Use of clic1 inhibitors in the preparation of a medicine for combating zika virus infection

Abstract

The application belongs to the field of biological medicine, and relates to application of a CLIC1 inhibitor in preparation of an anti-zika virus infection drug, wherein the CLIC1 inhibitor comprises at least one of the following two substances: I: a substance for inhibiting expression of a CLIC1 gene in a host cell; II: a substance for inhibiting a CLIC1 chloride channel function in the host cell. The application first discloses that the CLIC1 gene and protein are key host factors for zika virus replication, and zika virus replication can be blocked by inhibiting the expression or chloride channel function, thereby providing a new direction for development of an anti-zika virus drug targeting a host. Compared with traditional drugs targeting zika virus proteins, the anti-zika virus infection drug prepared by using the CLIC1 inhibitor targets a host factor, is more difficult to induce virus drug resistance, and has more persistent treatment potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, and in particular to the application of a Chloride Intracellular Channel 1 (CLIC1) inhibitor in the preparation of a drug for resisting Zika virus (ZIKV) infection. BACKGROUND

[0002] Zika virus is one of the potential "Disease X" candidate pathogens defined by the World Health Organization (WHO), and is an important object of virological research in the field of global public health. Important progress has been made in the study of its biological characteristics and pathogenic mechanism in recent years: in terms of transmission mode, in addition to the classic Aedes aegypti vector transmission route, the virus has been confirmed to be able to transfer between hosts through vertical transmission (mother-to-child) and sexual contact, etc. In terms of pathogenic mechanism, clinical and basic research shows that Zika virus infection is clearly associated with a variety of pathological phenotypes, including adult Guillain-Barre syndrome, neonatal microcephaly and meningoencephalitis, etc. nervous system damage, and specific tropism of the virus to testicular tissue leading to long-term persistence (more than 6 months) of viral RNA in semen and hematospermia, etc. reproductive system pathological changes, suggesting that it has the biological characteristics of multi-organ invasion.

[0003] Based on the prediction analysis of epidemiological models, it is pointed out that about half of the world's population is susceptible due to the lack of pre-existing immune protection, and the potential for virus variation, the risk of large-scale Zika virus epidemic in the future still needs to be highly concerned. Although the public health threat of Zika virus has been widely recognized by the international community, there is still a significant gap in the prevention and treatment of the virus. At present, there is still no approved vaccine or specific antiviral drug, and the development of anti-Zika virus infection drugs is imminent. SUMMARY

[0004] The inventors found that inhibiting the expression of CLIC1 gene can inhibit the replication of Zika virus in host cells, and known chloride channel inhibitors that inhibit the function of CLIC1 chloride channel in host cells can also inhibit the replication of Zika virus in host cells. Based on this finding, the present application provides the application of a CLIC1 inhibitor in the preparation of a drug for resisting Zika virus infection.

[0005] The technical solution provided by the present application is as follows:

[0006] The present application provides the application of a CLIC1 inhibitor in the preparation of a drug for resisting Zika virus infection, wherein the CLIC1 inhibitor comprises at least one of the following two substances:

[0007] I: a substance for inhibiting expression of a CLIC1 gene in a host cell;

[0008] II: a substance for inhibiting a CLIC1 chloride channel function in a host cell.

[0009] In some embodiments of the present application, the anti-zika virus infection drug comprises a CLIC1 inhibitor as an effective component for inhibiting replication of the zika virus in the host cell.

[0010] In some embodiments of the present application, the substance for inhibiting expression of a CLIC1 gene in a host cell comprises at least one of siRNA, shRNA, an siRNA plasmid, and an shRNA plasmid.

[0011] In some embodiments of the present application, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO: 1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO: 2.

[0012] In some embodiments of the present application, the anti-zika virus infection drug comprises a substance for inhibiting expression of a CLIC1 gene in a host cell and a carrier system thereof, and the carrier system is at least one of a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a liposome, a lipid nanoparticle, a cationic polymer, an exosome, an inorganic nanoparticle, and a polypeptide carrier.

