface. At the same time, note that a negative control should be set up.

[0109] (6) Place the 24-well plate after the operation into a 37°C, 5% CO2 incubator and incubate for 6 hours;

[0110] (7) After incubation, discard the original liquid in the well and replace it with 2% MEM medium (without antibiotics); collect cells and supernatant at appropriate time points according to experimental requirements;

[0111] (8) Collect the supernatant: Transfer all the liquid supernatant from the well plate to a new enzyme-free sterile centrifuge tube; Collect the cells: If the purpose of the subsequent experiment is to extract cellular RNA, add 600 μL of TRIzol reagent to each well, and repeatedly pipette 4-5 times to ensure that the cells are fully lysed and do not adhere to the plate, and transfer them to a new enzyme-free sterile centrifuge tube; Store all cells and supernatant at -80℃, and use qPCR and Western Blot techniques to detect the transfection efficiency.

[0112] (9) Cell inoculation after target gene knockdown: Cell count, calculate the required amount of virus solution for inoculation according to MOI=1, mix with culture medium, remove the original culture medium from the 24-well plate, add 500ul PBS for washing, add 500ul virus mixture to the 24-well plate, incubate in an incubator for 2 hours, discard the virus mixture, add 500ml PBS for washing twice, add 500ul 2% MEM culture medium (without antibiotics). Subsequently, samples were collected at time points and virus infection efficiency was detected by qPCR and Western Blot techniques;

[0113] 8. RNA Extraction

[0114] virus samples

[0115] (1) Place the sample on ice to thaw; place the 96-well reagent plate (Borgai MagaBio plus viral RNA purification kit) at room temperature for about 30 minutes in advance, and at the same time disinfect the nucleic acid extractor with ultraviolet light for 15-30 minutes;

[0116] (2) Invert the 96-well reagent plate to mix well, and shake off the droplets adhering to the wall;

[0117] (3) Remove the aluminum foil film from the sealing plate inside the biosafety cabinet, and add 300 μL of sample to the first and seventh columns of the reagent plate in the order of arrangement;

[0118] (4) After the sample is added, place the 96 reagent plate in the fully automated nucleic acid extraction and purification instrument, install the magnetic rod protective cover, close the instrument cover, and run the program;

[0119] (5) After the procedure is completed, remove the reagent plate, place it on a metal ice plate and move it into the biosafety cabinet. Collect the liquid in the fifth and eleventh column wells into RNase-free centrifuge tubes and immediately store them in a -80℃ freezer for later use.

[0120] Cell samples

[0121] Cellular RNA was extracted using the Kangwei Century Ultrapure RNA Extraction Kit.

[0122] (1) Thaw the sample on ice; disinfect the workbench and experimental supplies with ultraviolet light for 30 minutes; prepare enzyme-free pipette tips and centrifuge tubes in advance, and pre-cool the centrifuge at 4°C;

[0123] (2) Add 200 μL of chloroform to each sample, shake vigorously for 15 s, and let stand at room temperature for 2 min after shaking;

[0124] (3) After standing, transfer the sample to a centrifuge and set the program to 4℃, 12000rpm, 10min;

[0125] (4) After centrifugation, transfer the clear liquid from the top layer to a new enzyme-free centrifuge tube, add an equal volume of 70% ethanol solution to the supernatant, and mix by inverting.

[0126] (5) Transfer the above mixture to a centrifuge column, place it in a centrifuge, set the program to 12000 rpm, and the time to 20 s;

[0127] (6) After centrifugation, remove the centrifuge column, discard the liquid in the collection tube, add 700 μL Buffer RW1, put it back into the centrifuge, set the program to 12000 rpm, and the time to 20 s.

[0128] (7) After centrifugation, remove the centrifuge column, discard the liquid in the collection tube, add 700 μL of Buffer RW2, put it back into the centrifuge, set the program to 12000 rpm, and the time to 20 s; repeat this process twice.

[0129] (8) Discard all the liquid in the collection tube, put it into the centrifuge, set the program to 12000 rpm, and the time to 2 min; after the end, transfer the centrifuge column to a new centrifuge tube, open the lid and let it stand at room temperature for 2-5 min, and wait for the excess water on the filter membrane at the bottom of the centrifuge column to evaporate completely.

