KR101534462B1 - Pharmaceutical composition for prevention and treatment for Hepatitis C containing cyclin A2 expression or activity inhibitor - Google Patents

Abstract

BACKGROUND AND PURPOSE : Hepatitis C virus (HCV) requires host cell proteins for proliferation. In order to identify the cell factor essential for HCV proliferation, the siRNA library targeting the cell cycle was screened using HCV-infected cells grown by cell proliferation, and the gene encoding cyclin A2 was selected and characterized. CycA2 deregulation was associated with several types of tumors, including liver cancer.
Methods : The effects of CycA2 on HCV proliferation were studied by siRNA mediated knockdown analysis, Invitro and Invivo protein binding assay, Luciferase reporter gene analysis and immunoblot analysis.
Results : siRNA mediated CycA2 reduction significantly inhibited HCV replication in both HCV subgenomic replicon and HCVcc infected cells. In addition, HCV NS5B specifically interacted with CycA2 in Invitro and Invivo. Protein interactions were mediated through the cyclic box of CycA2 and the palm domain of NS5B. The R / HxL motif of the palm domain of HCV NS5B mediates protein interaction with CycA2, and this interaction is essential for HCV replication. Furthermore, tilophorin, a product of the natural plant that acts as a CycA2 inhibitor, inhibited HCV replication.
CONCLUSIONS: HCV regulates CycA2 through NS5B protein for autogenous proliferation, and tiloporpholine is a potential therapeutic agent for HCV.

Description

[0001] The present invention relates to a pharmaceutical composition for prevention and treatment of hepatitis C comprising a cyclin A2 inhibitor,

The present invention relates to a therapeutic agent for hepatitis C virus comprising a cyclin A2 inhibitor, and it has been confirmed that HCV replication is inhibited in an experiment using tylopholine, which is a kind of cyclin A2 inhibitor, to confirm the possibility as an HCV therapeutic agent.

Hepatitis C virus (HCV) is the first virus found in 1989 and is one of the major causes of liver disease, including acute or chronic hepatitis, hepatocellular acincosis (HCC) [Choo Q, et al, Science 244: 359-362, Kiyosawa et al., Hepatology 1990; 12; 671-675). Approximately 3% of the world's population is chronically infected with HCV. HCV belongs to the genus Hepacivirus in the Flaviviridae family, and HCV has an envelope in which the 9.6 kb genome is linear and in the form of a single stranded RNA. The HCV genome encodes a long polyprotein precursor of 3,000 amino acids or more. This polyprotein is processed into a protein with at least 10 functions by the host protease and viral protease in the endoplasmic reticulum. Core proteins and envelope proteins (E1, E2) are structural proteins that are cleaved by the signaling enzyme of the host cell [Hijikata M. et al., Proc Natl Acad Sci USA 1991; 88: 5547-5551; Matsuura Y. et al., Virology 1994; 205: 141-150; Lin C. et al., J Virol 1994; 68: 5063-5073. Nonstructural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are nonstructural proteins with a variety of enzymatic activities including serine proteases, RNA helicase and RNA-dependent RNA polymerase, Deg.] C (Grakoui A. et al., Proc Natl Acad Sci USA 1993; 90: 10583-10587; Bartenschlager R. et al., J Virol 1993; 67: 3835-3844; Tomei L. et al., J Virol 1993; 67: 4017-4026]. Although the hepatitis C virus is a very prevalent pathogen, effective therapeutic agents and vaccines have not yet been developed. Currently, interferon-α alone or PEG-conjugated interferon-α is used in combination with ribavirin for the treatment of hepatitis C virus. However, this drug therapy represents a significant therapeutic effect difference between genotype. Although boceprevir (Victrelis ™) and telaprevir (Incivek ™), inhibitors of HCV NS3 / 4A protease, have recently been approved by the US Food and Drug Administration, treatment strategies using viral proteins have been successful enough because of their susceptibility to errors in HCV RNA replication I can not. Therefore, there is a desperate need to develop more effective therapies using host factors related to the HCV life cycle.