[0013] In some embodiments of the present application, the substance for inhibiting a CLIC1 chloride channel function in a host cell is a chloride channel inhibitor.

[0014] In some embodiments of the present application, the chloride channel inhibitor is 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or R-(+)-methylindazolone (R(+)-IAA-94).

[0015] In some embodiments of the present application, the zika virus infection comprises upregulation of at least one of the following indicators: zika virus RNA level, zika virus structural protein E (E) expression level, and zika virus non-structural protein NS5 (NS5) expression level.

[0016] In some embodiments of the present application, the anti-zika virus infection drug is used for downregulating at least one of the following indicators: zika virus RNA level, zika virus structural protein E expression level, and zika virus non-structural protein NS5 expression level.

[0017] In some embodiments of the present application, the dosage form of the anti-zika virus infection drug includes at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, and transdermal administration.

[0018] In some embodiments of the present application, the anti-zika virus infection drug is used for preventing one of the following diseases caused by zika virus infection: congenital microcephaly, abnormal fetal brain development, placental inflammation, adult joint pain, or conjunctivitis.

[0019] Compared with the prior art, the present application has at least the following beneficial effects:

[0020] The present application first discloses that CLIC1 is a key host factor for zika virus replication, and the zika virus replication can be blocked by inhibiting the gene expression or the chloride ion channel function of CLIC1, thereby providing a new direction for developing an anti-zika virus drug targeting a host. Compared with the traditional drug targeting zika virus protein, the CLIC1 inhibitor targets a host factor, is more difficult to induce zika virus drug resistance, and has more persistent treatment potential. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0022] Figure 1 : Verification of the interaction between CLIC1 and zika virus NS5; wherein, A: flow chart of the immunoprecipitation-high resolution mass spectrometry combined technique for screening zika virus NS5 interacting host proteins; B: Co-IP verification in an exogenous overexpression system: HEK293T cells were co-transfected with pHA-CLIC1 plasmid and pFlag-NS5 plasmid, and after the lysate was precipitated by Flag antibody, Western blot detection confirmed that CLIC1 interacted with NS5; C: Interaction verification in an endogenous zika virus infection model: after zika virus infected HeLa cells for 48 h, Co-IP was performed using CLIC1 antibody, and Western blot detection of the interaction between zika virus NS5 and endogenous CLIC1; D: Subcellular co-localization analysis: HeLa cells co-transfected with pHA-CLIC1 plasmid and pFlag-NS5 plasmid were subjected to immunofluorescence staining, which showed that CLIC1 (red) and NS5 (green) presented significant co-localization (yellow combined signal) in the cytoplasm.

[0023] Figure 2: The regulatory role of CLIC1 in Zika virus replication; A: Zika virus (MOI = 1) infection for 24 h after transfection with siCLIC1, RT-qPCR showed that the viral RNA level in cells was significantly reduced; B: Western blot results showed that the expression of E and NS5 in cells transfected with siCLIC1 was significantly reduced, and CLIC1 was significantly reduced; C: Broad-spectrum verification (HeLa model): siCLIC1 treatment significantly inhibited the protein expression of E and NS5 in HeLa cells; D: After gradient transfection of pHA-CLIC1 (0, 0.5, 1.0 μg), the Zika virus RNA level showed a dose-dependent increase; E: Western blot results showed that the protein level of E and NS5 in HTR-8 cells overexpressing CLIC1 was significantly up-regulated; F: HeLa model verification: CLIC1 overexpression also significantly enhanced the protein expression of Zika virus E and NS5.

[0024] Figure 3 : Inhibitory effect of CLIC1 inhibitor on Zika virus replication; A: Chemical structure of NPPB; B: Antiviral effect of NPPB: In Zika virus-infected HTR-8 cells, NPPB treatment showed a dose-dependent inhibition of viral RNA replication; C: Chemical structure of R(+)-IAA-94; D: Antiviral effect of R(+)-IAA-94: Gradient concentration of R(+)-IAA-94 treatment significantly reduced the viral RNA level.

