Use of vps35 inhibitors in the preparation of drugs against zika virus infection

Abstract

The application belongs to the field of biological medicine, and discloses application of a VPS35 inhibitor in preparation of an anti-zika virus infection drug, wherein the VPS35 inhibitor comprises at least one of the following two substances: I: a substance for inhibiting VPS35 gene expression in a host cell; II: a substance for inhibiting retrograde transport function of VPS35 protein in the host cell. The application first discovers that the VPS35 protein is an important host factor in a zika virus replication cycle, and verifies through a zika virus infection model system that inhibition of VPS35 gene expression in the host cell or inhibition of the retrograde transport function of the VPS35 protein can inhibit zika virus replication in the host cell. Compared with traditional drugs targeting zika virus proteins, the VPS35 inhibitor provided in the application targets a host factor, is more difficult to induce zika virus drug resistance, and has more persistent treatment potential.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of a Vacuolar Protein Sorting-Associated Protein 35 (VPS35) inhibitor in preparation of a drug for resisting Zikavirus (ZIKV) infection. BACKGROUND

[0002] Zikavirus is transmitted by mosquitoes, and infection of the virus can cause nervous system disorders (such as microcephaly in newborns, Guillain-Barre syndrome), eye diseases and reproductive system damage and other serious complications. Notably, Zikavirus has the characteristics of breaking through immune-privileged sites such as the blood-brain barrier and placental barrier, and can be long-term stored in body fluids such as semen and saliva, leading to the spread of the virus through non-vector routes such as sexual transmission and vertical transmission, and there is a risk of large-scale epidemic. However, the current prevention and control measures for the virus are limited, and there is currently no approved vaccine or specific antiviral drug, and the development of an anti-Zikavirus infection drug is imminent. SUMMARY

[0003] In order to solve the problem of lack of anti-Zikavirus infection drugs in the prior art, the present application provides application of a VPS35 inhibitor in preparation of an anti-Zikavirus infection drug.

[0004] The applicant found that overexpression of the VPS35 gene can promote replication of Zikavirus in host cells, inhibition of expression of the VPS35 gene can inhibit replication of Zikavirus in host cells, and the existing VPS35 protein function inhibitors Brefeldin A (referred to as Brefeldin A), (E)-2-(((5-methylthiophene-2-yl) methylene) amino)-N-phenyl benzamide (referred to as Retro-2) and 6,8-difluoro-4-pyridin-3-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quinoline (referred to as Golgicide A) can also inhibit replication of Zikavirus in host cells, and the above findings show that the VPS35 inhibitor has good application prospects in preparation of an anti-Zikavirus infection drug.

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

[0006] The present application provides application of a VPS35 inhibitor in preparation of an anti-Zikavirus infection drug, and the VPS35 inhibitor comprises at least one of the following two substances:

[0007] I: a substance for inhibiting expression of a VPS35 gene in a host cell;

[0008] II: a substance for inhibiting retrograde transport function of a VPS35 protein in a host cell.

[0009] In some embodiments of the present application, the anti-zika virus infection drug comprises a VPS35 inhibitor as an effective component for inhibiting the replication of zika virus in host cells.

[0010] In some embodiments of the present application, the substance for inhibiting the expression of VPS35 gene in host cells comprises at least one of siRNA, shRNA, siRNA plasmid, shRNA plasmid.

[0011] In some embodiments of the present application, the siRNA comprises siVPS35-1 or siVPS35-2, wherein the sequence of the siVPS35-1 sense strand is shown in SEQ ID NO: 1, the sequence of the siVPS35-1 antisense strand is shown in SEQ ID NO: 2; the sequence of the siVPS35-2 sense strand is shown in SEQ ID NO: 3, and the sequence of the siVPS35-2 antisense strand is shown in SEQ ID NO: 4.

[0012] In some embodiments of the present application, the anti-zika virus infection drug comprises a substance for inhibiting the expression of VPS35 gene in host cells and a carrier system thereof, and the carrier system is at least one of lentiviral vector, adenoviral vector, adeno-associated viral vector, liposome, lipid nanoparticle, cationic polymer, exosome, inorganic nanoparticle, and polypeptide carrier.

