CN108210880B - Application of compound PS-341 in preparation of bunyaviridae phlebovirus inhibitor - Google Patents

Abstract

The invention discloses an application of a compound PS-341 in preparation of a bunyaviridae phlebovirus inhibitor, wherein the compound PS-341 has a structure shown in a formula (I):

Description

Application of compound PS-341 in preparation of bunyaviridae phlebovirus inhibitor

Technical Field

The invention relates to a new application of a compound PS-341, in particular to an application of the compound PS-341 in preparing a bunyaviridae phlebovirus inhibitor.

Background

In recent years, due to factors such as global climate change, several infectious diseases such as hand-foot-and-mouth disease, avian influenza, SARS, swine streptococcosis and the like which cause great harm to human health occur in China. The high-infectivity diseases pose a serious challenge to the society, economy and national safety of China, and have certain importance and urgency on effective prevention means of scientific research of the high-risk pathogens. The research of new infectious disease specific treatment medicines and vaccines is crucial to the prevention and control of infectious diseases in China.

Since 2009, it was reported that most of the outbreaks of tick bites in Henan et al caused epidemic diseases with fever as the main clinical feature, and the wide attention of various social circles was caused by various vectors. Experts in the department of health have conducted pathogen isolation and field investigation across regions, with the 2008 goal locked to Human Granulocytic Anaplasmosis (HGA). In 2011, The New England Journal of Medicine where The virus of The Chinese disease prevention and control center is located published relevant latest research results, a novel bunyavirus which causes fever with thrombocytopenia syndrome (SFTS) and is named as fever with thrombocytopenia syndrome virus (SFTS virus, SFTSV) and has an early morbidity of 30% is identified, and The true "Yuanjia" of tick bite in The middle of China since 2009.

Bunyaviruses are typically enveloped, spherical negative-strand RNA viruses, approximately 80-120nm in diameter, with glycoprotein protrusions on the surface. The bunyaviridae family has 350 virus members, is the largest among arboviruses, and is divided into 5 genera, 4 of which are capable of infecting humans and animals, including the Phlebovirus genus (Phlebovirus), the Bunyavirus genus (Bunyavirus) Hantavirus genus (Hantavirus), and the Nairovirus genus (Nairovirus), and a tomato spotted wilt virus genus (Tospovirus) in which only infected plants have been found so far. In addition to the spread of hantavirus by rodents and predators, 4 other genera were transmitted by arthropods such as mosquitoes, sand flies, ticks, midges, thrips, etc.

The major transmission pathway for SFTSV is tick bite, and some recent cases suggest that exposure may be one of its transmission pathways. The pathogenesis of SFTS is not clear, the virus has wide tropism, invades the internal organ digestive tract of the blood system and the like, and is mainly manifested by fever, remarkable reduction of platelets and leukopenia, and death due to exhaustion of multiple organs of severe patients. However, the disease caused by the SFTSV infection of the novel bunyavirus has the characteristics of rapid disease process and critical disease consequences, and can be transmitted through animal media and contact, so that outbreaks in local areas are very likely to be caused, and sudden public health events are caused. Due to the limited understanding of the pathogenic mechanism of the SFTSV infection of the novel bunyavirus, mature therapeutic means are lacking, and the virus is still emerging in parts of the domestic area and in Japan, Korea or even the United states. Therefore, research on specific therapeutic drugs or vaccines for the novel bunyavirus SFTSV is imminent.

The compound PS-341 is a 20S proteasome inhibitor with the molecular formula of C₁₉H₂₅BN₄O₄Is developed by millennium Pharmaceuticals in the United statesA dipeptide boronic acid derivative. The compound PS-341 is used as a proteasome inhibitor, and can strongly and reversibly inhibit the function of proteasome, so that a plurality of proteins which should be eliminated are accumulated in cells in a large amount, and then the cells are subjected to apoptosis. At present, the compound PS-341 has attracted attention for its antitumor effect by inducing apoptosis. However, it has not been reported that PS-341 inhibits the replication of a virus belonging to the genus phlebovirus of the family bunyaviridae.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of a compound PS-341 in preparation of a bunyaviridae phlebovirus inhibitor.

The technical scheme of the invention is summarized as follows:

the application of a compound PS-341 in preparing a bunyaviridae phlebovirus inhibitor is disclosed, wherein the compound PS-341 has a structure shown in a formula (I):

the virus of phlebovirus of bunyaviridae is fever-associated thrombocytopenia syndrome virus (SFTSV), UUUKV or Sicily phlebovirus (SFSV).