[0025] Figure 4 : Map of pHA-CLIC1 plasmid (i.e. pXJ40-HA-CLIC1 in the figure, pXJ40 is the name of the vector backbone of the plasmid). DETAILED DESCRIPTION

[0026] In order to make the objects, technical solutions and advantages of the present application clearer, the technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0027] In the present disclosure, the term "CLIC1" refers to a protein in the cell, mainly located in the nucleus and exhibiting nuclear membrane and plasma membrane chloride channel activity, which can regulate basic cell processes, including stabilizing cell membrane potential, trans-epithelial transport, maintaining intracellular pH, and regulating cell volume. The term "CLIC1 gene" refers to a gene encoding CLIC1; the term "CLIC1 mRNA" refers to a messenger RNA encoding CLIC1. The term "CLIC1 antibody" refers to a specific immunoglobulin against chloride channel protein 1.

[0028] In the present disclosure, the term "NS5" refers to a non-structural protein encoded by Zika virus, mainly composed of a methyltransferase domain and an RNA-dependent RNA polymerase domain. The term "NS5 antibody" refers to a specific immunoglobulin produced against Zika virus non-structural protein NS5; the term "Zika virus E antibody" refers to a specific immunoglobulin produced against Zika virus structural protein E.

[0029] In the present disclosure, the term "siRNA" refers to small interfering RNA, which is an exogenous artificially synthesized double-stranded RNA molecule with a length of about 21-23 nt, which can inhibit the expression of CLIC1 mRNA by degrading it.

[0030] In the present disclosure, the term "shRNA" refers to short hairpin RNA, which is an exogenous artificially designed single-stranded RNA molecule with a length of about 50-70 nt, which forms a "stem-loop" structure through its base complementarity, and finally inhibits gene expression by generating siRNA.

[0031] In the present disclosure, the term "Co-IP" refers to co-immunoprecipitation, which is an experimental method for verifying the interaction between proteins, by precipitating the target protein and its associated protein complex with specific antibodies.

[0032] In the present disclosure, the term "RT-qPCR" refers to reverse transcription-quantitative polymerase chain reaction, which is a molecular biology technique for detecting RNA levels in cells. The present disclosure uses this technique to detect viral RNA levels in cells for analysis of viral replication.

[0033] In the present disclosure, the term "MOI" refers to multiplicity of infection, which is an index for indicating the ratio of the number of viruses to the number of cells when the virus infects the cells. In the present disclosure, "MOI = 1" means that each cell is infected with an average of 1 virus particle.

[0034] In the present disclosure, the term "NPPB" refers to Figure 3 A chloride channel inhibitor of the structure shown in the present disclosure can directly bind to the pore region of the CLIC1 channel, thereby inhibiting the function of the CLIC1 chloride channel in host cells through steric hindrance, and thereby inhibiting Zika virus replication.

[0035] Herein, the term "R(+)-IAA-94" refers to Figure 3 C, which can target the gating region of the CLIC1 channel, stabilize the "closed conformation" of the channel, and thus inhibit the CLIC1 function and Zika virus replication.

[0036] Herein, the term "vector system" refers to a tool for delivering siRNA or shRNA, including lentiviral vectors, adenoviral vectors, liposomes, lipid nanoparticles, cationic polymers, etc., which can improve the delivery efficiency and stability.

[0037] Herein, the term "Western blot" refers to a protein blotting method, which is an experimental method for detecting the expression level of specific proteins (such as CLIC1, E, and NS5) in cells.

[0038] Herein, the term "plasmid" refers to a circular DNA vector that can carry exogenous genes (such as the CLIC1 gene) or substances that inhibit gene expression (such as the coding sequence of siRNA or shRNA) for overexpression or inhibition of specific genes in cells.

