CN111000981B

CN111000981B - Application of antiviral protein C19orf66 in targeting Zika virus non-structural protein NS3 antiviral drug

Info

Publication number: CN111000981B
Application number: CN201911275747.7A
Authority: CN
Inventors: 黎孟枫; 朱勋; 何振健; 吴珏珩; 吴云; 董信怀; 谭姹辉; 陈德林; 袁洁; 李隽�
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-12-12
Filing date: 2019-12-12
Publication date: 2021-08-13
Anticipated expiration: 2039-12-12
Also published as: CN111000981A

Abstract

The invention discloses application of an antiviral protein C19orf66 as an inhibitor of a Zika virus non-structural protein NS3 in antiviral drugs. The invention provides a new application of an antiviral protein C19orf66 in efficiently targeting a Zika virus non-structural protein NS3 and promoting the degradation of a lysosome pathway of NS3 so as to inhibit the proliferation of the Zika virus in a host. The method develops a new clinical application field of the humanized antiviral protein in the field of antiviral control, thereby providing a new thought and direction for the development of a medicament targeting the non-structural protein NS3 of Zika virus, providing a scientific basis for the development of medicaments resisting the Zika virus and providing a new thought for clinical antiviral treatment.

Description

Application of antiviral protein C19orf66 in targeting Zika virus non-structural protein NS3 antiviral drug

Technical Field

The invention belongs to the technical field of protein engineering, and particularly relates to application of an antiviral protein C19orf66 as a Zika virus non-structural protein NS3 inhibitor in antiviral drugs.

Background

Zika virus (ZIKV) belongs to the Flaviviridae family of Flaviviridae, a virus that is transmitted primarily by Aedes mosquitoes. In recent decades, mosquito-borne flaviviruses, including dengue virus (DENV), West Nile Virus (WNV), Yellow Fever Virus (YFV), and the recent new panda virus (Zika virus, ZIKV), have exploded into major epidemics that are prevalent in several areas throughout the world. At the end of 2013 or early 2014, Zika virus disease began to epidemic in America on a large scale, especially in Brazil. Statistics in 2018 show that there are 84 cases reported in countries and regions infected with ZIKV. Although most people are infected with ZIKV, the main clinical symptoms are manifested as self-limited fever, rash, arthralgia, conjunctivitis, etc., unlike diseases caused by other flaviviridae viruses (e.g., DENV and YFV), ZIKV may also cause other congenital abnormalities of newborn infants such as microcephaly, and cause adult symptoms such as Guillain-barre syndrome (GBS), meningoencephalitis, multiple organ failure and Thrombocytopenia (thrombocytopentopenia), so ZIKV has been classified as a serious threat to global Health pathogens by World Health Organization (WHO) and announces outbreak of the kaiviridae as a "sudden public Health event of global concern".

At present, no effective vaccine or medicament aiming at ZIKV is clinically used for preventing or treating related diseases caused by ZIKV. Patients mainly rely on self innate immunity and adaptive immunity to cope with ZIKV infection. The Innate immune system (lnnate immune system) is considered the first line of defense against viral infection in humans, while Type I interferons (IFN-I) are key cytokines of the Innate immune system. The rapid and wide production of IFN-I by a host is an important event of the antiviral response of the innate immune system of the host. FN-I itself has no direct antiviral effect, but acts on the interferon-alpha/beta receptor (IFNAR) of the target cell in an autocrine or paracrine manner, thereby activating the corresponding downstream signaling pathway, ultimately leading to the production of a large number of interferon-stimulated genes (ISGs). It has been reported that different proteins encoded by ISGs can directly exert antiviral effects through different mechanisms, including inhibition of viral transcription, translation and replication, and alterations affecting viral nucleic acid degradation and host own metabolism. The reported anti-Zika virus ISGs include IFITM1, IFITM3, PARP12 and Viperin, but the specific antiviral mechanism of many ISGs is still unclear and needs to be further studied.