The invention has the advantages that:

experiments prove that the compound PS-341 can inhibit the fever-associated thrombocytopenia syndrome virus, Wukuh virus or Sicily phlebovirus in phlebovirus of bunyaviridae. The invention discovers that the SFTSV nonstructural protein NSs specifically binds to retinoic acid (retinoic acid) induced gene protein I (RIG-I) in a host cell interferon pathway and degrades the RIG-I through a ubiquitination pathway. The specific combination and degradation can inhibit the activity of a host interferon-beta promoter, block the generation of host interferon-beta and enable SFTSV to escape from a natural immune defense mechanism related to the host interferon. PS-341, a 20S proteasome inhibitor, can be degraded by inhibiting the RIG-I ubiquitination pathway mediated by SFTSV NSs, thereby activating the interferon antiviral pathway of host cells during viral infection and significantly inhibiting replication and proliferation of viruses.

Drawings

FIG. 1 shows that NSs, nonstructural proteins of fever with thrombocytopenia syndrome virus (SFTSV), UUUKV, West Sicily sandfly virus (SFSV), inhibit the activity of interferon-beta promoter.

FIG. 2: NSs of SFTSV and SFSV degrade retinoic acid (retinoic acid) induced gene protein I (RIG-I), a natural immune key protein of the host.

FIG. 3 PS-341 inhibits SFTSV SFSV NSs-mediated degradation of RIG-I.

FIG. 4: PS-341 inhibits UUUKV NSs-mediated RIG-I degradation.

FIG. 5: PS-341 inhibits intracellular SFTSV replication.

Detailed Description

The present invention will be further described with reference to the following specific examples.

Example 1 plasmid construction

Plasmid construction by a method comprising the following steps

Synthesis of nonstructural protein cDNA of fever with thrombocytopenia syndrome Virus (SFTSV), UUUKV, Sicily sandfly virus (SFSV)

Searching gene sequences according to GENBANK gene sequence numbers SFTSV (GI:747024328), SFSV (GI:334035) and UUUKV (GI:38371709), respectively finding out cDNA sequences of the SFSV and the UUKV, and synthesizing the cDNA sequences of the SFTSV, the SFSV and the UUKV by Suzhou Jinzhi Biotechnology Limited.

Amplification of target Gene

Designing virus non-structural protein (NSs) primers according to gene sequences SFTSV (GI:747024328), SFSV (GI:334035) and UUUKV (GI:38371709), wherein the upstream primer enzyme cutting site is Sal1, the downstream primer enzyme cutting site is selected from BamH1, and an HA tag is added at the tail end of the downstream primer. The target gene was amplified using cDNA as a template according to the PCR protocol system.

PCR product gel recovery

The PCR product was added to 10. mu.L of 6XDNA loading buffer, followed by nucleic acid electrophoresis. The agarose gel containing the gene fragment of interest was carefully excised under an ultraviolet lamp, weighed into a previously weighed clean EP tube, and weighed again to calculate the weight of the excised agarose (100 mg-100. mu.L). Adding three times volume of Buffer QG, incubating in 50 deg.C metal bath for 10min, and shaking gently once every 2min until the gel is completely dissolved. Add one volume of isopropanol to the gel and mix well. The QIA flash column was placed in a 2ml centrifuge tube. Transferring the gel solution into QIA fast adsorption column, centrifuging for 1min at 17,900 × g, transferring and centrifuging twice if the volume is more than 800 μ L, discarding waste liquid in the collection tube, and returning the adsorption column to the collection tube. The column was then centrifuged at 17,900 Xg for 1min with 500. mu.L Buffer QG, and the filtrate was discarded. Adding 750 μ L Buffer PE, centrifuging for 1min, pouring off waste liquid in the collecting tube, and placing the adsorption column back into the collecting tube. Centrifuging the adsorption column again at 17,900 Xg for 1min, discarding the waste liquid in the collection tube, returning the adsorption column to the collection tube, and allowing the column to stand at room temperature for 3min for complete air drying. The column was loaded into a fresh 1.5ml centrifuge tube. Suspending 50 μ L Buffer EB in the middle of the adsorption column, standing at room temperature for 2min, centrifuging at 17,900 Xg for 2min to elute DNA solution, and storing at-20 deg.C for subsequent enzyme digestion.