[0039] Herein, the term "exogenous overexpression system" refers to an experimental system in which the target protein is overexpressed in cells by introducing an exogenous gene (such as a plasmid encoding CLIC1 or NS5) into host cells (such as HEK293T cells), which is used to study the interaction or functional regulation between proteins.

[0040] The technical solutions of the present application are described in detail as follows:

[0041] The infection cycle of Zika virus begins with the entry of viral particles into cells through endocytosis mediated by various host receptors (such as AXL, TIM-1, DC-SIGN, etc.), followed by the release of genomic RNA into the cytoplasm. Viral RNA is translated to generate a polyprotein precursor, which is cleaved by host proteases and the viral NS2B-NS3 complex to produce 3 structural proteins (referred to as C, prM / M, and E, respectively) and 7 non-structural proteins (referred to as NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, respectively). Therefore, these viral RNAs, structural proteins, and non-structural proteins can serve as markers for Zika virus replication, which can be used to study the mechanism of CLIC1 inhibitors inhibiting Zika virus replication in host cells.

[0042] The present application found that CLIC1 and NS5 directly bind in cells in host cells to form a complex, and that inhibition of CLIC1 gene expression in host cells and inhibition of the chloride channel function of CLIC1 in host cells can inhibit Zika virus replication in host cells.

[0043] Based on this, the application provides the use of a CLIC1 inhibitor in the preparation of an anti-zika virus infection drug, wherein the CLIC1 inhibitor comprises at least one of the following two substances: I: a substance that inhibits the expression of a CLIC1 gene in a host cell; II: a substance that inhibits the function of a CLIC1 chloride channel in a host cell.

[0044] The application is verified by Co-IP in an exogenous overexpression system, and it is found that CLIC1 is directly combined with NS5, and it is speculated that the zika virus combines with CLIC1 through NS5, and uses CLIC1 as a host protein for promoting virus replication.

[0045] The application is verified by a zika virus infection model, and it is found that knocking down CLIC1 by using an siRNA gene silencing technology can significantly inhibit zika virus replication (P<0.01). Figure 2 In addition, treating cells by using a substance (NPPB or R(+)-IAA-94) that inhibits the function of a CLIC1 chloride channel in a host cell can also significantly reduce zika virus replication (P<0.01). Figure 3 The above experimental results show that CLIC1 is an important host factor for zika virus replication, and inhibiting the expression of a CLIC1 gene in a host cell and inhibiting the function of a CLIC1 chloride channel in a host cell can both inhibit the replication of zika virus in a host cell, thereby slowing down the infection process of zika virus and achieving the prevention, alleviation and / or treatment of zika virus infection.

[0046] In some embodiments of the application, the anti-zika virus infection drug comprises a CLIC1 inhibitor as an effective component for inhibiting the replication of zika virus in a host cell. The drug can also comprise other components for inhibiting the replication of zika virus in a host cell or components for inhibiting inflammation for the synergistic treatment of zika virus infection.

[0047] In some embodiments of the present invention, the substance that inhibits CLIC1 gene expression in host cells includes at least one of siRNA, shRNA, siRNA plasmid, and shRNA plasmid. These substances ultimately inhibit CLIC1 gene expression by generating siRNA and activating the RNAi pathway, thereby degrading CLIC1 mRNA. Specifically, siRNA refers to an exogenously synthesized double-stranded RNA molecule approximately 21-23 nt in length, which directly enters the cytoplasm of the host cell through transfection or infiltration; siRNA plasmid refers to an artificially constructed circular DNA vector containing the coding sequence of siRNA and a promoter, including the RNA polymerase III promoter U6 or H1; shRNA refers to an exogenously designed single-stranded RNA molecule approximately 50-70 nt in length, forming a "stem-loop" structure through self-base complementarity, with the stem complementing CLIC1 mRNA and the loop being a non-complementary sequence; and shRNA plasmid refers to a circular DNA vector carrying the shRNA coding sequence, containing the shRNA DNA template and a promoter, where the shRNA DNA template refers to the double-stranded DNA corresponding to the stem-loop structure.