C19orf66 (also named IRAV, UPF0515, RyDEN, Shiftless) is a recently discovered ISG that inhibits replication of DENV, WNV, Hepatitis C Virus (HCV), Chikungunya virus (CHIKV), and the like in cultured cells in vitro. Suzuki Y et al reported that C19orf66 could interfere with DENV transcription by binding to viral RNA and its regulatory proteins PABPC1 and LARP 1. Balinsky et al reported that C19orf66 could bind to DENV replication complex, thereby interfering with DENV RNA processing, affecting viral replication. Recent studies have shown that C19orf66 can bind to ribosomes and mRNA during translation, inhibiting programmed-1 ribosomal frame shifting (1 PRF) of mRNA, and thus affect mRNA translation levels. A patent which we have filed also claims that the antiviral protein C19orf66 is used in the preparation of drugs for resisting Zika virus infection, but the precise drug target of C19orf66 in the preparation of drugs for resisting Zika virus infection is still unknown.

Therefore, the research on the biological function, the related molecular mechanism and the target protein of C19orf66 in the Zika virus infection process can not only provide new theoretical contents for the occurrence, development and mechanism of Zika virus infection, but also provide a new strategy for the prevention and treatment of Zika virus.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides application of antiviral protein C19orf66 as an inhibitor of Zika virus non-structural protein NS3 in antiviral drugs.

An object of the present invention is to provide a use of C19orf66 in the preparation of a zika virus inhibitor.

In order to achieve the purpose, the invention is realized by the following technical scheme:

c19orf66 is located in 3 band 2 sub band of short arm 1 region of chromosome 19, cDNA full length is 768bp, there are two transcription spliceosomes, the expressed protein product contains 255 amino acid residues, Gene ID:55337, GenBank: AAH 35817.1. The cell level of the invention proves that C19orf66 has the capacity of inhibiting Zika virus, and shows that the C19orf66 can be used as a medicine for inhibiting the Zika virus.

Specifically, in the invention, an immunofluorescence experiment is adopted to determine that C19orf66 and Zika virus nonstructural protein NS3 have co-localization in cells; the co-immunoprecipitation experiment proves that the C19orf66 and the Zika virus non-structural protein NS3 have interaction; transient expression system and Western blotting are used for proving that C19orf66 can promote the degradation of the non-structural protein NS3 of the Zika virus in cells; using inhibitors of different degradation pathways of the protein, it was demonstrated that C19orf66 mediates the degradation of the non-structural protein NS3 of zika virus through the lysosomal pathway.

The invention therefore claims the following:

use of the antiviral protein C19orf66 in the preparation of an inhibitor of a non-structural protein of zika virus.

Preferably, the Zika virus inhibitor is a Zika virus non-structural protein NS3 inhibitor.

Preferably, the Zika virus inhibitor is an inhibitor of Zika virus proliferation in a host cell.

Compared with the prior art, the invention has the following beneficial effects:

the antiviral protein C19orf66 provided by the invention has the novel application of efficiently targeting the Zika virus non-structural protein NS3 and promoting the degradation of the lysosome pathway of NS3 so as to inhibit the proliferation of the Zika virus in host cells. The method develops a new clinical application field of the humanized antiviral protein in the field of antiviral control, thereby providing a new thought and direction for the development of a medicament targeting the non-structural protein NS3 of Zika virus, providing a scientific basis for the development of medicaments resisting the Zika virus and providing a new thought for clinical antiviral treatment.

Drawings

FIG. 1 is a graph showing the results of confirmation of the co-localization of C19orf66 and Zika virus nonstructural protein NS3 in cells using immunofluorescence assay.

FIG. 2 is a graph showing the results of co-immunoprecipitation experiments demonstrating the interaction between C19orf66 and Zika virus nonstructural protein NS 3.

FIG. 3 is a graph showing the intracellular degradation results of C19orf66 promoting the non-structural protein NS3 of Zika virus by using a transient expression system and Western blotting.

FIG. 4 is a graph showing the results of the degradation of non-structural protein NS3 of Zika virus mediated by the lysosomal pathway using inhibitors of different degradation pathways of the protein, confirming that C19orf 66.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified.