Double enzyme digestion of NSs target gene fragment and VR1012 vector (Wuhan vast Ling Biotech Co., Ltd.)

Enzyme cutting conditions are as follows: enzyme digestion is carried out for 90min at 37 ℃, and inactivation is carried out for 5min at 65 ℃. Adding 10 mu L of 6xDNA loading buffer into the enzyme digestion product, uniformly mixing, carrying out 1% agarose gel electrophoresis detection, and then purifying and recovering the target fragment by using a QIA rapid agarose gel recovery kit.

The NSs enzyme cutting product is connected with a VR1012 carrier

And (3) slightly and uniformly mixing the enzyme digestion product of the target gene and the VR1012 vector according to the molar ratio of 3:1, and connecting for 2 hours at room temperature, wherein the system refers to the specification.

Transformation of ligation products and identification of positive clones

mu.L of DH 5. alpha. competence was placed on ice for 10min, after it was slowly melted, 5. mu.L of ligation product was added, gently mixed and ice-cooled for 30 min. The EP tube was placed in a metal bath previously warmed to 42 ℃ and heat-shocked for 90s without shaking the tube. The tube was quickly transferred to ice and allowed to stand for 2min to cool the cells. Add 500. mu.L of non-resistant LB medium to each tube, incubate for 45min in a 37 ℃ constant temperature shaker to resuscitate the bacteria and express the plasmid-encoded antibiotic resistance gene. The bacterial liquid is coated on a solid culture plate with kanamycin resistance by a coating rod, and after the bacterial liquid is completely absorbed, the bacterial liquid is placed in an incubator at 37 ℃ for overnight culture. mu.L of LB medium containing kanamycin resistance was taken and added to 1.5ml of EP tubes, and single colonies on the plates were picked and placed in the EP tubes and shaken at 220rpm in a constant temperature shaker at 37 ℃ for 3 hours. Then carrying out PCR identification on the bacterial liquid. The PCR product was subjected to 1% agarose gel electrophoresis to identify positive clones. Obtaining the NSs expression vector of the SFTSV, the SFSV and the UUUKV non-structural proteins.

Extraction of recombinant plasmids

The bacterial culture corresponding to the identified positive clone was inoculated into 5ml of LB medium containing resistance for overnight culture. And (5) preserving the strains. 500 μ L of overnight cultured bacterial liquid was put in a 1.5ml EP tube, and equal volume of sterilized 50% glycerol was added thereto, and after mixing, the strain was stored in a refrigerator at-80 ℃ for use. Column equilibration step: 500. mu.L of the equilibration solution BL was added to the adsorption column CP3 (the adsorption column was placed in the collection tube), and the tube was centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to remove the waste solution from the collection tube, and the adsorption column was replaced in the collection tube. 1-5ml of overnight-cultured bacterial liquid is taken and added into a centrifuge tube, and is centrifuged for 1min at 12,000rpm (13,400 Xg) by using a conventional desktop centrifuge, and the supernatant is removed as much as possible (when the bacterial liquid is more, the bacterial precipitate can be collected into one centrifuge tube by centrifuging for many times). To the centrifuge tube where the pellet of bacteria was left, 250. mu.L of the solution P1 (RNaseA was added) was added, and the pellet of bacteria was thoroughly suspended using a pipette or a vortex shaker. 250 μ L of the solution P2 was added to the centrifuge tube, and the tube was gently turned upside down 6 to 8 times to lyse the cells sufficiently. Add 350. mu.L of solution P3 into the centrifuge tube, turn gently up and down 6-8 times immediately, mix well, at which time white flocculent precipitate will appear. Centrifuge at 12,000rpm (. about.13,400 Xg) for 10 min. The supernatant collected in the previous step was pipetted into adsorption column CP3 (the adsorption column was put into the collection tube), taking care not to aspirate the pellet as much as possible. Centrifuge at 12,000rpm (13,400 Xg) for 60sec, remove waste liquid from the collection tube, and place the adsorption column CP3 in the collection tube. 500 μ L of deproteinized liquid PD was added to the adsorption column CP3, centrifuged at 12,000rpm (. about.13,400 Xg) for 60sec, the waste liquid in the collection tube was discarded, and the adsorption column CP3 was replaced in the collection tube. 600. mu.L of the rinsing solution PW (absolute ethanol added) was added to the adsorption column CP3, centrifuged at 12,000rpm (. about.13,400 Xg) for 60sec, the waste liquid in the collection tube was discarded, and the adsorption column CP3 was placed in the collection tube. And repeating the operation step k. The adsorption column CP3 was placed in a collection tube and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min in order to remove the residual rinse from the adsorption column. The adsorption column CP3 was placed in a clean centrifuge tube, 100. mu.L of elution buffer EB was added dropwise to the middle of the adsorption membrane, and the mixture was left at room temperature for 2min and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min to collect the plasmid solution in the centrifuge tube.