[0130] (9) Add 40 μL of RNase-Free Water to the filter membrane at the bottom of the centrifuge column, put the centrifuge column into the centrifuge, set the program to 12000 rpm, and the time to 2 min;

[0131] (10) After centrifugation, remove the centrifuge column and discard it. All the liquid obtained in the centrifuge tube is the RNA of the cell sample required for the experiment. After quickly marking the time and basic information, place the RNA in a -80℃ refrigerator for later use.

[0132] Reverse transcription to synthesize cDNA

[0133] (1) Remove the RNA from the -80°C freezer and thaw it on ice;

[0134] (2) Prepare the reverse transcription reaction mixture on the ice plate inside the biosafety cabinet:

[0135]

[0136] (3) Mix the reaction mixture thoroughly and centrifuge briefly;

[0137] (4) Set the reaction procedure as follows:

[0138]

[0139] (5) After the reaction is complete, store the cDNA sample in a -20℃ refrigerator for later use.

[0140] Real-time quantitative PCR (RT-qPCR)

[0141] 1. Probe method

[0142] Viral load was detected using RT-qPCR with cDNA as a template. A negative control group was also established using sterile water as a template.

[0143] (1) Take the DNA, 2×Pro Taq HS Probe Premix, and upstream and downstream primers of the TAMV-S fragment out of the -20 freezer and thaw them on ice;

[0144] (2) The detection reaction system is as follows:

[0145]

[0146] (3) The reaction system procedure is as follows:

[0147]

[0148] (4) Primer sequence synthesis

[0149] The upstream and downstream primers and probes for the TAMV-S fragment were purchased from Shanghai Sangon Biotech Co., Ltd.

[0150]

[0151] 2. Dye method

[0152] The knockdown efficiency of the target gene was determined using cDNA as a template and dye-based RT-qPCR. A negative control group was also established using sterile water as a template.

[0153] (1) Take the DNA, 2×SYBR Green Pro Taq HS Probe Premix, and upstream and downstream primers of the target gene out of the -20 freezer and thaw them on ice;

[0154] (2) The detection reaction system is as follows:

[0155]

[0156] (3) The reaction system procedure is as follows:

[0157]

[0158] (4) Primer sequence synthesis:

[0159]

[0160] Example 2, Experimental Results

[0161] To investigate the molecular basis of TAMV infection, we constructed TAMV glycoproteins Gn and Gc, and nucleocapsid protein Np plasmids tagged with flags, using empty plasmids as controls. Figure 1 Viral protein A was transiently expressed in HEK293T cells, which support TAMV infection, have high transfection efficiency, and have annotated human proteome databases for identification. After Western blotting confirmed successful viral protein expression, Co-IP was performed on cell lysates using Flag magnetic beads, and differential bands were observed using gel electrophoresis and silver staining. The differences from the control group were clearly observed visually. Figure 1 (B) Differential bands were cut and identified by mass spectrometry. Based on the mass spectrometry results and the cut position of the bands, and the annotation information of related protein functions and cell localization in the database (UniProt, DisGeNET), we initially selected 29 proteins from the interacting proteins of TAMV derived from HEK293T cells (Table 11). Subsequently, the interaction between the proteins selected in the table and TAMV was verified by CO-IP.

[0162]

[0163] 2. Human-derived protein TCP1 interacts with TAMV envelope glycoprotein.

[0164] Based on mass spectrometry identification results, silver staining differential band positions, and annotation information on protein function and cellular localization from the UniProt and DisGeNET databases, candidate molecules were screened from interacting proteins of TAMV-infected host cells (Table 11). Transient transfection was performed in HEK293T cells, which support viral protein expression, using Flag-tagged TAMV glycoproteins (Gn, Gc) and nucleocapsid protein (Np) as controls, with an empty vector plasmid as a control. After Western blotting confirmed successful viral protein expression, Co-IP experiments were performed on cell lysates using Flag magnetic beads to verify the interactions between candidate proteins and viral proteins. Results showed that TAMV-Gc protein could form a complex with endogenous TCP1 (T-Complex Protein 1) (gene ID: 6950) (the TCP1 band was significant in the IP:Flag lane), while TAMV-Gn and Np showed no significant interaction with hTCP1; the Input lane verified the integrity of protein expression. Therefore, the TAMV-Gc protein interacts with human hTCP1 cells (Figure 2A).

