CN111803485B

CN111803485B - Application of compound AN-329 in inhibiting hantavirus release diffusion

Info

Publication number: CN111803485B
Application number: CN202010554072.6A
Authority: CN
Inventors: 吴兴安; 应旗康; 王芳; 张晓晓; 刘梓谕; 董宇航; 刘蓉蓉
Original assignee: Fourth Military Medical University FMMU
Current assignee: Fourth Military Medical University FMMU
Priority date: 2020-06-17
Filing date: 2020-06-17
Publication date: 2022-12-20
Anticipated expiration: 2040-06-17
Also published as: CN111803485A

Abstract

The invention discloses application of a compound AN-329 to inhibition of hantavirus release and diffusion, wherein the compound AN-329 can inhibit the interaction of hantavirus Gn protein and a host ESCRT; lays a certain foundation for researching the life cycle of the hantavirus and the interaction mechanism of the hantavirus and the host, and provides a thought for developing anti-hantavirus medicaments. Experiments prove that: after AN-329 is added, the amount of HTNV antigen in the cells secondarily infected by the supernatant is obviously reduced; the nucleic acid level of HTNV in cells secondarily infected by the supernatant is obviously lower, and the small molecule compound AN-329 effectively inhibits the release and diffusion of hantavirus.

Description

Application of compound AN-329 in inhibiting hantavirus release diffusion

Technical Field

The invention belongs to the technical field of microbial immunity, and particularly relates to application of a compound AN-329 in inhibiting hantavirus release and diffusion.

Background

HTNV belongs to Bunyaviridae (Bunyaviridae), hantavirus (Hantavirus). In 1976, korean scholars Li Gaowang successfully isolated the virus for the first time in lung tissues of Uighur melanogaster mice in Hantaan river basin in epidemic areas of the country. The Hantaan virus HTNV is taken as a representative of Hantaan virus to be researched, the process of cell entry and intracellular replication of the HTNV is known and researched to a certain extent at present, and the life cycle of the HTNV is divided into five stages of adsorption, penetration, genome release, replication, translation and budding release. Early in viral infection, budding release is very vigorous, which is the key to expanding the infection range, but there is no conclusion about how the virus releases progeny virus.

The hemorrhagic fever of renal syndrome caused by hantavirus is acute, has serious symptoms and high mortality rate, and lacks of specific and effective treatment medicines. At present, the research at home and abroad considers that hantaan virus can be extracellularly in two modes, one mode is that through an endoplasmic reticulum-Golgi body secretion pathway, virus mRNA translates a precursor GPC of envelope Glycoprotein (GP) on the endoplasmic reticulum, and after being cut, the virus forms Gn protein and Gc protein two parts (Gn is reserved at the N end of GPC, and Gc is reserved at the C end), and then the Gn protein and the Gc protein are secreted and released to the outside of cells through Golgi body; and the Gn protein and the Gc protein of the virus are expressed and then positioned on the surface of a cell membrane, so that the host ESCRT is hijacked to directly bud from the cell plasma membrane. However, in either method, the support of the host ESCRT is required. In recent years, domestic and foreign studies show that the HTNV Gn cytoplasmic tail region can interfere with the release of viruses after mutation, and the previous studies of the applicant find that part of key molecules in host ESCTs interact with Gn to be the key for completing the release of the viruses.

Disclosure of Invention

The invention aims to provide application of a compound AN-329 in inhibiting hantavirus release and diffusion, which can effectively inhibit the release and diffusion of hantavirus.

The first technical scheme adopted by the invention is the application of the compound AN-329 in inhibiting the release and diffusion of hantavirus.

The present invention is also characterized in that,

the structure of the compound AN-329 is AN-329/43450194:

in particular to application of a compound AN-329 in inhibiting the release and diffusion of hantaan virus.

The compound AN-329 can inhibit the interaction of hantavirus Gn protein and host ESCR.

The second technical scheme adopted by the invention is that the compound AN-329 inhibits the release and diffusion of hantavirus and is applied to the preparation of the anti-hantavirus medicament.

The beneficial effects of the invention are:

the invention proves that the release of the hantaviruses is due to the interaction of the cytoplasmic tail region of the hantaviruses Gn and the host ESCR, and the small molecular compound AN-329 can interfere the interaction of the cytoplasmic tail region of the hantaviruses Gn and the host ESCR and inhibit the release and diffusion of the hantaviruses; lays a certain foundation for researching the life cycle of the hantavirus and the interaction mechanism of the hantavirus and the host, and provides a thought for developing anti-hantavirus medicaments.

Drawings

FIG. 1 shows Western blot detection results of HTNV antigens of HTNV infected cells added with AN-329;

FIG. 2 shows the result of immunofluorescence assay of HTNV antigen of HTNV infected cells added with AN-329;

FIG. 3 shows the results of the detection of the viral nucleic acid level of HTNV-infected cells with AN-329.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

1. Screening of key epitope sequences affecting the cytoplasmic tail of HTNV-related Gn

(one) prediction

Firstly, a bioinformatics technology is used to design small molecules capable of blocking the interaction of Gn and ESCRT, and 45 small molecule compounds with structures capable of entering the active center of the ESCRT are selected from a small molecule compound library.