Mass preparation of endotoxin-removing plasmids

100ml of overnight-cultured broth was centrifuged at 10,000rpm (11,500 Xg) at room temperature for 3min to collect the bacteria. The supernatant was removed as much as possible, and 10ml of the solution P1 (RNaseA had been added) was added to the centrifuge tube containing the bacterial cell pellet, and the bacterial cell pellet was suspended by using a pipette and sufficiently rinsed. Adding 10ml of solution P2 into the centrifuge tube, immediately turning gently up and down for 6-8 times, mixing well, and standing at room temperature for 5 min. Adding 10ml of solution P4 into a centrifuge tube, immediately and gently turning up and down for 6-8 times, and fully mixing until the solution appears white dispersed flocculent precipitate. Then, the mixture is placed at room temperature for about 10 min. Centrifuge at 10,000rpm (. about.11,500 Xg) for 10min to allow the white precipitate to settle to the bottom of the tube, carefully pour the entire solution into filter CS1, filter by slowly pushing the push handle and collect the filtrate in a clean 50ml tube. To the filtrate were added 0.35 times the filtrate volume of isopropanol and 1/2 times the isopropanol volume of 5M NaCl, and mixed well by inverting upside down. Centrifuge at 10,000rpm (. about.11,500 Xg) for 30min at 4 ℃ and decant the supernatant and invert it onto absorbent paper. The pellet was rinsed thoroughly by adding 6ml 70% ethanol to the tube, centrifuged 10min at 10,000rpm (. about.11,500 Xg) at 4 ℃ and the supernatant decanted and inverted on absorbent paper. The tube was left open at room temperature for 20min to allow ethanol to evaporate sufficiently, and 1.5ml of elution buffer TB was added to dissolve the precipitate sufficiently. The DNA products-SFTSV, SFSV, UUKV nonstructural protein NSs expression plasmids were stored at-20 ℃ for subsequent transfection.

Example 2 cell culture, transfection, detection of Interferon promoter Activity

Cell culture

1)293T cells (

CRL-3216^TM) Culture of (2)

293T cells are cultured in a DMEM liquid medium containing 10% FBS and 1% penicillin-streptomycin, incubated and cultured in an incubator at 37 ℃ and containing 5% C02, after the cells are fully grown, the cells are digested by pancreatin containing 0.25% EDTA, after about 2-3min, 10% FBS fresh medium is added for termination, a room temperature centrifuge is used for 1200rpm and 3min, supernatant is removed, a proper amount of fresh medium is taken for re-suspension, one third of the cells are returned to a culture bottle, and the rest is transferred to a 24-well cell culture plate.

2) HeLa cell(s) ((s))

CCL-2^TM) Culture of (2)

HeLa cells were cultured in DMEM liquid medium containing 10% FBS and 1% penicillin-streptomycin, incubated at 37 ℃ in an incubator containing 5% C02. After about 2-3 days, digesting with pancreatin containing 0.25% EDTA, after about 3-5inin, adding 10% FBS fresh culture medium to terminate, centrifuging at 1200rpm for 3min at room temperature, removing supernatant, taking a proper amount of fresh culture medium to resuspend, placing one third of the fresh culture medium in a culture flask to continue culturing, and transferring the rest to a 24-well cell culture plate. .

Sendai virus (a)

VR-907^TM) Culturing

1) Preparation of chick embryos

Selecting SPF-grade 10-day-old chick embryos (Meidiyautong laboratory animal technology Co., Ltd.), marking an air outlet chamber by using an egg candler, and marking a gap between two blood vessels in an area with obvious blood vessels below the air chamber to serve as an inoculation part; wiping with 75% alcohol twice, drying, sterilizing, perforating at the inoculation position, and vertically placing on the egg rack with the air chamber facing upwards.