[0165] To further confirm the interaction between TAMV glycoprotein and hTCP1, the Myc-hTCP1 plasmid was constructed and co-transfected with the Flag-TAMV-Gn / Gc recombinant plasmid into HEK293T cells. The cells were then used with Flag magnetic beads (… Figure 2 B, Figure 2 (D) and Myc magnetic beads ( Figure 2 C, Figure 2 A two-way Co-IP experiment was conducted to verify the interaction between Myc-hTCP1 and Flag-TAMV-Gn and Gc proteins. The results showed that Myc-hTCP1 and Flag-Gn interacted: co-precipitation of Myc-hTCP1 and Flag-Gn was detected in the IP lanes, confirming their interaction. Figure 2 B, Figure 2 Interaction between Myc-hTCP1 and Flag-Gc: Co-precipitation of Myc-hTCP1 and Flag-Gc was detected in the IP lane, further confirming the interaction between Gc-hTCP1. Figure 2 D, Figure 2 (E); The input lane uses GAPDH as an internal control to verify the uniformity of protein loading. Therefore, forward and reverse CO-IP experiments demonstrate that TAMV-Gn and Gc interact with hTCP1.

[0166] 3. Knockdown of human-derived protein TCP1 significantly inhibits TAMV infection.

[0167] To investigate the role of the human protein TCP1 (its amino acid sequence is shown in SEQ ID No. 1, and its DNA sequence is shown in SEQ ID No. 2) in TAMV infection, this study constructed a knockdown cell line to verify the changes in viral infection and deeply elucidate its regulatory mechanism: a TCP1-targeted knockdown (siTCP1) cell line was constructed, with non-targeted interference (siNC) as a control. In this embodiment, the siRNA sequence targeting TCP1 was: GCAAGAUCACUUCUUGUUA (SEQ ID No. 7), and the cell line used was human RD cells. Verification was performed using a combination of qPCR and Western blotting: qPCR results showed that the expression level of TCP1 mRNA in the siTCP1 group was significantly reduced, with a knockdown efficiency of 87% (…). Figure 3 (A); Protein level: Western blotting was used to detect TCP1 protein expression. The results showed that the TCP1 protein band in the siTCP1 group was significantly weaker than that in the NC group. The internal control GAPDH was used to verify the uniformity of sample loading. Figure 3 (A)

[0168] SEQ ID No.1:

[0169] megplsvfgdrstgetirsqnvmaaasianivksslgpvgldkmlvddigdvtitndgatilklllevehpaakvlceladlqdkevgdgttsvviiaaellknadelvkqkihptsvisgyrlackeavryinenlivntdelgrdclinaaktsmsskiigingdffanmvvdavlaikytdirgqprypvnsvnilkahgrsqmesmlisgyalncvvgsqgmpkrivnakiacldfslqktkmklgvqvvitdpekldqirqresditkeriqilatganvilttggiddmclkyfveagamavrrvlkrdlkriakasgatilstlanlegeetfeaamlgqaeevvqericddelelikntkartsasiilrandfmcdemerslhdalcvvkrvlesksvvpggavaveaalsiylenyatsmgsreqlaiaefarsllvipntlavnaaqdstdlvaklrafhneaqvnperknlkwigldlsngkprdnkqagvfeptivkvkslkfateaaitilriddliklhpeskddkhgsyedavhsgalnd

[0170] SEQ ID No. 2:

[0171]

[0172] Cells in the siTCP1 group and NC group were infected with TAMV (MOI=1) respectively. Infection phenotype was detected at different levels of viral nucleic acid load and protein expression at 48 h (2 dpi) and 96 h (4 dpi) post-infection. Viral nucleic acid levels were detected by qPCR, and it was found that the viral load in the cell supernatant of the siTCP1 group was significantly lower than that of the NC group at both 2 dpi and 4 dpi. Figure 3 (B); while at 2 dpi and 4 dpi, the relative expression level of TAMV mRNA in the siTCP1 group was significantly lower than that in the NC group ( Figure 3 (C). Subsequently, Western blotting was used to detect intracellular TAMV-NP protein (viral nucleocapsid protein, representing viral replication level): at 4 dpi, the TAMV-NP band intensity in the siTCP1 group was significantly weaker than that in the NC group, and GAPDH was used to verify the uniformity of sample loading. Figure 3 (D).