[0013] In some embodiments of the present application, the substance for inhibiting the retrograde transport function of VPS35 protein in host cells comprises at least one of brefeldin A, (E)-2-(((5-methylthiophen-2-yl) methylidene) amino)-N-phenyl benzamide, and 6,8-difluoro-4-pyridin-3-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quinoline.

[0014] In some embodiments of the present application, the dosage form of the anti-zika virus infection drug comprises at least one of tablet, granule, pill, powder, capsule, injection, and oral liquid.

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

[0016] In some embodiments of the present application, the anti-zika virus infection drug is used for down-regulating 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 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.

[0018] Compared with the prior art, the present application has at least the following beneficial effects:

[0019] The present application first discovers that VPS35 protein is a key host factor for Zika virus replication, and by inhibiting the expression of VPS35 gene or inhibiting the retrograde transport function of VPS35 protein, the replication of Zika virus can be blocked, thereby providing a new direction for developing anti-Zika virus infection drugs targeting host factors. Compared with traditional drugs targeting Zika virus proteins, the VPS35 inhibitor provided by the present application targets host factors, is more difficult to induce Zika virus drug resistance, and has more persistent treatment potential. BRIEF DESCRIPTION OF DRAWINGS

[0020] 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. 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.

[0021] Figure 1 : VPS35 protein interacts with NS5 protein and participates in Zika virus replication; wherein:

[0022] A: technical flow chart of IP-MS experiment for screening host proteins interacting with NS5 protein in A549 cells. Based on NS5 antibody, candidate interacting proteins are identified by liquid chromatography-tandem mass spectrometry, and 211 host proteins that can interact with NS5 protein are identified;

[0023] B: RT-qPCR experiment detects that the gene knockdown effect of the candidate protein is significant after transfecting siRNA to knock down the candidate protein in A549 cells;

[0024] C: After adding Zika virus (1 MOI) to the A549 cells with knockdown of the candidate protein and infecting for 24 h, RT-qPCR experiment detects the expression level of Zika virus RNA in the cells, which shows that knockdown of VPS35 can significantly down-regulate the expression level of Zika virus RNA in the cells.

[0025] Figure 2 : Knockdown of VPS35 reduces the replication level of Zika virus; wherein:

[0026] A: Transfect siRNA in A549 cells, add Zika virus (1 MOI) infection for 24 h, detect by Western blot experiment, show that VPS35 protein expression is down-regulated, and the expression of E protein and NS5 protein is reduced;

[0027] B: Transfect siRNA in A549 cells, add Zika virus (1 MOI) infection for 24 h, detect by RT-qPCR experiment, show that the intracellular Zika virus RNA level is significantly reduced;

[0028] C: Transfect siRNA in A549 cells, add Zika virus (1 MOI) infection for 24 h, detect by RT-qPCR experiment, show that the intracellular Zika virus RNA level is significantly reduced;

[0029] D: Transfect siRNA in HTR-8 cells, add Zika virus (1 MOI) infection for 24 h, detect by Western blot experiment, show that VPS35 protein expression is down-regulated, and the expression of E protein and NS5 protein is reduced;

[0030] E: Transfect siRNA in HTR-8 cells, add Zika virus (1 MOI) infection for 24 h, detect by RT-qPCR experiment, show that the intracellular Zika virus RNA level is significantly reduced;

[0031] F: Transfect siRNA in mouse primary testicular supporting cells, add Zika virus (1 MOI) infection for 24 h, detect by Western blot experiment, show that VPS35 protein expression is down-regulated, and the expression of E protein and NS5 protein is reduced;

[0032] G: Transfect siRNA in mouse primary testicular supporting cells, add Zika virus (1 MOI) infection for 24 h, detect by RT-qPCR experiment, show that the intracellular Zika virus RNA level is significantly reduced.