2) Preparation of virus inoculation material. Sterilizing the sterilized injector and the elbow tweezers by high pressure and moist heat.

3) Injecting and inoculating in allantoic cavity: sucking Sendai virus with 1ml syringe with No. 6 needle, inserting needle into the chick embryo direction for 0.5-1cm from the punched small hole, injecting 200 μ L, sealing the small hole with adhesive tape, adding melted solid paraffin, sealing, placing back into incubator, incubating, turning egg twice every day, observing once, and discarding the dead in 24 hr.

4) And (5) harvesting the virus. Sendai virus material, harvested 48 hours after inoculation. Before harvesting, chicken embryo should be refrigerated in refrigerator at 4 deg.C overnight to avoid blood vessel rupture during dissection, eggshell is sterilized with 75% alcohol, eggshell and allantoic membrane are removed with forceps, urine sac liquid is carefully sucked into centrifugal tube with pipettor, 8ml can be collected, centrifuging at 3000rpm for 30min, removing impurities, subpackaging, and storing at-80 deg.C.

Cell transfection and double reporter Gene detection

293T cells are inoculated into a 24-well plate, and the cells are cultured for about 12h and transfected after the cells grow to 70-80% confluence. SFTSV, SFSV, UUKV NSs expression plasmids or a control vector VR1012 were mixed with 0.5. mu.g of pIFN-. beta. -luc plasmid (constructed by inserting the full-length human IFN-. beta.promoter into pGL3-Luc (Promega) vector) and 0.5ng of pRL-TK (Wuhan vast Ling Biotech Co., Ltd.) using TransLipid HL transfection reagent, diluted in 50. mu.L, Opti-MEM medium, 3. mu.L TransLipid HL was diluted in 50. mu.L-MEM medium, and allowed to stand at room temperature for 5 min. Mixing the above two solutions gently, and standing at room temperature for 20 min. The plasmid-TransLipid HL mixture was added to 293T cells in 24-well plates and the 293T cells were co-transfected. After 24h of transfection, Sendai virus (SEV, 20HA/ml) was added for stimulation, and cells were harvested 18h after virus stimulation. Centrifuging, removing supernatant, gently rinsing with PBS twice, adding 1 × passive lysine buffer (1 ml of 5 × passive lysine buffer into 4ml of deionized water), and subjecting to room temperature rotary shaking table for 30min for complete lysis. The cell lysate was pipetted into an EP tube and centrifuged at 8.000rpm for 5 min. 40 μ L of the supernatant was added to a 96-well plate. According to the standard operation steps of Dual-luciferase-assay (Promeg, Cat No: E1910), an LB960 multifunctional detector is used for detecting the value of the reporter gene, and the data after the treatment of the firefly luciferase/renilla luciferase is analyzed to obtain the activation value of the reporter gene.

Immunoblotting (Western-Blot)

Sample preparation

48h after transfection, the supernatant culture was discarded and 1ml PBS (8 g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH₂PO₄Tween-201 ml, water to 1L, stored at room temperature) the 293T cells were blown up and collected in 1.5ml EP tubes. Centrifuge at 8000rpm for 5min, and discard the supernatant. 80 μ of LRAPI, 20 μ L of 5 XSDS loading buffer was added and the cells were lysed by repeatedly pipetting with a pipette. Heating at 95 deg.C for 30min, vortex shaking, and centrifuging at 8000rpm for 5 min. Storing at-40 deg.C for use. Western-Blot

The denatured protein samples prepared were sequentially added to 12% polyacrylamide gel sample wells at 10. mu.L per well, and 1 XSDS gel loading buffer was added to the unused sample wells. Connecting the electrophoresis device with a power supply, running for 30min at 90V, increasing the voltage to 120V when the sample runs to the interface of the lamination glue and the separation glue, continuing electrophoresis until the bromophenol blue reaches the bottom of the separation glue, and then closing the power supply. And after electrophoresis is finished, taking out the glass plate, prying the glass plate, and cutting off the laminated layer glue. And (3) soaking the pre-cut NC membrane and the membrane-transferring filter paper in a culture dish containing 1 multiplied by semi-dry membrane-transferring buffer solution. And (3) placing the film transfer filter paper on the bottom layer, placing the NC film on the film transfer filter paper, placing the protein glue on the NC film, and finally covering a layer of film transfer filter paper on the NC film. And (3) placing the sandwich formed by the filter paper and the NC film into a semi-dry film converter, and paying attention to the fact that the NC film is close to the anode and the protein glue is close to the cathode. The film transfer condition was 18V for 25 min. And after the membrane conversion is finished, taking out the NC membrane, and sealing for 60min by using 5% milk sealing liquid. anti-HA tag (Sigma) or anti-Tublin (Sigma) antibody was incubated as primary antibody and incubated overnight at 4 ℃. After overnight incubation, membranes were washed three times with PBST for 10min each. Then, a corresponding horseradish peroxidase-labeled secondary antibody (Abcam) was attached and incubated for one hour. The membrane was then washed three times with PBST for 10min each. And (3) uniformly mixing the ECL developing solution according to the ratio of 1:1, dripping the ECL developing solution onto an NC membrane, carrying out a light-shielding reaction for 3min, and placing the mixture into a Chemi Doc (TM) XRS + gel imager for developing.