[0173] Therefore, based on the combined results of intracellular viral nucleic acid and protein levels and viral load in the supernatant, it can be clearly concluded that knocking down TCP1 expression significantly inhibits the replication ability and release efficiency of TAMV in host cells.

[0174] 4. The TCP1 protein cannot bind to the glycoproteins of Bunyaviruses CCHFV, YEZV, and SFTSV.

[0175] Nairoviruses (such as CCHFV, TAMV, YEZV, SGLV, etc.) are mainly transmitted through tick bites. Their invasion of host cells depends on the interaction between envelope glycoproteins and receptors on the host cell surface. To clarify whether TCP1 protein is a universal host factor for Nairoviruses, CCHFV glycoproteins Gn and Gc, YEZV glycoproteins Gn and Gc were selected to construct Flag-tagged CCHFV-Gn, CCHFV-Gc, and YEZV-Gn, YEZV-Gc, and Myc-tagged human TCP1 protein, which were co-expressed in 293T cells. After 48 hours, the interaction was verified by CO-IP combined with Western blotting using His, Flag, and Myc magnetic beads. The current experiment shows that only Flag-tagged CCHFV / YEZV Gn and Gc proteins were detected in the complex captured by the Flag magnetic beads, and Myc-TCP1 was not enriched. Figure 4 China A Figure 4 (B); In the Myc magnetic bead-captured complex, only the Myc-tagged TCP1 protein was detected, and the Flag-tagged CCHFV / YEZV Gn and Gc proteins were not enriched ( Figure 4B, Figure 4 (C). The Gn and Gc proteins of Myc-TCP1 and Flag-CCHFV / YEZV were successfully expressed in cells, and GAPDH was used to verify the uniformity of loading. The above experiments show that TCP1 did not interact with the glycoproteins (Gn and Gc) of CCHFV and YEZV. Figure 4 China A Figure 4 (B)

[0176] In addition, we selected another Bunyavirus causing a serious infectious disease—Severe Fever with Thrombocytopenia Syndrome Virus (SFTSV)—to verify whether its glycoprotein interacts with TCP1. A His-tagged SFTSV-Gn recombinant plasmid was constructed and co-expressed with the human Myc-tagged TCP1 protein in 293T cells. The interaction was verified by His magnetic bead CO-IP combined with Western blotting. Only the His-SFTSV-Gn band was detected in the His-bead-captured complex; Myc-TCP1 was not enriched. Myc-TCP1 and His-SFTSV-Gn proteins were successfully expressed in cells, and GAPDH was used to verify the uniformity of loading. Current experiments indicate that TCP1 does not interact with SFTSV (Gn protein). Figure 4 (C)

[0177] 5. There is an interaction between tick TCP1 and TAMV envelope glycoprotein.

[0178] The natural tick host of TAMV is the Asian glass tick (Tectus australis). Hyalomma asiaticum ), and the virus can be found in tiny fan-shaped ticks ( Rhipicephalus microplus The CTVM23 cell line derived from [the tick species] proliferated stably. Therefore, we constructed recombinant plasmids Myc-HyTCP1 and Myc-RmTCP1 for the TCP1 of the two tick species mentioned above; among them, *Hylocereus Asiana* ([the tick species])... Hyalomma asiaticum The amino acid sequence of TCP1 (HyTCP1) in *Hypericum micranthum* is shown in SEQ ID No. 3, and its DNA sequence is shown in SEQ ID No. 4; *Hypericum micranthum* (… Rhipicephalus microplus The amino acid sequence of TCP1 (RmTCP1) in the sample is shown in SEQ ID No. 5, and its DNA sequence is shown in SEQ ID No. 6. Myc-HyTCP1 and Myc-RmTCP1 were co-transfected with the recombinant plasmids Flag-TAMV-Gn and Flag-TAMV-Gc, respectively, into HEK293T cells; and then transfected with Flag magnetic beads (… Figure 5 China A Figure 5 C, Figure 5 Chinese E, Figure 5 (G) and Myc magnetic beads ( Figure 5 B, Figure 5 D, Figure 5 China F, Figure 5 A two-way Co-IP experiment was conducted to verify the interaction between TCP1 and TAMV-Gn and Gc proteins of two tick species. The results showed that both HyTCP1 and RmTCP1 can interact with TAMV-Gn and Gc.