[0048] Preferably, the substance that inhibits CLIC1 gene expression in host cells is siRNA, which can directly act as an effector molecule to rapidly silence target gene mRNA in a short period. In some embodiments of the present invention, the siRNA is siCLIC1; wherein:

[0049] Nucleotide sequence of the positive strand of siCLIC1:

[0050] 5'-GUGGAUGAAACCAGUGCUGAA-3' (SEQ ID NO: 1);

[0051] Nucleotide sequence of the siCLIC1 antisense strand:

[0052] 5'-UUCAGCACUGGUUUCAUCCAC-3' (SEQ ID NO: 2).

[0053] As short-chain RNA molecules, siRNA is easily degraded by nucleases and has difficulty actively crossing the cell membrane (due to negative charge and cell membrane repulsion). Therefore, it requires delivery aids via vector systems to improve stability, cellular uptake efficiency, and targeting. In some embodiments of the present invention, the anti-Zika virus infection drug includes a substance that inhibits CLIC1 gene expression in host cells and its vector system. The vector system is at least one of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, liposomes, lipid nanoparticles, cationic polymers, exosomes, inorganic nanoparticles, and peptide carriers.

[0054] In some embodiments of the present application, the substance that inhibits the function of CLIC1 chloride channel in host cells is a chloride channel inhibitor, which can reduce the opening frequency of CLIC1 channel. For example, it is known that NPPB can directly bind to the pore region of CLIC1 channel, especially the positively charged amino acid residues (such as arginine, lysine) at the entrance of the channel, and has a steric hindrance effect; R(+)-IAA-94 targets the gating region of CLIC1 channel, which can stabilize the "closed conformation" of the channel. Experiments have shown that they can inhibit the replication of Zika virus.

[0055] In some embodiments of the present application, the Zika virus infection includes upregulation of at least one of the following indicators ('upregulation' refers to an increase in the level of the corresponding indicator in the body of a healthy subject not infected with Zika virus): Zika virus RNA level, Zika virus structural protein E expression level, Zika virus non-structural protein NS5 expression level.

[0056] In some embodiments of the present application, the anti-Zika virus infection drug is used to downregulate (‘downregulation’ refers to a decrease in the level of the corresponding indicator in the body of a subject infected with Zika virus before using the drug) at least one of the following indicators: Zika virus RNA level, Zika virus structural protein E expression level, Zika virus non-structural protein NS5 expression level.

[0057] In some embodiments of the present application, the anti-Zika virus infection drug is used to prevent one of the following diseases caused by Zika virus infection: congenital microcephaly, fetal brain development abnormalities, placental inflammation, adult joint pain or conjunctivitis.

[0058] In some embodiments of the present application, the anti-Zika virus infection drug can be prepared into a suitable dosage form according to the needs of the clinic, and the dosage form includes but is not limited to tablets, granules, pills, powders, capsules, injections, oral liquids.

[0059] In some embodiments of the present application, the anti-Zika virus infection drug can be administered according to the needs of the clinic by using a suitable administration route, and the administration route includes but is not limited to intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, transdermal administration.

[0060] The technical solutions of the present application are described in detail below through specific embodiments. In the following examples, the experimental methods are all conventional methods unless otherwise specified. In the following examples, the materials, reagents, etc. used are commercially available unless otherwise specified. The sources of reagents used in the examples are as follows:

[0061] Table 1

[0062]

[0063] pHA-CLIC1 plasmid was constructed by the inventors using the plasmid construction method commonly used in the art, and the plasmid map is shown in Figure 4 , which is used for overexpression of CLIC1 with an influenza virus hemagglutinin (HA) tag in cells.

[0064] The construction method of pFlag-NS5 plasmid is referred to reference PBMD:32117232, which is used for overexpression of NS5 with a Flag tag in cells. The Flag tag is a short peptide composed of 8 amino acids (sequence DYKDDDK).