Statistical analysis

Data mean and standard error were obtained by GraphPad Prism (version 5.01) analytical processing. All experiments were repeated at least 3 times and all data were evaluated by the T test for P values, which were considered statistically significant when less than 0.05.

Example 3 inhibition of IFN- β promoter Activity by SFTSV SFSV UUUKV NSs

Experiments such as cell transfection, fluorescence double-reporter assay and Western blotting were performed according to the procedures specified (the same procedure as in example 2)

200ng RIG-I-Flag plasmid (Addge) was co-transfected with 200ng VR1012 vector, 50ng SFTSV NSs and 500ng SFSV or UUKV NSs, respectively, into 293T cells, along with 500ng IFN-. beta. -Luc and 50ng pRL-TK plasmid. 48 hours after transfection, the expression of the reporter gene was detected by a double reporter gene detection method. NSs protein expression levels are shown in FIG. 1 by Western-Blot results. The double-reporter gene detection result shows that the NSs protein of the virus obviously inhibits the activation of the RIG-I induced interferon beta promoter, and the result also indicates that the NSs protein inhibits the production of host type I interferon.

Example 4, SFTSV SFSV NSs induce degradation of RIG-I-Flag

Experiments such as cell transfection, fluorescence double-reporter assay and Western blotting were performed according to the procedures specified (the same procedure as in example 2)

200ng RIG-I-Flag plasmid (Addge) was co-transfected with 200ng VR1012 vector, 50ng SFTSV NSs and 500ng SFSV NSs into 293T cells, along with 500ng IFN-. beta. -Luc and 50ng pRL-TK plasmid, respectively. 48 hours after transfection, the expression of the reporter gene was detected by a double reporter gene detection method. NSs protein expression levels are shown in FIG. 2 by Western-Blot results. The double-reporter gene detection result shows that the NSs protein of the virus obviously inhibits the activation of RIG-I induced interferon beta promoter, and when SFTSV and SFSV NSs are expressed, the RIG-I-Flag level in cells is obviously reduced, so that the induction of RIG-I degradation by SFTSV and SFSV NSs is prompted.

Example 5: PS-341 inhibits SFTSV SFSV NSs-mediated RIG-I degradation

Experiments such as cell transfection, fluorescence double-reporter assay and Western blotting were performed according to the procedures specified (the same procedure as in example 2)

To explore whether RIG-I degradation occurs through direct activation of the NSs protein or through non-direct degradation via the proteasome pathway. Next we analyzed the effect of NSs on RIG-I expression and interferon-beta promoter activation at the cellular level upon addition of PS-341. 200ng RIG-I-Flag and VR1012,50ng SFTSV NSs and 500ng SFSV NSs were co-transfected into 293T cells, respectively, and simultaneously transfected with 500ng reporter gene plasmid IFN-. beta. -Luc and 50ng Renilla luciferase pRL-TK plasmid. 36 hours after transfection, the culture broth was supplemented with PS-341 to a final concentration of 50 nM. After 12 hours, the expression of the reporter gene was detected by the double reporter gene detection method. NSs protein expression levels are shown in FIG. 3 by Western-Blot results. The experimental results are shown in fig. 3: PS-341 apparently stabilized the accumulation of RIG-I in cells. It can be seen that intracellular RIG-I levels were essentially consistent with each other regardless of the presence of NSs protein, and that the interferon promoter activity was restored in transfected SFSV NSs cells.