[0033] Figure 3 : Overexpression of VPS35 promotes Zika virus replication; wherein:

[0034] A: Transfect 0, 0.5 or 1 μg of pHA-VPS35 plasmid in A549 cells, add Zika virus (1 MOI) infection for 24 h, detect by Western blot experiment, show that VPS35 is gradiently overexpressed, and the expression of E protein and NS5 protein is increased;

[0035] B: Transfect 0, 0.5 or 1 μg of pHA-VPS35 plasmid in A549 cells, add Zika virus (1 MOI) infection for 24 h, detect the cell samples by RT-qPCR experiment, and show that the expression level of Zika virus RNA in the cells is significantly increased;

[0036] C: Transfect 0, 0.5 or 1 μg of pHA-VPS35 plasmid in A549 or HTR-8 cells, add Zika virus (1 MOI) infection for 24 h, detect the cell culture supernatant samples by RT-qPCR experiment, and show that the expression level of Zika virus RNA in the supernatant is significantly increased;

[0037] D: Transfect 0, 0.5 or 1 μg of pHA-VPS35 plasmid in HTR-8 cells, add Zika virus (1 MOI) infection for 24 h, detect by Western blot experiment, and show that the expression of VPS35 gradient overexpression, E protein and NS5 protein is increased;

[0038] E: Transfect 0, 0.5 or 1 μg of pHA-VPS35 plasmid in HTR-8 cells, add Zika virus (1 MOI) infection for 24 h, detect by RT-qPCR experiment, and show that the expression level of Zika virus RNA in the cells is significantly increased.

[0039] Figure 4 : NS5 protein interacts with VPS35 protein; wherein:

[0040] A: Transfect pHA-VPS35 plasmid and pFlag-NS5 plasmid into HEK293T cells, collect and lyse the cells, use Flag antibody as the primary antibody, and Co-IP experiment shows that NS5 protein interacts with VPS35 protein;

[0041] B: Add Zika virus (1 MOI) to A549 cells for 24 h infection, collect and lyse the cells, use VPS35 antibody as the primary antibody, and Co-IP experiment shows that NS5 protein interacts with endogenous VPS35 protein in the cells;

[0042] C: Transfect pHA-VPS35 plasmid and pFlag-NS5 plasmid into HeLa cells, fix the cells and stain, and observe by laser confocal microscope that NS5 protein and VPS35 protein co-localize in the cells, indicating that they interact with each other;

[0043] D: Use HDOCK program to dock NS5 protein and VPS35 protein, obtain a complex model. Use PyMOL software (Version 3.0.3) to display the results, indicating that they interact directly with each other.

[0044] Figure 5 Knockdown of VPS35 gene accelerates NS5 protein degradation; wherein:

[0045] A: Equal amount (0.2 μg) of pFlag-NS5 plasmid and dose gradient (0, 0.2, 0.4 μg) of pHA-VPS35 plasmid were transfected into A549 cells, and Western blot experiment showed that the expression of NS5 protein was up-regulated in a VPS35 gradient-dependent manner;

[0046] B: A549 cells were transfected with siNC or siVPS35, and then transfected with pFlag-NS5 plasmid, 24 h later, CHX (10 μg / mL) was added to treat the cells for 24 h, and Western blot experiment showed that VPS35 protein delayed the degradation of NS5 protein;

[0047] C: Image J software was used to analyze the gray scale of Western blot results in B. Figure 5

[0048] Figure 6 Inhibition of retrograde transport function of VPS35 protein can inhibit Zika virus replication. A549 cells were pretreated with drug inhibitors Brefeldin A, Retro-2 or Golgicide A (DMSO as control) at the indicated concentrations for 1 h, and then infected with Zika virus (1 MOI) in the presence of the corresponding inhibitors for 24 h. The cells were collected and subjected to Western blot experiment, which showed that the expression of E protein and NS5 protein in the cells treated with the three inhibitors was reduced.

[0049] Figure 7 Map of pHA-VPS35 plasmid (i.e. pCAGGS-HA-VPS35 in the figure, pCAGGS is the vector backbone name of the plasmid). DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme 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.

[0051] ​Herein, the term "VPS35 protein" is short for vacuolar protein sorting-associated protein 35, which is a core component of the retromer complex. The term "retromer complex" refers to a protein complex composed of subunits such as VPS35, VPS26, and VPS29, which is responsible for the retrograde transport of cargo proteins from endosomes to the Golgi apparatus or plasma membrane, avoiding their degradation by lysosomes, and plays a key role in the sorting and transport of intracellular substances.