Example 1 cell co-localization of C19orf66 and zika virus nonstructural protein NS 3.

First, experiment method

1. And (4) cell transfection.

(1) Taking hNPC as a cell model, inoculating a proper amount of cells on a circular cover glass subjected to ultraviolet sterilization treatment for culturing 24 hours before transfection;

(2) adding 200ng of each of a plasmid pCEDF-C19orf66-myc highly expressing C19orf66 and a plasmid pCDEF-NS3-flag highly expressing NS3 into 25 mu l of Opti-MEM, adding P3000TM 1 mu l, and mixing softly;

(3) add 2. mu.l Lipofectamine TM 3000 reagent to 25. mu.l Opti-MEM, mix gently, and leave at room temperature for 5 minutes;

(4) mixing the liquids (2) and (3), flicking the tube wall, and standing at room temperature for 5 min;

(5) adding the (4) into a culture medium of hNPC cells, and culturing in an incubator at 37 ℃;

(6) and changing the liquid after 4-6 h, and continuing culturing.

2. Cellular immunofluorescence.

(1) 48 hours after transfection, the cell culture medium was aspirated and washed twice with 1 × PBS;

(2) fixing cells with 4% paraformaldehyde for 30s, adding ice-precooled methanol to fix cells, placing in a refrigerator at-20 deg.C, and fixing for 10 min;

(3) the fixative was discarded and washed 3 times with 1 × PBS;

(4) adding 0.2% Triton-X100, standing at room temperature for 5min to increase permeability of cell membrane;

(5) discarding Triton-X100, washing 3 times with 1 × PBS;

(6) adding 10% BSA, placing on a horizontal shaking bed, and sealing for 30 min;

(7) incubating primary antibody: diluting with 1 × PBS according to the concentration recommended by the antibody specification, and incubating for 1h at room temperature;

(8) washing with 1 × PBS for 3 times, each for 10 min;

(9) hatching a secondary antibody: according to the recommended concentration of 1: diluting with 500, 1 XPBS, and incubating at room temperature for 30-45 min in a dark place after all processes begin after incubating the secondary antibody;

(10) washing with 1 × PBS for 3 times, each for 10min, and keeping out of the sun;

(11) staining nuclei with DAPI (1: 10000, 1 × PBS dilution), 10min, and keeping out of the sun;

(12) washing with 1 × PBS for 3 times, each for 10min, and keeping out of the sun;

(13) dry the slide in the dark overnight, seal the cover slip on the slide with Anti-fade;

(14) and (5) observing and photographing under a fluorescence microscope.

Second, experimental results

The results are shown in FIG. 1, and immunofluorescence analysis shows that C19orf66 and Zika virus nonstructural protein NS3 are co-localized in cells, i.e., the binding between the two is proved.

Example 2 interaction of C19orf66 and Zika virus nonstructural protein NS 3.

First, experiment method

1. Appropriate amount of cells were plated in cell culture dish P100, and after 24 hours, Zika virus (1MOI) was infected, and 48 hours later, the cells were used for co-immunoprecipitation, and the corresponding proteins were detected by immunoblotting.

2. The method of co-immunoprecipitation is as follows:

(1) taking out the treated cells, discarding the original culture medium, and washing with precooled 1 × PBS for 2 times;

(2) adding precooled 6ml of 1 XPBS, scraping cells from a culture dish, transferring cell sap into a 15ml centrifuge tube, and centrifuging at 2000rpm and 4 ℃ for 5 min;

(3) discarding the supernatant, adding 500 μ l of 1 × IP lysis buffer (containing protease inhibitor) into the cell precipitate, transferring the cell suspension into a 1.5ml centrifuge tube, placing on ice, shaking once every 10min for 30s each time, maintaining for 30min, and ensuring the cells to be fully lysed;

(4) centrifuging at 12000rpm for 30min, collecting supernatant, and repeating the step for 1 time;

(5) taking 30 mu l of lysate as Input, adding the rest lysate into protein A/G-beads pre-crosslinked with NS3 antibody, and incubating overnight at 4 ℃ by slow shaking;