[0179] SEQ ID No. 3:

[0180] msfgtglsvsgkrssgasvrtqnvmaacsianivksslgpvgldkmlvddigdvtitndgatilkllevehpaakvlveladlqdqevgdgttsvvliaaellknadelvkckihptsiisgyrlackeacryiqeh lttsveelghecvvnaaktamsskligpdsdffanmvvdaasairvsdgkggyrypikavnvlkahgrsaresvlvqgyalnctvasqamtkkvtgakiacldfslqkakmhmgvqvlvtdpekleairqremditk eriskilsaganvilvtggiddlclkyfveagamavrrckkqdlrriakatgaqllvslanmegeesfdatmlgeaeevvqekicddelilikkpkvqtassiivrgandffvdeversihdalcvvrrvlesktvv pgggaveaalsiylenfatslssreqlaiaeyarsllvipktlavnaakdatdlvsklrayhnssqtkqdhaqlkwvgldlyegtvrdnqkagvleptvskikslkfateaaitilriddlikleptpsshddrdecm

[0181] SEQ ID No. 4:

[0182]

[0183] SEQ ID No.5:

[0184] msfgtglsvsgkrssgasvrtqnvmaacsianivksslgpvgldkmlvddigdvtitndgatilkkllevehpaakvlveladlqdqevgdgttsvvlvaaellknadelvkckihptsiisgyrlackeacryiqehlttsveelghesvvnaaktamsskligpdsdffasmvvdaanavkvsdgkggfrypikavnvlkahgrsaresvlvqgyalnctvasqamtkkvlgakiacldlslqkakmhmgvqvlvtdpekleairqremditk eriskilgaganvilvtggiddlclkyfveagamavrrckkqdlrriakatgaqllvslanmegeesfdasmlgdaeevvqekvcddelilikkpkvqtassiiiirgandffvdeversihdalcvvrrvlesktvvpggaveaalsiylenfatslssreqlaiaeyarsllvipktlavnaakdatdlvsklrayhnssqtkqdhaqlkwvglgdlyegttvrdnqkagvleptvskikslkfateaaitilriddliklepspsshddrdecm

[0185] SEQ ID No. 6:

[0186]

[0187] In summary, TCP1 specifically participates in the TAMV infection process in human cell lines, and TCP1 knockdown significantly inhibits TAMV infection. TCP1 can specifically bind to both Gn and Gc of the TAMV envelope glycoprotein. TCP1 proteins from *Hypericum aizoon* and *Fish-headed Tetranychus spp.* also interact with TAMV Gn and Gc. These findings suggest that TCP1 may serve as an important universal host factor for TAMV infection across different species and could be a potential candidate target for blocking cross-species transmission of TAMV.

[0188] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A method for inhibiting Tamud virus infection, wherein the method is a method for inhibiting Tamud virus infection of host cells, characterized in that, The method includes the step of reducing or inhibiting the expression level and / or activity of the TCP1 gene or its encoded protein in host cells, and the method is an in vitro or ex vivo method; the host is a human, *Hypericum asiaticum*, or *Fernula microphylla*.

2. Use of a TCP1 inhibitor in the preparation of a medicament for inhibiting host infection with Tamud virus, said inhibitor being capable of reducing or inhibiting the expression level and / or activity of the TCP1 gene or its encoded protein, said inhibitor being siRNA, said siRNA having the sequence shown in SEQ ID No. 7, said host being human, Asian water hyacinth tick, or miniature fan-head tick.

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