Several evidences suggest that cell cycle deregulation in hepatocytes contributes to malignant transformation and tumorigenesis. HCV core proteins, NS3, NS4B and NS5A proteins appear to potentiate transformation into oncogene [Banerjee A. et al., Viruses 2010; 2: 2108-2133]. These HCV proteins promote cell growth, cell cycle uncontrollability when stably expressed in cells or in transgenic mice. Nevertheless, there is no evidence of transformation in the context of HCV replication and infection. Unlike HBV, hepatocellular carcinoma requires HCV infection prior to cirrhosis, and HCV mediated liver cancer is associated with immunological pathogenicity. However, the precise role of HCV in cell cycle regulation is still uncertain. To gain insight into the mechanism of HCV replication, we identified the factors involved in HCV proliferation using an siRNA library targeting cell cycle genes.

Cyclin A2 (which is used in combination with "CycA2 " in the following description of the invention and the like) can activate two other cyclin-dependent kinases (CDKs), CDK1 and CDK2, It is a cell cycle control protein that can be. CycA2 is mostly present in the nucleus and is slightly present in the endoplasmic reticulum when HCV replication occurs [Tsang WY et al., J. Cell Biol. 2007; 178: 621-633]. Uncontrolled CycA2 has been detected in several types of tumors, including liver cancer, and thus uncontrolled CycA2 appears to contribute to cancer development [Yam CH et al., Cell Mol. Life Sci. 2002; 59: 1317-1326]. It has been reported that upregulation of CycA2 expression by HBV pre-S2 induces nodule proliferation in hepatocytes [Wang HC et al., Hepatology 2005; 41: 761-770]. Furthermore, the disruption of the cyclin A gene by insertion of HBV DNA is associated with liver cancer [Wang J. et al., Nature 1990; 343: 555-557].

Accordingly, it is an object of the present invention to solve the problem that there is no effective therapeutic agent for hepatitis C in the past, and to provide an effective preventive and therapeutic agent for hepatitis C infection.

The present inventors have found that CycA2 specifically interacts with the NS5B protein, which plays a crucial role in HCV proliferation. In addition, low concentrations of tilophorin in HCVcc cells significantly inhibited HCV proliferation and exogenous expression of CycA2 in the presence of tilapolin restored HCV replication. These results suggest that CycA2 plays a crucial role in HCV proliferation and therefore can be used as a therapeutic agent for HCV.

Therefore, inhibiting the expression or activity of CycA2 may inhibit the proliferation of HCV, and thus a composition containing a substance that inhibits the expression or activity of CycA2 can be used as a preventive and therapeutic agent for hepatitis C virus.

The present invention provides a pharmaceutical composition for the prevention and treatment of hepatitis C comprising a CycA2 expression or activity inhibitor.

The present invention also relates to a composition, wherein the CycA2 expression inhibitor is any one selected from the group consisting of an antisense nucleotide complementary to the mRNA of the CycA2 gene, short hairpin RNA (shRNA) and short interfering RNA (siRNA) to provide.

In addition, the present invention is characterized in that the siRNA for CycA2 is any one of SEQ ID NOS: 1 to 4. The siRNA for CycA2 can be easily prepared by those skilled in the art based on the CycA2 gene sequence, and the number of possible siRNAs is not limited, and the above four siRNAs are exemplified by Only. It is also apparent to those skilled in the art that siRNAs against the CycA2 gene can inhibit the expression of CycA2 by binding to the CycA2 gene.

The shRNA for CycA2 can also be easily prepared by those skilled in the art based on the CycA2 gene sequence. There is no particular limitation on the number of possible shRNAs, and the shRNA for the CycA2 gene is the CycA2 gene Can inhibit the expression of CycA2. It is apparent to those skilled in the art that the present invention can be used to inhibit the expression of CycA2.

Also, the present invention provides a composition, wherein the CycA2 protein activity inhibitor is tylophorine.

In addition, the present invention provides a composition, wherein the CycA2 protein activity inhibitor is an antibody binding to the CycA2 protein.

The present invention also provides a composition, wherein said antibody is an antibody that binds to a cyclin box of CycA2.

The composition for the prevention and treatment of hepatitis C comprising the CycA2 protein expression or activity inhibitor of the present invention as an active ingredient comprises 0.0001 to 50% by weight of the effective ingredient based on the total weight of the composition.

The composition of the present invention may contain one or more active ingredients which exhibit the same or similar functions in addition to the expression or activity inhibitor of the CycA2 protein. The composition of the present invention can be administered orally or parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation.