[0065] Nucleotide information of siCLIC1:

[0066] Nucleotide sequence of the sense strand of siCLIC1:

[0067] 5'-GUGGAUGAAACCAGUGCUGAA-3' (SEQ ID NO: 1);

[0068] Nucleotide sequence of the antisense strand of siCLIC1:

[0069] 5'-UUCAGCACUGGUUUCAUCCAC-3' (SEQ ID NO: 2).

[0070] Nucleotide information of siNC:

[0071] Nucleotide sequence of the sense strand of siNC:

[0072] 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 3),

[0073] Nucleotide sequence of the antisense strand of siNC:

[0074] 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 4).

[0075] Example 1: Construction of Zika virus infected cell model

[0076] 1. Amplification of Zika virus:

[0077] C6 / 36 mosquito cells were inoculated in T175 culture flask, when the cell confluence reached 90%, the culture medium was replaced with 20 mL MEM medium containing 2% fetal bovine serum (FBS), and 1 mL Zika virus suspension was added to the flask, and then the flask was placed in a 28°C incubator for continuous culture; 3 days after inoculation of Zika virus, 5-10 mL of MEM medium containing 2% FBS was added to the culture flask, and the cells were continuously cultured; about 30% of the cells showed obvious pathological changes under an inverted microscope on the 5th-6th day after inoculation of Zika virus; the supernatant in the culture flask was transferred into a 50 mL centrifuge tube, filtered using a sterile filter with a pore size of 0.22 μm, and then stored for use.

[0078] 2. Titration of Zika virus (virus plaque method):

[0079] The supernatant stored in step (1) was gradiently diluted using MEM medium to obtain Zika virus suspensions with different dilution degrees; BHK-21 hamster kidney cells were inoculated into a 12-well plate, and when the cell confluence reached about 70%, the original culture medium was removed, and the cells were washed with 1 mL PBS in each well; 500 μL of Zika virus suspension with different dilution degrees was added to each well, and the plate was incubated in a 37°C incubator for 4 h to allow the virus to enter the cells. After incubation, the virus suspension in each well was discarded, and the cells were washed twice with serum-free medium; then 1 mL of MEM medium containing 1.2wt% methyl cellulose and 2wt% FBS was added to each well; 4-5 days after inoculation of the virus, the 12-well plate was removed, and the bottom of the cell plate was observed under natural light, and plaques were observed; an appropriate amount of crystal violet fixing and staining solution was added to each well, and the plate was fixed and stained at room temperature overnight; the 12-well plate was gently washed with running water to wash away the methyl cellulose in the wells, and the number of plaques was counted, and the virus titer was calculated: virus titer = number of plaques × dilution factor × 2 (PFU / mL).

[0080] 3. Detection of infection and replication indicators of Zika virus:

[0081] The Zika virus suspension was taken out from a -80°C refrigerator and quickly dissolved in a 37°C water bath, and the cells were infected with 1 MOI of Zika virus for 4 h, then the virus suspension was discarded, the cells were washed with PBS for 3 times, and new culture medium was added for continuous culture. The cells and cell supernatant were collected 24 h after infection, and the detection of Zika virus replication indicators was performed, RT-qPCR experiment was used to detect the replication of viral RNA in the cells, and Western blot experiment was used to detect the expression of NS5 and E in the cells.

[0082] Example 2: Interaction between CLIC1 and NS5

[0083] 1. Screening of NS5 interacting host proteins by co-immunoprecipitation-high resolution mass spectrometry technology: as follows:Figure 1 As shown in A, using Flag antibody and HA antibody for immunoprecipitation, combined with liquid chromatography-tandem mass spectrometry to identify candidate interacting proteins, a total of 88 host proteins that can interact with NS5 were identified, among which the interaction score of CLIC1 with NS5 was higher and the reliability was high, indicating that the binding ability of the two was strong, and CLIC1 was the key candidate interacting protein.