[0052] Herein, the term "VPS35 gene" refers to the gene encoding the "VPS35 protein".

[0053] Herein, the term "VPS35 inhibitor" refers to a substance that can inhibit the expression of the VPS35 gene or inhibit the function of the VPS35 protein, including two categories: one is a substance that inhibits the expression of the VPS35 gene in host cells (such as siRNA, shRNA, etc.), and the other is a substance that inhibits the retrograde transport function of the VPS35 protein in host cells (such as Brefeldin A, Retro-2, Golgicide A, etc.). The "retrograde transport function" refers to the process of the intracellular substance being transported in the opposite direction from the endosome to the Golgi apparatus or plasma membrane, which is mediated by the retromer complex, can recover specific proteins and maintain their functional stability, and avoid their degradation.

[0054] Herein, the term "siRNA" refers to small interfering RNA, which is a type of double-stranded RNA molecule with a length of about 20-25 nucleotides, which can specifically bind to the target gene mRNA and mediate the RNA interference (RNAi) process, thereby silencing the expression of the target gene, and is commonly used for gene function research and antiviral therapy.

[0055] Herein, the term "Co-IP" refers to co-immunoprecipitation, which is a protein interaction research technique based on the specific binding of antigen-antibody, which can precipitate target proteins and their interacting proteins by antibodies, and is commonly used to verify the existence of protein complexes in vivo.

[0056] Herein, the term "RT-qPCR" refers to reverse transcription-quantitative polymerase chain reaction, which is a technique for quantitative amplification after reverse transcription of RNA to cDNA, and is used to detect gene expression levels or viral RNA replication amounts.

[0057] Herein, the term "Western blot" refers to a protein blotting method, which separates proteins by gel electrophoresis and transfers them to a membrane, and uses specific antibodies to detect the expression level of target proteins, which is a commonly used method for protein analysis.

[0058] In the present document, the term "lentiviral vector" refers to a gene delivery tool based on lentivirus modification, which can integrate exogenous genes into the host cell genome to achieve long-term stable expression, and is widely used in gene therapy and cell engineering.

[0059] Zika virus is a single-stranded positive-sense RNA virus with an envelope, and the single polyprotein precursor encoded by its genome can be processed into three structural proteins (hereinafter referred to as C protein, M protein and E protein, respectively) and seven non-structural proteins (hereinafter referred to as NS1 protein, NS2A protein, NS2B protein, NS3 protein, NS4A protein, NS4B protein and NS5 protein, respectively) by viral and host proteases. Among them, the NS5 protein, as a core component of the viral replication complex, has multiple enzyme activities, including RNA-dependent RNA polymerase (RdRp) activity and methyltransferase (MTase) activity. It is known that the NS5 protein directly participates in the replication of viral RNA through RdRp activity, ensuring accurate replication and amplification of the viral genome; its MTase activity plays a key role in the formation of the 5' cap structure of viral RNA, which is crucial for the stability and translation efficiency of viral RNA. More and more studies have shown that the process of viral infection of host cells to complete its own replication is greatly dependent on various factors in the host cell, and the virus can adjust the intracellular environment to facilitate its infection by "using" and "hijacking" host factors. The NS5 protein-host factor interaction plays a key role in the replication and pathogenesis of Zika virus, and has attracted the attention of researchers. However, current research on the use of host factors by NS5 protein to regulate efficient viral replication is still insufficient, and it is not possible to determine the key target in the host cell that inhibits Zika virus.

[0060] As shown in Figure 1 The present application has screened 211 potential host factors that can bind to NS5 protein with higher reliability through immunoprecipitation-mass spectrometry technology, and further found through siRNA library screening that knocking down the VPS35 gene in the cell can significantly inhibit Zika virus replication. Experiments have shown that inhibiting the expression of VPS35 gene or inhibiting the retrograde transport function of VPS35 protein can block Zika virus replication.

