KR100875088B1 - Screening Methods of Antiviral Agent By Using The Movement of Mitochondria - Google Patents

Abstract

The present invention relates to a method for screening HBV virus antiviral agent by using mitochondrial migration, and more specifically, to inhibit the migration of mitochondria around the nuclear membrane by the contact of HuH7 cells containing HBV mutants with potential antiviral agents. It relates to a high-efficiency antiviral screening method characterized in that the identification.

Description

Screening methods of antiviral agent by using the movement of mitochondria

Hepatitis B virus (HBV; Hepadnaviridae family) is a potent source of liver disease, with an estimated 350 million chronic HBV carriers worldwide. HBV infection causes acute and chronic hepatitis, which can further progress to cirrhosis and hepatocelluar carcinoma (HCC) (6, 29). Although epidemiological evidence is a major etiological evidence of hepatocellular carcinoma (HCC), the importance of chronic HBV infection is strongly supported, but the molecular mechanisms of disease development and cancer development are not clearly known.

Hepadnaviruses are small, membrane-like DNA viruses that replicate primarily in liver cells and are involved in hepatocellular carcinoma. Hepatitis B virus (HBV) is the prototype of hepadnaviruses and has a partially unfolded circular DNA genome. After entering the hepatocytes and transferring the virus to the nucleus, the viral DNA is converted into covalently closed circular (ccc) DNA, which is used as a template for viral messenger RNA (mRNA) transcription. . Pregenomic RNA (pgRNA) serves as mRNA for capsid (C) and polymerase (P) proteins and serves as a template for reverse transcription. Subgenomic 2.4 kb RNA serves as mRNA for the expression of large HBsAg, 2.1 kb RNA serves as mRNA for medium and small HBsAg, and 0.7 kb mRNA serves as mRNA for pgRNA. pgRNA and reverse transcriptase are packed into viral core particles in the cytoplasm to form immature core particles. Encapsidated pgRNA is reverse transcribed by reverse transcriptase and partially transcribed into double unpaired circular DNA. Mature core particles containing partially unfolded circular DNA are further processed or recycled to the nucleus for virion assembly in the endoplasmic reticulum (ER) and Golgi apparatus. The recycling mechanism is believed to be important for maintaining the cccDNA pool in the nucleus. Such behavior of HBV core particles in the host cytoplasm is important in HBV life history, but little is known about this mechanism (Blum HE, Gerok W and Vyas GN: The molecular biology of hepatitis B virus. Trends Genet 5: 154- 8, 1989).

During the life cycle of a virus, the virus modifies the host's system in several ways. Viruses induce reorganization of cytoskeleton and distribution of organelles in host cells to facilitate dispersal during invasion and release from the host (Cudmore et al., Trends Microbiol 5: 142-8, 1997) Dramsi S, Auun Rev Cell Dev Biol 14: 137-66 1998; Ploubidou A et al EMBO J 19: 3932-44, 2000).

Takada et al. Reported that the binding of HBV X protein and mitochondria causes aggregation of mitochondria around the nuclear membrane and caused cell death (Takada et al, Oncogene, 18, pp6965-6973 (1999)). However, Takada et al. Have not reported that mitochondrial migration is inhibited when mutations in other genes essential for HBV propagation other than HBV X protein are inhibited.

U.S. Patent No. 5968781, to which Dongwha Chemical has patented, relates to HBV polymerase and an antiviral screening method using the same, and provides an antiviral screening method using histidine tagged HBV polymerase and RNase H. However, such a screening method can detect only inhibitors related to HBV polymerase and RNase H, and there was a limit in the search for virus inhibitors throughout the virus life cycle.

The present inventors have conducted comparative studies of HBV core protein deficient mutants, HBV reverse transcriptase deficient mutants, HBV priming deficient mutants, HBV RNase H deficient mutants or HBV polymerase deficient mutants with wild type HBV. If the core protein, reverse transcriptase, HBV priming reaction deficiency, HBV RNase H reaction deficiency, mitochondria do not move around the nuclear membrane, wild type HBV with normal proliferative capacity confirmed that the mitochondria move around the nuclear membrane The invention has been completed.