(6) centrifuging the overnight co-incubated protein-antibody mixture at 3000rpm and 4 ℃ for 5min, washing with a 0.1ml syringe and a 1 × IP wash buffer for 5-6 times, and taking care that the movement is gentle to avoid damaging the protein binding effect

3. The specific operation of the immunoblotting experiment is as follows:

carrying out reducing SDS-PAGE on the protein sample, and stopping electrophoresis after bromophenol blue runs out of the gel; preparing two pieces of 3M filter paper and a PVDF membrane, immersing the filter paper and the PVDF membrane into methanol deionized water for 5 minutes, and then immersing the filter paper and the fiber pad into a 1 Xrotary membrane buffer solution; stripping off the gel, removing the concentrated gel part, and cutting the filter paper and the PVDF membrane into the size of the gel; the membranes were transferred according to the sandwich method, in the following order: the negative electrode, the fiber pad, 1 piece of special filter paper, the gel, the PVDF membrane, 1 piece of special filter paper and the fiber pad positive plate are placed in a membrane transferring groove; ice-bath, 300mA film-turning for 2 hours; taking out the PVDF membrane, and sealing with sealing liquid for 1 hour; primary antibody 4 degree overnight incubation; recovering primary antibody, washing membrane with TBST for 10min each time, and repeating for 3 times; incubating the secondary antibody at normal temperature for 60 min; recovering secondary antibody, washing membrane with TBST for 10min each time, and repeating for 3 times; developing with ECL luminescence in a dark room.

Second, experimental results

The results are shown in FIG. 2, and the co-immunoprecipitation experiment demonstrates that non-structural protein NS3 forms a complex with C19orf66 in hNPC cells after Zika virus infection, further demonstrating that the two bind to each other.

Example 3 effect of C19orf66 on the intracellular content of the nonstructural protein NS3 of zika virus.

First, experiment method

1. The exogenous high expression method is described in step 1 of example 1. Wherein the amounts of the C19orf66 plasmid were 0, 0.2, 0.4, 0.6, 0.8, 1ug, the amount of the plasmid NS3-Flag was 2ug, and the amounts of P3000TM and Lipofectamine were changed accordingly.

2. 48 hours after transfection, the proteins were collected and detected by immunoblotting.

3. Immunoblotting procedure as in example 2.

The results are shown in FIG. 3, and when the expression level of C19orf66 in the cells is increased, the content gradient of NS3 is reduced, which shows that C19orf66 promotes the degradation of the Zika virus nonstructural protein NS 3.

Example 4 the effect of C19orf66 on the intracellular content of the zika virus nonstructural protein NS3 was caused by the lysosomal pathway mediated degradation of NS 3.

First, experiment method

1. The exogenous high expression method is described in step 1 of example 1. Since the cells were plated in 6-well plates, the amounts of plasmid, P3000TM, Lipofectamine were varied accordingly.

2. Lysosomal inhibitors (ammonium chloride, chloroquine phosphate) were added 24 hours after transfection to inhibit lysosomal function, proteasome inhibitors (MG132) inhibited the proteasomal pathway and autophagy inhibitors (3-methyladenine) inhibited the autophagy pathway. Protein was recovered after 24 hours. And the corresponding protein was detected by immunoblotting. Immunoblotting procedure as in example 2.

Second, experimental results

The results are shown in FIG. 4: when a lysosome inhibitor (ammonium chloride and chloroquine phosphate) is added to inhibit lysosomes, the degradation of the Zika virus nonstructural protein NS3 is slowed, but the same effect is not achieved when the proteasome inhibitor (MG132) inhibits proteasomes and the autophagy inhibitor (3-methyladenine) inhibits autophagy, which indicates that C19orf66 mediates the degradation of the Zika virus nonstructural protein NS3 through a lysosome pathway.

Claims

1. Application of antiviral protein C19orf66 in preparing an inhibitor of Zika virus non-structural protein NS 3.

2. The use according to claim 1, wherein said inhibitor of Zika virus is an inhibitor of the proliferation of Zika virus in a host cell.