[0061] Based on the above experimental results, the application provides a use of a VPS35 inhibitor in the preparation of an anti-zika virus infection drug, wherein the VPS35 inhibitor comprises at least one of the following two substances: I: a substance for inhibiting the expression of a VPS35 gene in a host cell; II: a substance for inhibiting the retrograde transport function of a VPS35 protein in a host cell. Both of the two substances inhibit zika virus replication by targeting and inhibiting host factors, and the genetic variation rate of the host factors is much lower than that of the virus genes and the functions are conservative, so compared with targeting and inhibiting the zika virus itself, targeting and inhibiting the host factors can more durably inhibit the virus replication and is not easily affected by virus mutations.

[0062] In some embodiments of the application, the anti-zika virus infection drug comprises the VPS35 inhibitor as an effective component for inhibiting the replication of the zika virus in the host cell. The anti-zika virus infection drug can comprise the VPS35 inhibitor as the only effective component for inhibiting the replication of the zika virus in the host cell, which can significantly reduce the levels of NS5 protein, E protein and zika virus RNA in the host cell.

[0063] In some embodiments of the application, the substance for inhibiting the expression of the VPS35 gene in the host cell comprises at least one of siRNA, shRNA, siRNA plasmid and shRNA plasmid. These substances all target and degrade target gene mRNA, block protein synthesis and reduce the expression level of the VPS35 protein. Preferably, siRNA is used, which can directly act as an effector molecule and quickly silence target gene mRNA in a short period of time. In some embodiments of the application, the siRNA comprises siVPS35-1 or siVPS35-2, wherein the sequence of the sense strand of siVPS35-1 is shown in SEQ ID NO: 1, the sequence of the antisense strand of siVPS35-1 is shown in SEQ ID NO: 2, the sequence of the sense strand of siVPS35-2 is shown in SEQ ID NO: 3, and the sequence of the antisense strand of siVPS35-2 is shown in SEQ ID NO: 4. Experiments have proved that siVPS35-1 or siVPS35-2 can effectively reduce the expression level of the VPS35 protein.

[0064] In some embodiments of the application, the anti-zika virus infection drug comprises the substance for inhibiting the expression of the VPS35 gene in the host cell and a carrier system thereof, wherein the carrier system is at least one of a lentivirus carrier, an adenovirus carrier, an adeno-associated virus carrier, a liposome, a lipid nanoparticle, a cationic polymer, an exosome, an inorganic nanoparticle and a polypeptide carrier. siRNA and shRNA generally need to be delivered into a host cell by a carrier system to play a role, and the carrier system can overcome biological barriers and efficiently and targetedly deliver active substances siRNA and shRNA to target cells.

[0065] In some embodiments of the present application, the substance that inhibits the retrograde transport function of VPS35 protein in the host cell comprises at least one of brefeldin A, (E)-2-(((5-methylthiophen-2-yl) methylidene) amino)-N-phenylbenzamide, 6,8-difluoro-4-pyridin-3-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline. These substances are known to inhibit the retrograde transport function of VPS35 protein in the host cell, and experiments show that they can inhibit the replication of Zika virus through this mechanism.

[0066] In some embodiments of the present application, the dosage form of the anti-Zika virus infection drug comprises at least one of tablets, granules, pills, powders, capsules, injections, oral liquids.

[0067] In some embodiments of the present application, the Zika virus infection comprises up-regulation of at least one of the following indicators (‘up-regulation’ refers to an increase in the level of the corresponding indicator in the body of a healthy subject who has not been infected with Zika virus): Zika virus RNA level, Zika virus structural protein E expression level, Zika virus non-structural protein NS5 expression level.

[0068] In some embodiments of the present application, the anti-Zika virus infection drug is used to down-regulate (‘down-regulation’ 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.

[0069] 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.

[0070] In some embodiments of the present application, the anti-Zika virus infection drug can be administered according to the needs of the clinic, using appropriate administration routes, including but not limited to intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, oral administration, sublingual administration, nasal administration, transdermal administration.

[0071] 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.

[0072] Table 1 Sources of reagents used in Examples 1-6

[0073]

[0074] The pHA-VPS35 plasmid is constructed by the inventors using the plasmid construction method commonly used in the art. The map of the pHA-VPS35 plasmid is shown in Figure 1. Figure 7 which is used for overexpression of the VPS35 protein with an influenza virus hemagglutinin (HA for short) tag in cells.