It is to develop a method and system that can effectively screen HBV antiviral agents in a short time.

For the foregoing purpose, the first aspect of the present invention provides a method for screening an HBV antiviral agent, characterized in that mitochondria are inhibited from moving around the nuclear membrane. More specifically, it is an antiviral screening method characterized in that mitochondria in the cytoplasm are inhibited from moving around the nuclear membrane by potential antiviral administration in HuH7 cells in which the transfected HBV is stably maintained. More specifically, culturing the cells transfected and stably maintained with wild-type hepatitis B virus having a normal proliferative capacity; Contacting the potential antiviral agent with the cell; Supplying a detection object for identifying the position of the mitochondria; And hepatitis B virus inhibitor screening method comprising the step of confirming that the movement of mitochondria around the nuclear membrane is inhibited. More specifically, culturing PUB9 cells, which are stabilized cells in which 1 × 10 ⁴ to 1 × 10 ⁸ HBVs proliferate, in a culture dish; Contacting the PUB9 cells with a potential antiviral agent; Hepatitis B virus inhibitor screening method comprising the step of confirming that the movement of mitochondria to the periphery of the nuclear membrane through the immunofluorescence analysis that can determine the location of the mitochondria.

Potential antiviral agents according to the present invention refer to substances that are effective for inhibiting the growth of viruses.

The method of confirming that mitochondria migrates around the nuclear membrane can be performed by using a detector. The detection object can be any material that can easily measure the position of the mitochondria. Suitable detectors are well known to those skilled in the art. Water Examples detected mitochondrial specific Mito Tracker Red ^TM (MitoTrackerRed ^TM), an analogue and not when loader (rhodacyanine analog) MKT-077, tetramethyl rhodamine ethyl ester (tetramethylrhodamine ethyl ester), rhodamine (Rhodamine) 123 (Johnson et al J. Cell Biol. 88 , 526-535 (1981); Waggoner, AS, in: Applications of Fluorescence in the Biomedical Sciences (L. Taylor, A. Waggoner, R. Murphy, F. Lanni, R. Birge, eds.), pp-3-28, Alan Liss, 1986), 6G rhodaman (Felkai et al, The EMBO Journal 18 , 1783-1792 (1999)), but are not limited to these. Detection of the detectable is well known to those skilled in the art. Where a detectable is detected by enzymatic activity, generally it comprises the step of providing the enzyme with its appropriate substrate and detecting the reaction product (eg the light produced by luciferase). Another method may include quantification of the detectant expression. The method of quantification can be absolute quantification or can be quantified compared to the housekeeping level. Such detection may be performed manually or may be performed automatically as in a high-throughput screening system. High throughput test methods for the presence, absence, or quantification of detectables are well known to those skilled in the art.

A second aspect of the invention is to provide an antiviral screening system. More specifically, wild-type hepatitis B virus with normal proliferative ability is stably maintained; A container for contacting the cell with a potential antiviral agent; And hepatitis B virus inhibitor screening system comprising a polarimeter. As a cell for stably maintaining the virus, all cells in which the hepatitis B virus proliferate can be used. Preferably, HuH7 cells, HepG2 cells are used. Keeping stable means that the virus remains in the cell after generations. In most cases, to ensure this stability, antibiotic resistance genes are added to ensure that the virus remains stable even after cell division. In contrast, a transient transfection method is used. Temporally transfected viral genes disappear as the host cell continues to divide.

The polarimeter may be any polarizer known in the art. Preferably, a confocal microscope using a mito tracker can be used.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the following examples.