[0084] 2. Co-IP verification in exogenous overexpression system: HEK293T cells were co-transfected with pHA-CLIC1 plasmid and pFlag-NS5 plasmid, and the lysate was precipitated by HA-CLIC1. Then, Flag-NS5 was used for Western blot detection of Flag-NS5 enrichment. Figure 1 B). The results showed that NS5 could be co-precipitated by HA-CLIC1 and detected, indicating that CLIC1 and NS5 had direct physical interaction in cells.

[0085] 3. Interaction verification in endogenous Zika virus infection model: After Zika virus infected HeLa cells for 48 h, Co-IP was performed using CLIC1 antibody, and NS5 antibody was used for Western blot detection of endogenous Zika virus NS5 co-precipitation signal, which confirmed that CLIC1 combined with NS5 to form a complex under the condition of Zika virus infection. Figure 1 C).

[0086] 4. Subcellular co-localization analysis: HeLa cells co-transfected with pHA-CLIC1 plasmid and pFlag-NS5 plasmid were immunofluorescence stained, showing that CLIC1 (red) and NS5 (green) showed significant co-localization in cytoplasm (yellow merged signal), indicating that CLIC1 interacted with NS5 in cells. Figure 1 D).

[0087] Example 3: siRNA knockdown of CLIC1 inhibits Zika virus replication

[0088] This example uses siCLIC1 to knock down CLIC1 gene in human placental trophoblast cells HTR-8, and sets up a blank control group to explore the regulatory effect of CLIC1 on Zika virus replication.

[0089] siCLIC1 group: HTR-8 cells were inoculated in a 24-well culture plate, and siCLIC1 targeting CLIC1 gene was transfected by subculturing. After 24 h of transfection, the cells were infected with Zika virus at 1 MOI. After 24 h of Zika virus infection, the cell supernatant and cells were collected, and the Zika virus replication indicators were detected.

[0090] siNC group: HTR-8 cells were inoculated in 24-well culture plates, and siNC for control was transfected by subculture. After 24 h of transfection, the cells were infected with Zika virus at 1 MOI. After 24 h of Zika virus infection, the cell supernatant and cells were collected, and the Zika virus replication index was detected.

[0091] (1) RT-qPCR experiment was used to detect the replication of Zika virus RNA in cells. As shown in FIG. A, knocking down CLIC1 gene can significantly down-regulate the level of Zika virus RNA in cells. Figure 2

[0092] (2) Western blot experiment was used to detect the expression of E and NS5 in cells using Zika virus E antibody and NS5 antibody. As shown in FIG. B, compared with the siNC group, the expression level of CLIC1 in the siCLIC1 group was significantly down-regulated, and the expression of E and NS5 was also significantly down-regulated. Figure 2

[0093] 2. HeLa cells were inoculated in 24-well culture plates, and siCLIC1 or siNC targeting CLIC1 was transfected by subculture; after 24 h of transfection, the cells were infected with Zika virus at 1 MOI; after 24 h of Zika virus infection, the cells were collected, and Western blot experiment was used to detect the expression of E and NS5 in cells using Zika virus E antibody and Zika virus NS5 antibody. As shown in FIG. C, compared with the siNC group, the expression level of CLIC1 in the siCLIC1 group was significantly down-regulated, and the expression level of E and NS5 was also significantly down-regulated. Figure 2

[0094] The results of this example show that knocking down CLIC1 can inhibit Zika virus replication.

[0095] Example 4: Overexpression of CLIC1 promotes Zika virus replication

[0096] In order to confirm the regulatory effect of CLIC1 on Zika virus replication, this example uses a dose gradient of pHA-CLIC1 plasmid to explore the effect of CLIC1 overexpression on Zika virus replication.

[0097] 1. HTR-8 cells were inoculated in 24-well culture plates, and pHA-CLIC1 plasmid was transfected at a dose gradient (doses were 0 μg, 0.5 μg, and 1 μg), and pHA empty plasmid was used to make up; after 24 h of transfection, the cells were infected with Zika virus at 1 MOI; after 24 h of Zika virus infection, the cells were collected, and the Zika virus replication index was detected:

[0098] (1) RT-qPCR experiment was used to detect the replication of Zika virus RNA in cells. As shown in FIG.​​​Figure 2 As shown in D, gradient expression of CLIC1 can significantly up-regulate the intracellular Zika virus RNA level.