The composition of the present invention may further comprise at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and one or more of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. In addition, it can be formulated into injection formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. And can be formulated preferably according to the respective ingredients using appropriate methods in the art or using the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The composition of the present invention may comprise a freeze-dried composition, particularly for the composition of an injectable solution upon addition of an isotonic sterile solution or sterile water or suitable physiological saline. The dosage of the active ingredient to be used may vary depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, severity of disease, etc. and the daily dosage is about 0.0001 to 100 mg / kg, preferably 0.001 to 10 mg / kg, and can be administered once or several times a day.

The composition of the present invention may be used alone or in combination with radiation therapy, chemotherapy, and a method using a biological response modifier.

HCV replication is highly dependent on host cell factors. The development of effective therapeutics for HCV has not yet been fully successful, as HCV produces similar species due to the unique nature of RNA replication. Therefore, identification of cellular factors involved in HCV proliferation may provide a new strategy for developing anti-HCV therapies. The present inventors have suggested that CycA2 is essential for HCV proliferation by showing that siRNA-mediated CycA2 knockdown significantly reduces intracellular HCV RNA, viral protein and extracellular HCV RNA levels. Interestingly, cyclin A1 / B1 or CDK1 / CDK2 was also not essential for HCV replication (Figure 14). This suggests that CycA2 is specifically involved in HCV replication, and that this role can not be replaced by members of other cyclin families.

CycA2 specifically interacts with NS5B as revealed by GST pulldown and common immunoprecipitation assays. Protein interactions between CyvA2 and NS5B are mediated through the cyclic box of CycA2 and the palm domain of NS5B. The R / HxL motifs of viruses and cellular proteins have been proposed to be essential for interaction with CycA2 [Wohlschlegel J.A. et al., Mol Cell Biol 2001; 21: 4868-4874). In addition, the present inventors have shown that the R / HxL motif of the NS5B palm domain mediates protein interaction with CycA2. It is noteworthy that the H254R mutation does not bind CycA2 while the H254R mutation has the ability to bind to CycA2. As a result, HCV proliferation is significantly reduced in the H254A mutation compared to the wild type. This suggests that protein interactions between CycA2 and NS5B can create a good intracellular environment that enhances the effect of viral replication rather than modulating viral hygromycin activity. A cell-based RNA-dependent RNA polymerase assay system developed by Ranjith-Kumar et al. [Ranjith-Kumar C.T. et al., PloS One 2011; 6: e22575], the inventors have shown that CycA2 has no effect on the hygroscopic activity of NS5B (data not shown). In addition, the inventors have demonstrated that siRNA-mediated CycA2 knockdown significantly reduces HCV replication in Huh7 cells. These data support the notion that protein interactions between CycA2 and NS5B create an intracellular environment that increases HCV replication. In this case, it was expected that the NS5B-CycA2 interaction would activate cell proliferation and partially explain the increased HCV replication by CycA2. However, if this is so, this effect is small and HCV replication is dependent on cell growth in Huh7 cells, whereas in other cells such as Huh6 cells, HCV replication will be limited to Huh7 cells because it is independent of cell growth [Scholle F. et al , J Virol 2004; 78: 1513-1524, Windisch MP et al., J Virol 2005; 79: 13778-13793). Indeed, the inventors have found that cell cycle progression is not altered by protein interactions between NS5B and CycA2 in HCV infected Huh7.5 cells (Fig. 9). In addition, treatment with tilolol for a short period of time was sufficient to inhibit CycA2 expression and HCV replication, but had no effect on cell growth. Therefore, there is a need for studies to determine the multifunctional effect of NS5B-CycA2 interaction on HCV replication.