Example 1 Confirmation of Proliferation of Hepatitis B Virus and Rearrangement of Mitochondria

(1) Preparation of HBV Inhibitory Mutant DNA and Stable Cell Line

HBV wild-type strains are HBV subtypes subcloned with pcDNA3 (Invitrogen) to express HBV under the cytomegalovirus early (CMV IE) promoter (Kim et al., Virology 322.22-30, 2004). pPB was constructed from 1.3 kb of adw R9 (Blum et al., Trends Genet 5. 154-158, 1989). The priming response deficient TP Tyr65Phe mutant replaced Tyr65 with Phe in the TP domain of the P protein (Kim et al., Virology 322. 22-30, 2004). P protein deficient mutations were prepared by changing the AUG start codon and frame-shifting. Reverse transcriptase deficient RT YMHA mutations were prepared by retrofitting the conserved YMDD reverse transcriptase motif with YMHA. C protein deficiency mutation of HBV was prepared by introducing a stop codon (TAA) to the C protein 8th amino acid (Glu, GAA). RNase H response deficient mutations were prepared by substituting Val for Glu750 in the RH domain of the P protein ( FIG. 1 ). HBV proliferative stable cell line PUB9 was obtained by transfection of pPB. The HBV non-proliferative stable cell lines PUF12, YMHA46, RH27 and P-def30 were transfected with TP Tyr65Phe, RT YMHA, RNase H and P-deficient mutations, respectively. Stable cell lines were selected with 1 mg / ml G418 sulfate (Geneticin ^R ) and maintained at 200 ug / ml G418 sulfate. PEB8 as a control was prepared by transducing pPB into HepG2 cell line and maintained at 200 ug / ml G418 sulfate.

(2) cell culture and transfection

HuH7 hepatoma cell line was used for transfection of HBV wild type and mutant clones. HuH7 cells were maintained in Dulbecco's modified eagle's medium supplemented with penicillin, streptomycin and 10% fetal calf serum. HBV wild type and mutant plasmids (8 ug) were transfected onto 10 cm plates using polyethyleneimine (1 ug / ul) with HuH7 cells. HBV proliferative stable cell line PUB9 was obtained by transfection of pPB. Stable cell lines selected with 1 mg / ml G418 sulfate (Genetisin ^R ) are maintained in Dulbecco's Modified Eagles medium supplemented with 200 ug / ml G418 sulfate, penicillin and streptomycin and 10% fetal calf serum.

 Example 2 Differences in Mitochondrial Rearrangement in Wild-type Hepatitis B Virus and Inhibited HBV Mutants.

(1) Immunofluorescence Analysis

Stably transfected cells were incubated for 24-48 hours on a cover slip, washed with PBS and fixed with 4% formaldehyde for 7 minutes. And it is permeated for 5 minutes with 0.15% Triton X-100 dissolved in PBS. Samples are incubated for 1 hour with 1% bovine serum albumin ( BSA ) and incubated with the first antibody. Anti-HBc antibody (DAKO) is used to detect core particles. The first antibody dissolved in 1% bovine serum albumin is incubated at room temperature for 1 hour. Cells are incubated for 1 hour with FITC conjugated anti-mouse antibody or rhodamine conjugated anti-mouse antibody. Mito-trackers were used to detect mitochondria. Cover slips with proliferative stabilized cells grown on cover slips are placed upside down on slides and observed with a confocal laser scanning microscope (LSM510, Zeiss).

(2) mitochondrial clustering

Clustering around the nuclear membrane of the mitochondria is judged by two criteria. If the area occupied by the mitochondria in the cell was less than 40%, crescent or annular mitochondrial staining was observed. Obvious mitochondrial aggregation was used as the first criterion and the analysis was performed in a blind manner by two people. As a second standard of amorphous mitochondrial aggregation, the LSM5 image browser (Zeiss Inc.) occupied by mitochondria in cells is automatically calculated and compartmentalized and compared with the entire cell area. For the sake of simplicity, the nuclear domain is always included in the calculation. If the area of mitochondria per cell is less than 60%, the mitochondria were determined to be aggregated cells, whereas if the value of the mitochondria was higher than 60%, the mitochondria were determined to be non-aggregated cells.

(3) results

In order to observe the distribution of intracellular microorganisms in cells expressing HBV wild type and mutant, the location of mitochondria was examined using mito-tracker and confocal microscopy. 2 , it can be seen that mitochondria are gathered in a ring shape around the nuclear membrane in PUB9, an HBV proliferating cell. In contrast, in HBV mutants, which are non-proliferative cells, mitochondria are stably expressed and distributed throughout the cytoplasm.