[0099] (2) Using Zika virus E antibody and Zika virus NS5 antibody, the expression of E and NS5 in the cells was detected by Western blot experiment. As shown in Figure 2 As shown in E, gradient expression of CLIC1 can also significantly up-regulate the expression levels of E and NS5.

[0100] 2. HeLa cells were inoculated in 24-well culture plates, and when the cell confluence reached 70%, the pHA-CLIC1 plasmid was transfected in a dose gradient (the dose was 0 μg, 0.5 μg, 1 μg, respectively), and the pHA empty plasmid was supplemented; 24 h after transfection, the cells were infected with 1 MOI of Zika virus; 24 h after Zika virus infection, the cells were collected, and the expression of E and NS5 in the cells was detected by Western blot experiment using Zika virus E antibody and Zika virus NS5 antibody. As shown in Figure 2 As shown in F, gradient expression of CLIC1 can also significantly up-regulate the expression levels of Zika virus structural protein E and non-structural protein NS5.

[0101] The results of this example show that overexpression of CLIC1 can promote Zika virus replication.

[0102] Example 5: CLIC1 inhibitor blocks Zika virus replication

[0103] In order to explore the regulatory effect of CLIC1 inhibitor on Zika virus replication, the CLIC1 inhibitors used in this example are commercially available chloride channel inhibitors NPPB and R(+)-IAA-94, and their chemical structural formulas are as shown in Figure 3 A, C.

[0104] Dimethyl sulfoxide (abbreviated as DMSO) was used to dissolve the chloride channel inhibitors NPPB or R(+)-IAA-94, and then the culture medium was used to dilute NPPB to 100 μM, 20 μM, 4 μM, 0.8 μM, and R(+)-IAA-94 to 200 μM, 100 μM, 10 μM, 1 μM.

[0105] HTR-8 cells were cultured with culture medium containing different concentrations of NPPB or R(+)-IAA-94, and 1 MOI of Zika virus was added to the cells for infection, and the cells were collected 24 h after infection.

[0106] RT-qPCR experiment was used to detect the replication of Zika virus RNA in the cells, as shown in Figure 3As shown in B, D, the chloride channel inhibitor NPPB or R(+)-IAA-94 can significantly down-regulate the intracellular Zika virus RNA level.

[0107] The embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacements and shall be included in the protection scope of the present application.

Claims

1. The application of CLIC1 inhibitors in the preparation of drugs against Zika virus infection, characterized in that: The CLIC1 inhibitor includes at least one of the following two substances: I: A substance that inhibits the expression of the CLIC1 gene in host cells; the substance that inhibits the expression of the CLIC1 gene in host cells is siRNA, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO:1, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO:2; II: A substance that inhibits the function of CLIC1 chloride ion channel in host cells; the substance that inhibits the function of CLIC1 chloride ion channel in host cells is a chloride ion channel inhibitor; the chloride ion channel inhibitor is R-(+)-methylindazole.

2. The application according to claim 1, characterized in that: The anti-Zika virus infection drug includes a substance that inhibits the expression of the CLIC1 gene in host cells and its vector system, wherein the vector system is at least one of lentiviral vector, adenovirus vector, adeno-associated virus vector, liposome, lipid nanoparticle, cationic polymer, exosome, inorganic nanoparticle, and polypeptide vector.

3. The application according to claim 1, characterized in that: The Zika virus infection includes the upregulation of at least one of the following indicators: Zika virus RNA level, Zika virus structural protein E expression level, and Zika virus non-structural protein NS5 expression level.

4. The application according to claim 1, characterized in that: The anti-Zika virus infection drug is used to downregulate at least one of the following indicators: Zika virus RNA level, Zika virus structural protein E expression level, and Zika virus non-structural protein NS5 expression level.

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