The present inventors evaluated the effect of thyllofurin on HCV proliferation. In the present invention, tilaphorin significantly inhibited HCV proliferation at the levels of intracellular HCV RNA, viral proteins, and extracellular HCV RNA in HCV infected cells. Tylophorin selectively inhibited CycA2 expression, and low doses (0.06 μM) of tilophorin were sufficient to inhibit HCV proliferation. Furthermore, overexpression of CycA2 in the presence of tilaphorin substantially restored HCV replication. This indicates the possibility of inhibiting HCV replication by interfering with various signal pathways. Tilophorin has been reported to exert anti-inflammatory activity by inhibiting iNOS (inducible nitric-oxide synthase) and cyclooxygenase-II [Yang CW et al., Mol Pharmacol 2006; 69: 749-758]. This is very interesting because some HCV strains can affect onset through upregulation of proinflammatory genes. Taken together, the present inventors have shown that CycA2 plays a crucial role in HCV proliferation through interaction with NS5B. Furthermore, the present inventors have shown that thylophorin inhibits the proliferation of HCV, and thus has potential as an HCV therapeutic agent.

siRNA mediated CycA2 reduction significantly inhibited HCV replication both in HCV subgenomic replicon cells and in HCVcc infected cells. In addition, HCV NS5B specifically interacted with CycA2 in Invitro and Invivo. Protein interactions were mediated through the cyclic box of CycA2 and the palm domain of NS5B. The R / HxL motif of the palm domain of HCV NS5B mediates protein interaction with CycA2, and this interaction is essential for HCV replication. Furthermore, tilophorin, a product of the natural plant that acts as a CycA2 inhibitor, inhibited HCV replication.