Example 3 Hepatitis B Virus Inhibitor Screening Confirmation

HuH7 hepatoma cell line is used for transfection of HBV wild type. HuH7 cells are maintained in Dulbecco's modified eagle's medium supplemented with penicillin and streptomycin and 10% fetal bovine serum. HBV wild-type plasmids (8 μg) are transfected onto 10 cm plates with polyethylenimine (1 μg / μl) into HuH7 cells. HBV proliferative stabilized cell line PUB9 is prepared by transfection of pPB. Stabilized cell lines were selected with 1 mg / ml G418 sulfate (Geneticin ^R ) and maintained in Dulbecco's Modified Eagle's Medium supplemented with 200 ug / ml G418 sulfate, penicillin and streptomycin and 10% fetal calf serum.

PUB9 cells transfected with HBV proliferative stabilized cell lines were incubated for 24 hours on coverslips prepared in 6-well plates, followed by Hepaguard ™ from the latent antiviral company Hepaguard Company Group ( HCG ). After 3 days of treatment with 350 μg / ml, Adefovir ™ was treated with 100 μM and immunofluorescence was performed after 4 days. In order to observe the mitochondria, it was observed by a confocal laser scanning microscope (LSM510, Zeiss) using a mito-tracker.

As a result , PUB9 cells treated with nothing as shown in the panel (A) of FIG. 3 showed a pattern in which mitochondria were gathered around the nuclear membrane. In the experimental group (B) treated with adefovir of FIG. 3 and the experimental group (C) treated with hepagard, mitochondria were inhibited from moving around the nuclear membrane, and hepatitis B virus was suppressed.

The present invention is a screening method that can easily and efficiently search for inhibitors of hepatitis B virus. Such a screening method can be a high throughput method to search for antiviral agents with a large potential. In addition, since it is confirmed that the intracellular virus is inhibited by treating the cell directly, there is an advantage in that a drug delivered to the intracellular virus can be selected when the cell is actually treated outside the cell.

1 shows a schematic of the preparation of HBV wild type and mutants. HBV sequences are shown as thick lines and ε sequences are indicated at the 5 'and 3' ends. C, P, S and X genes are shown as open boxes. Each domain of P protein (TP, Spacer, RT and RNase H) is shown. The location of the point mutations is indicated by closed arrows.

2 shows the mitochondrial distribution in cells expressing the hepatitis B virus wild type and proliferative mutants. Mito-Tracker and anti-alpha-tubulin antibodies observed through confocal microscopy are stained mitochondria (red) and microtubules (green), respectively. (A) Wild type (B) RNase H response deficient proliferation inhibition mutant (C) Priming reaction deficient proliferation inhibition mutant (D) Reverse transcript deficiency proliferation inhibition mutant

FIG. 3 is an immunofluorescence assay after incubating stable PUB9 cells in which 2 × 10 ⁵ hepatitis B viruses are propagated on a coverslip prepared in a 6-well plate for 24 hours, and then treated with hepatitis B virus inhibitor. : IFA). (A) No treatment on stable PUB9 cells in which hepatitis B virus proliferates. (B) 4 days after treatment with adefovir (100 uM) (C) 3 days after treatment with hepagard (350 μg / ml) (D) PUF12 cells that do not proliferate hepatitis B virus

Claims (3)

Transfecting the cells with wild-type hepatitis B virus having normal proliferative capacity and culturing the cells; Contacting the potential antiviral agent with the cell; Supplying a detection object for identifying the position of the mitochondria; And Hepatitis B virus inhibitor screening method comprising the step of confirming that the movement of mitochondria around the nuclear membrane is inhibited. Cells transfected with wild-type hepatitis B virus with normal proliferative capacity; A container for contacting the cell with a potential antiviral agent; And Hepatitis B virus inhibitor screening apparatus comprising a polarimeter. The method of claim 1, further comprising: culturing the stabilized cells in which 1 × 10 ⁴ to 1 × 10 ⁸ HBVs proliferate in a culture dish; Contacting the cell with a potential antiviral agent; And Hepatitis B virus inhibitor screening method comprising the step of confirming that the movement of mitochondria to the periphery of the nuclear membrane through the immunofluorescence analysis that can determine the location of the mitochondria.