[0075] The pFlag-NS5 plasmid is constructed according to the method in the reference PBMD:32117232, which is used for overexpression of the NS5 protein with a Flag tag in cells. The Flag tag is a short peptide composed of 8 amino acids (the sequence is DYKDDDK).

[0076] The information of siVPS35-1, siVPS35-2 and siNC used in the following examples is shown in Table 2:

[0077] Table 2 siRNA sequences used in the examples

[0078]

[0079] Example 1: Construction of a Zika virus infected cell model

[0080] 1. Amplification of Zika virus:

[0081] C6 / 36 mosquito cells are inoculated in a T125 culture flask. When the cell confluence reaches 90%, the culture medium is replaced with 20 mL of MEM medium containing 2% fetal bovine serum (FBS), and 1 mL of Zika virus suspension is added to the flask, which is then placed in a 27°C incubator for further culture. After 3 days of inoculation of Zika virus, 5-10 mL of MEM medium containing 2% FBS is supplemented to the culture flask for further culture of the cells. On the 5th-6th day after inoculation of Zika virus, about 30% of the cells show obvious pathological changes under an inverted microscope. The supernatant in the culture flask is transferred into a 50 mL centrifuge tube, filtered using a sterile filter with a pore size of 0.22 μm, and stored for use.

[0082] 2. Determination of the titer of Zika virus (virus plaque method):

[0083] The supernatant reserved in step (1) is gradient diluted using MEM medium to obtain Zika virus suspensions with different dilution degrees; BHK-21 hamster kidney cells are inoculated into a 12-well plate, and when the cell confluence is about 70%, the original culture medium is removed, and the cells are washed with 1 mL of PBS in each well; 500 μL of Zika virus suspension with different dilution degrees is added to each well, and the cells are 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 is discarded, and the cells are washed twice with serum-free medium; then 1 mL of MEM medium containing 1.2wt% methyl cellulose and 2wt% FBS is added to each well; after 4-5 days of virus inoculation, the 12-well plate is removed, and the cell plate bottom is observed under natural light, and plaques can be seen; an appropriate amount of crystal violet fixing and staining solution is added to each well, and the cells are fixed and stained at room temperature overnight; the 12-well plate is gently washed with running water to wash away the methyl cellulose in the wells, and the number of plaques is counted, and the virus titer is calculated: virus titer = number of plaques x dilution factor x 2 (PFU / mL).

[0084] 3. Detection of Zika virus infection and replication indicators:

[0085] The Zika virus suspension is taken out from a -80°C refrigerator and quickly dissolved in a 37°C water bath, and the cells are infected with 1 MOI of Zika virus for 4 h, then the virus suspension is discarded, the cells are washed with PBS 3 times, and new culture medium is added for continued culture. At 24 h after infection, the cells and cell supernatant are collected for detection of Zika virus replication indicators, RT-qPCR experiment is used to detect the replication of viral RNA in the cells, and Western blot experiment is used to detect the expression of NS5 protein and E protein in the cells.

[0086] Example 2: siRNA knockdown of VPS35 inhibits Zika virus replication

[0087] To explore the regulatory effect of VPS35 protein on Zika virus replication, the VPS35 gene in A549 cells is knocked down by siRNA in this embodiment, denoted as the siRNA group; and a blank control group is set up, denoted as the siNC group.

[0088] siRNA group: A549 cells are inoculated in a 24-well culture plate, and siRNA targeting the VPS35 gene (siVPS35-1 or siVPS35-2) is transfected while the cells are passaged. After 24 h of transfection, the cells are infected with 1 MOI of Zika virus. After 24 h of Zika virus infection, the cell supernatant and cells are collected for detection of Zika virus replication indicators.

[0089] siNC group: A549 cells were inoculated in 24-well culture plates, and siNC 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.

[0090] The expression of E protein and NS5 protein in Zika virus infected A549 cells in the siRNA group and the siNC group was detected by Western blot experiment using Zika virus E antibody and Zika virus NS5 antibody, and GAPDH was used as an internal reference protein to correct the loading amount, as shown in Figure 2 As shown in A, compared with the siNC group, the level of VPS35 protein in the siRNA group was significantly down-regulated, and the expression of E protein and NS5 protein was also significantly down-regulated.