Example 6: PS-341 inhibits UUUKV NSs-mediated RIG-I degradation

Experiments such as cell transfection and Western blotting were carried out according to the procedures (the same as in example 2)

To determine if the NSs protein of UUUKV is identical to other phleboviruses, it interacts with RIG-I. The plasmid expressing RIG-I-Flag protein and the plasmid carrying HA-tagged UUUUKV NSs protein are transfected into 293T cells together, and PS-341 is supplemented in No. 3 culture solution 36 hours after transfection till the final concentration is 50 nM. After 12 hours, NSs protein expression levels are shown in FIG. 4 by Western-Blot results. It can be seen from the results that PS-341 treatment restored RIG-I levels in UUUKV NSs expressing cells (compared to cell No. 2). We found that the NSs protein of UUUKV overexpressed also affected the accumulation of RIG-I in cells. The effect of PS-341 was tested in the case of UUUKV NSs protein overexpression, and similarly, PS-341 restored RIG-I expression and enhanced UUKV NSs protein stable expression, compared to cells transfected with empty vector. These results demonstrate that the NSs protein of UUKV induces degradation of RIG-I through the ubiquitinated proteasome pathway. UUUKV is used as a tick-borne virus model, has an important role in researching the interaction between host cells and viruses, and is used as an important model in researching the interaction mechanism between viruses and hosts. UUKV NSs protein has been recently reported to inhibit interferon-beta production, and we found for the first time that its NSs protein degrades RIG-I.

Example 6 inhibition of intracellular SFTSV replication by PS-341

293T cells or HeLa cells were cultured in 24-well cell culture plates. When the cell density reaches 70-80%, the cells are counted. The SFTSV HB29 strain infected the cells at 1 MOI. The first group of cells is used as a blank control, and the second group of cells is infected with virus and is supplemented with PS-341 to a final concentration of 50nM in the culture solution; 1 hour before the third group was infected, the culture broth was supplemented with PS-341 to a final concentration of 50nM (FIG. 5). Culturing the three groups of cells for 24 hours or 48 hours, taking 100 mu l of culture supernatant, and extracting virus RNA by using a Qiaamp Viral RNA Mini Kit according to the instruction; the virus content in the culture supernatant is detected by one-step Real-time PCR. Chinese disease prevention and control center virus disease prevention and control. The reagents used were: TaqMan Real-Time PCR Master mixers (ThermoFisher, Cat No:4444557), according to the instructions;

primer probes used in Real-time PCR are shown in the following table:

SEQUENCE LISTING

<110> Tianjin university

Application of <120> compound PS-341 in preparation of bunyaviridae phlebovirus inhibitor

<130>

<160> 9

<170> PatentIn version 3.3

<210> 1

<211> 34

<212> DNA

<213> Artificial Synthesis

<400> 1

acgcgtcgac accatgtcgc tgagcaaatg ctcc 34

<210> 2

<211> 59

<212> DNA

<213> Artificial Synthesis

<400> 2

cgggatccct acgcgtaatc tgggacgtcg taagggtaga cctccttcgg gaggtcacc 59

<210> 3

<211> 38

<212> DNA

<213> Artificial Synthesis

<400> 3

acgcgtcgac accatgatga acagccgata catgtttg 38

<210> 4

<211> 62

<212> DNA

<213> Artificial Synthesis

<400> 4

cgggatccct acgcgtaatc tgggacgtcg taagggtaaa agtcagaatc agacgagctc 60

tc 62

<210> 5

<211> 40

<212> DNA

<213> Artificial Synthesis

<400> 5

acgcgtcgac accatgtctt acttcactat ccagaacgag 40

<210> 6

<211> 58

<212> DNA

<213> Artificial Synthesis

<400> 6

cgggatccct acgcgtaatc tgggacgtcg taagggtaca gtgatcctac gactggcc 58

<210> 7

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 7

gggtccctga aggagttgta aa 22

<210> 8

<211> 24

<212> DNA

<213> Artificial Synthesis

<400> 8

tgccttcacc aagactatca atgt 24

<210> 9

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 9

ttctgtcttg ctggctccgc gc 22

Claims (1)

1. The application of a compound PS-341 in preparing a bunyaviridae phlebovirus inhibitor is disclosed, wherein the compound PS-341 has a structure shown in a formula (I):

the virus of phlebovirus of bunyaviridae is fever-associated thrombocytopenia syndrome virus, Wukuh virus or Sicily phlebovirus.

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