(II) screening

A549 cells are digested and then are passaged to a 6-pore plate cell culture plate, HTNV is infected according to MOI =1 when the cell density reaches 60%, cell culture supernatant is added 6h after infection according to different compound concentrations, the mixture is uniformly mixed, and then CO is placed at 37 DEG C ₂ The cells were cultured in a cell incubator for 24h, and on the same day, the A549 cells were digested and then passaged to a new 96-well plate. Cell supernatants were aspirated from 6-well plates 24h after infection and diluted 10-fold in a gradient into 96-well plates. After the 96-well plate is cultured for 4d, collecting cells, performing immunofluorescence assay on the HTNV nucleocapsid antigen to determine the virus amount. Screening to obtain 1 of the compounds with the best effect of inhibiting HTNV release: AN-329.

2. Identification of inhibition of HTNV release at AN-329 cellular level

Wsternblot detection

Digesting A549 cells, transferring to a 6-pore plate cell culture plate, infecting HTNV according to MOI =1 when the cell density reaches 60%, adding cell culture supernatant 6h after infection according to different compound concentrations, uniformly mixing, and placing CO at 37 DEG C ₂ Culturing in a cell incubator for 24h, digesting the A549 cells on the same day, and then passaging toA new 6-well plate was used to collect cells after further 4 days of culture. The cell samples obtained were added to a Western blot sample buffer, boiled for 5min, cooled and then electrophoresed for 50min in 12% SDS-PAGE at 160V, followed by transferring to PVDF membrane at a constant voltage of 100V. PVDF membrane is blocked by 5% bovine serum albumin blocking solution BSA for 1h at room temperature, then HTNV specific monoclonal antibody is added, and incubation is carried out overnight at 4 ℃. The excess antibody was washed away with TBST buffer, an infrared-labeled anti-mouse secondary antibody was added, incubated at room temperature for 2h, and the TBST washed away unbound secondary antibody, scanned using an oddesal infrared imager and analyzed for results. The results are shown in FIG. 1 as a positive control, and the amount of HTNV antigen in cells infected twice with supernatant was significantly reduced after AN-329 was added.

(II) immunofluorescence assay

A549 cells are digested and then are passaged to a 6-pore plate cell culture plate, HTNV is infected according to MOI =1 when the cell density reaches 60%, cell culture supernatant is added 6h after infection according to different compound concentrations, the mixture is uniformly mixed, and then CO is placed at 37 DEG C ₂ Culturing in a cell incubator for 24h, digesting the A549 cells on the same day, then transferring to a new 6-pore plate, continuously culturing for 4d, and then collecting the cells. Cells were fixed with 0.4% paraformaldehyde and permeabilized with Triton X-100. The treated cells were incubated for 1h at 37 ℃ with Gn-specific monoclonal antibodies. Excess antibody was washed away from PBST, cy 3-labeled anti-mouse fluorescent secondary antibody was added, incubated at 37 ℃ for 1h, and unbound secondary antibody was washed away from PBST. After staining the nuclei with DAPI solution, the distribution and intensity of red fluorescence was observed under a fluorescence microscope. As shown in FIG. 2, the fluorescence area in the cells was significantly reduced by the addition of AN-329.

(III) qRT-PCR detection

A549 cells are digested and then are passaged to a 6-pore plate cell culture plate, HTNV is infected according to MOI =1 when the cell density reaches 60%, cell culture supernatant is added according to different compound concentrations for 6h after infection, the mixture is uniformly mixed and then is cultured in a CO2 cell culture box at 37 ℃ for 24h, the A549 cells are digested and then are passaged to a new 6-pore plate on the same day, the cells are collected after continuous culture for 3d, total RNA is extracted, and 2000ng of the total RNA is taken to be reversely transcribed into cDNA. And performing qRT-PCR by using SYBR green as a fluorescent dye, an HTNV S fragment as a target and beta-actin as an internal reference. The results are shown in FIG. 3, where nucleic acid levels of HTNV were significantly lower in cells secondarily infected via supernatant in FIG. 3.

According to the content, the invention proves that the release of the hantavirus is due to the interaction of the Gn cytoplasmic tail region of the hantavirus and the host ESCR, so that the small molecular compound AN-329 capable of inhibiting the release and diffusion of the hantavirus is screened out, and the release and diffusion of the hantavirus are inhibited; is beneficial to the study of the life cycle of the virus and provides an idea for developing anti-hantavirus medicines.

Claims

1. The application of a compound AN-329 in preparing a medicament for inhibiting hantavirus release diffusion, wherein the structure of the compound AN-329 is AN-329/43450194:

。

2. the use of claim 1, in particular to the use of compound AN-329 for inhibiting the spread of hantavirus release.

3. The use of claim 1, wherein said compound AN-329 inhibits the interaction of hantavirus Gn protein with host ESCRT.

4. The application of a compound AN-329 in inhibiting hantavirus release and diffusion in preparing a hantavirus-resistant medicament, wherein the structure of the compound AN-329 is AN-329/43450194:

。