[0091] The replication of Zika virus RNA in Zika virus infected A549 cells was detected by RT-qPCR experiment, as shown in Figure 2 As shown in B, the siRNA knockdown of VPS35 gene can significantly down-regulate the level of Zika virus RNA in cells.

[0092] The replication of Zika virus RNA in the cell supernatant was detected by qPCR experiment, as shown in Figure 2 As shown in C, the siRNA knockdown of VPS35 gene can significantly down-regulate the level of Zika virus RNA in the cell supernatant.

[0093] Referring to the above experiments, siRNA targeting VPS35 gene (siVPS35-1 or siVPS35-2) was used to silence the expression of VPS35 gene in HTR-8 cells (Fig. Figure 2 D and Figure 2 E) or mouse primary testicular Sertoli cells (Fig. Figure 2 F and Figure 2 G), and Zika virus infection was performed, and the Zika virus replication related index was detected. It was found that knockdown of VPS35 gene in cells can significantly inhibit Zika virus replication.

[0094] Example 3: Overexpression of VPS35 can promote Zika virus replication

[0095] In order to confirm the regulatory effect of VPS35 protein on Zika virus replication, the effect of overexpression of VPS35 on Zika virus replication was further studied. A549 cells and HTR-8 cells were transiently transfected with a dose gradient of pHA-VPS35 plasmid, and 1 MOI of Zika virus was added to infect the cells. The overexpression of VPS35 protein and the expression of E protein and NS5 protein were detected by Western blot experiment (Fig. Figure 3 A and Figure 3D), and the Zika virus RNA level in cells (E) and cell culture supernatant (F) was detected by RT-qPCR to reflect the replication of Zika virus. The results showed that overexpression of VPS35 significantly promoted the expression of E protein and NS5 protein, and the Zika virus RNA level in cells and cell culture supernatant of the cells overexpressing VPS35 was higher than that of the control group. Figure 3 B and Figure 3 E) and cell culture supernatant (F) to reflect the replication of Zika virus. The results showed that overexpression of VPS35 significantly promoted the expression of E protein and NS5 protein, and the Zika virus RNA level in cells and cell culture supernatant of the cells overexpressing VPS35 was higher than that of the control group. Figure 3 C) was detected by RT-qPCR to reflect the replication of Zika virus. The results showed that overexpression of VPS35 significantly promoted the expression of E protein and NS5 protein, and the Zika virus RNA level in cells and cell culture supernatant of the cells overexpressing VPS35 was higher than that of the control group.

[0096] The above experimental results show that the VPS35 protein can promote the replication of Zika virus.

[0097] Example 4: VPS35 interacts with Zika virus NS5

[0098] 1. Co-IP verification in exogenous overexpression system: HEK293T cells were co-transfected with pHA-VPS35 plasmid and pFlag-NS5 plasmid, and the lysate was precipitated with Flag antibody. The enrichment of NS5 protein was detected by Western blot using HA antibody to confirm that VPS35 protein interacts with NS5 protein in cytoplasm (A). Figure 4 A).

[0099] 2. Interaction verification in endogenous Zika virus infection model: Zika virus infected A549 cells for 48 h, and then Co-IP verification was performed using VPS35 antibody. Western blot was used to detect the co-precipitation signal of endogenous NS5 protein using Zika virus NS5 antibody to confirm that VPS35 protein interacts with NS5 protein under the condition of Zika virus infection (B). Figure 4 B).

[0100] 3. Subcellular co-localization analysis: HeLa cells co-transfected with pHA-VPS35 plasmid and pFlag-NS5 plasmid were immunofluorescence stained, showing that VPS35 protein (red) and NS5 protein (green) showed significant co-localization (yellow combined signal) in cytoplasm, indicating that VPS35 protein interacts with NS5 protein in cells (C). Figure 4 C).

[0101] 4. Molecular docking analysis: Molecular docking simulation analysis was performed on the protein structure of NS5 protein and VPS35 protein, and the results suggested that NS5 protein directly interacts with VPS35 protein (D). Figure 4 D).

[0102] Example 5: VPS35 can delay the degradation of NS5 protein

[0103] It is known that VPS35 protein is a core component of the retromer complex, responsible for sorting and recognizing cargo proteins and transporting them from endosomes to TGN or plasma membrane, avoiding their degradation by lysosomes. To explore the effect of VPS35 protein on the expression of NS5 protein, equal amounts of pFlag-NS5 plasmid and dose gradient of pHA-VPS35 plasmid were transfected into A549 cells, and the effect of VPS35 protein gradient expression on the expression of NS5 protein was detected by Western blot experiment (A). Figure 5 The experimental results show that the expression level of NS5 protein is positively correlated with the expression level of VPS35 protein.

[0104] To further explore the effect of VPS35 protein on NS5 protein, siRNA (siVPS35-1 group: siVPS35-1; siVPS35-2 group: siVPS35-2; siNC group: siNC) was transfected into A549 cells, followed by transfection of pFlag-NS5 plasmid, and the cells were treated with cycloheximide (CHX) for protein synthesis inhibition. The expression of NS5 protein was detected by Western blot. The experimental results are shown in Figure 5 B and Figure 5 C, the degradation rate of NS5 protein in siVPS35-1 group and siVPS35-2 group is faster than that in siNC group, indicating that VPS35 protein can delay the degradation of NS5 protein.

[0105] Example 6: Inhibition of the retrograde transport function of VPS35 protein can inhibit Zika virus replication

[0106] To explore the effect of VPS35 retrograde transport function inhibitor, dimethyl sulfoxide (DMSO) was used to dissolve VPS35 retrograde transport function inhibitors Brefeldin A, Retro-2 or Golgicide A, and a culture medium containing 40 ng / mL Brefeldin A, 10 ng / mL Retro-2 or 50 ng / mL Golgicide A was prepared for culturing A549 cells, and 1 MOI of Zika virus was used to infect the cells. After 24 h of infection, the cells were collected. Zika virus E antibody and Zika virus NS5 antibody were used to detect the expression levels of E protein and NS5 protein in the cells by Western blot, as shown in Figure 6 After the retrograde transport pathway of the host cells was inhibited, the replication ability of Zika virus was weakened, indicating that the existing VPS35 retrograde transport function inhibitors can inhibit the replication of Zika virus.

[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. Use of a VPS35 inhibitor in the preparation of a drug for treating Zika virus infection, characterized in that: The VPS35 inhibitor includes at least one of the following two substances: I: a substance for inhibiting the expression of VPS35 gene in host cells; the substance for inhibiting the expression of VPS35 gene in host cells is siRNA, and the siRNA includes siVPS35-1 or siVPS35-2, wherein the sequence of the siVPS35-1 positive strand is shown as SEQ ID NO: 1, the sequence of the siVPS35-1 negative strand is shown as SEQ ID NO: 2, the sequence of the siVPS35-2 positive strand is shown as SEQ ID NO: 3, and the sequence of the siVPS35-2 negative strand is shown as SEQ ID NO: 4; II: a substance for inhibiting the retrograde transport function of VPS35 protein in host cells; the substance for inhibiting the retrograde transport function of VPS35 protein in host cells includes at least one of (E)-2-(((5-methylthiophene-2-yl) methylene) amino)-N-phenyl benzamide and 6,8-difluoro-4-pyridin-3-yl-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quinoline.

2. Use according to claim 1, characterized in that: The anti-zika virus infection drug includes a substance for inhibiting the expression of VPS35 gene in host cells 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.

3. Use according to claim 1, characterized in that: The dosage form of the anti-zika virus infection drug includes at least one of tablets, granules, pills, powders, capsules, injections, and oral liquids.

4. Use according to claim 1, characterized in that: The zika virus infection includes up-regulation of at least one of the following indexes: zika virus RNA level, zika virus structural protein E expression level, and zika virus non-structural protein NS5 expression level.

5. The use according to claim 1, characterized in that: The anti-zika virus infection drug is used for down-regulating at least one of the following indexes: zika virus RNA level, zika virus structural protein E expression level, and zika virus non-structural protein NS5 expression level.

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