Figure 1 is data showing that cyclin A2 is required for HCV replication. (A) Huh7.5 cells were transfected with 20 nM negative (Ne), cyclin A2 or positive (Po) siRNA for 48 h and then infected with Jc1 for four hours. Two days later, HCV infectivity was determined by the focus formation analysis method. (B) Huh7.5 cells were transfected with the indicated siRNA. After 96 hours, cell viability was determined by MTT assay. (C) Intracellular HCV RNA (left) and CycA2 mRNA (right) were separated from treated cells as described in panel A and analyzed by qPCR. (D) Total cell lysate was immunoblotted with antibody. (E) Cell culture supernatant was collected and extracellular HCV RNA was quantified by qPCR. (F) Subgenomic replicon (genotype 1b) cells were transfected with siRNA and protein expression after 96 hours was determined by immunoblot analysis. Ne: negative control siRNA; Po: siRNA targeting 5 'NTR of HCV as a positive control. The asterisk indicates a significant difference (p <0.05).
Figure 2 is data showing that cyclin A2 interacts with the HCV NS5B protein. (A) HEK293T cells were transfected with Myc-NS5A or Myc-NS5B. After 48 hours, total cell lysate was collected and incubated with GST or GST-CycA2. The complexes were precipitated with glutathione beads and the bound proteins were analyzed by immunoblotting using anti-Myc antibodies (top panel). Expression of Myc-NS5A and Myc-NS5B was confirmed by anti-Myc antibody (middle panel). GST and GST-CysA2 protein expression was confirmed by immunoblotting with anti-GST antibody (panel below). (B) HEK293T cells were transfected in combination with an expression plasmid. After 48 hours, the cell lysate was immunoblotted with anti-Myc monoclonal antibody, and the bound protein was immunoblot analyzed with anti-Flag monoclonal antibody (upper panel). (C) HEK293T cells were co-transfected with Flag-CycA2 and myc-NS5B of genotype 1b or Myc-NS5B of genotype 2a. After 48 hours, the cell lysate was immunoprecipitated with anti-Myc monoclonal antibody and the bound protein was detected by immunoblot analysis with anti-Flag monoclonal antibody (upper panel). (D) HEK293T cells were transfected with the Myc-NS5B expression plasmid. After 48 hours, the cell lysate was immunoprecipitated with anti-CycA2 polyclonal antibody, and the bound protein was detected by immunoblotting with anti-Myc monoclonal antibody (upper panel). (E) Immunoprecipitated with anti-CycA2 polyclonal antibody was treated with IFN or total cell lysate from HCV subgenomic replicon cells. Bound proteins were detected by immunoblotting with anti-NS5B monoclonal antibody (top panel) or anti-NS5A polyclonal antibody (middle panel). (F) Naive Huh7 cells and HCV replicon cells were fixed with 4% paraformaldehyde and incubated with anti-NS5B monoclonal antibody, anti-CyA2 polyclonal antibody and FITC-conjugated goat anti-mouse IgG, and cultured with rabbit anti-CycA2 polyclonal antibody and TRITC-conjugated goat anti-rabbit IgG to detect CycA2 (red). Double staining was expressed in yellow fluorescence in the combined image of the positions of NS5B and CycA2. Cells were stained with DAPI (4'6-diamidino-2-phenylindole) to label the nuclei. The box portion of the merged image is cropped to the right. (G) HCV replicon cells were fixed and incubated with anti-NS5B monoclonal antibody and incubated with FITC- or TRITC-conjugated anti-mouse antibody (rod # 1). HCV replicon cells were fixed and incubated with rabbit anti-CycA2 polyclonal antibody and then incubated with FITC or TRITC-conjugated anti-rabbit antibody (bars 2 and 3). The common location of NS5B and CycA2 (rod 4) was quantified by the Manders' overlap coefficient [Lim YS et al., J. Biol. 2011; 85: 8777-8788].
Figure 3 is data showing that protein interaction between CycA2 and NS5B is essential for HCV proliferation. (A) Amino acid sequence of wild type and mutant type of HxL in NS5B (genotype 2a). (B) HEK293T cells were cotransfected with wild type or mutant forms of HxL motifs in NS5B expression plasmid and Flag-CycA2. Total cell lysates were pooled and immunoprecipitated with anti-Flag monoclonal antibody 48 hours later, and the bound proteins were immunoblotted with anti-Myc monoclonal antibody (upper panel). (C) Huh7.5 cells were transfected with 10 [mu] g of wild-type or HxL mutants of Jc1. After 4 days, extracellular HCV RNA was separated from cell culture supernatant and quantified by qPCR. (D) Naive Huh7.5 cells were mock-infected or infected with wild-type or mutant forms of Jc1 collected in (C). Two days after infection, the total cell lysate collected was immunoblotted with the indicated antibody. (E) Two days after infection, culture supernatants and extracellular HCV RNA were quantitated by qPCR in cells treated as described in Panel D. (F) Naive Huh7.5 cells were infected with wild type or HxL mutants of Jc1. Two days after infection, cells were fixed and incubated with anti-NS5A polyclonal antibody. After washing with PBS, the samples were incubated with FITC-conjugated goat anti-rabbit IgG. Cells were stained with DAPI to label the nuclei. Samples were analyzed with a Zeiss LMS 700 laser confocal microscope system. (G) Polymerase activity of both NS5B wild type and mutant was analyzed as shown in Figure 7E. Data are the mean values of the results obtained from two independent experiments in duplicate, and the error bars represent the standard deviation of the mean. The asterisk indicates a significant difference (p <0.05).
Figure 4 is data showing that CycA2 is involved in the genomic replication step of the HCV life cycle. (A) A schematic view of the structure used in the present invention. (B) Huh7.5 cells were transfected with the indicated siRNAs. After 24 hours, the cells were cotransfected with 0.5 [mu] g of pRL-HL and 0.5 [mu] g of [beta] -galactosidase plasmid. Forty-eight hours post-transfection, the cell lysate was used to determine luciferase activity and normalize using β-galactosidase activity (top panel). Silent effects were confirmed by immunoblot analysis (bottom panel). GAPDH was included as a loading control. (C) Huh7.5 cells were cotransfected with 0.5 [mu] g of pRL-HL and 0.5 [mu] g of [beta] -galactosidase plasmid. After 4 hours, the cells were treated with the indicated amount of tylophorin for 48 hours and the luciferase activity was measured. (D) Huh7.5 cells were electroporated with 10 μg of JFH1-luc RNA and then incubated with DMSO (carrier) or 0.2 μM of tilaphorin. In parallel experiments, cells were electroporated with 10 μg of JFH1-luc GNN to determine input RNA translation. Cells were collected at indicated times and luciferase activity was measured. The asterisk indicates a significant difference from the DMSO control activity (Student's t test; _* , P <0.05; _** , P <0.01).
Figure 5 shows that CycA2 interacts with the HCV NS5B protein and the palm domain of NS5B through the cyclin box of CycA2. (A) Flag-CycA2 and mutants. aa: Amino acid. (B) CycA2 interacts with NS5B through a cyclin box. HEK293T cells were co-transfected with Myc-NS5B and mutants of the Flag-CycA1 expression vector. After 48 hours, the cell lysate was immunoprecipitated with anti-Myc monoclonal antibody and the bound protein was immunoblotted with anti-Flag monoclonal antibody (upper left panel). Myc-NS5B and Flag-CycA2 protein expression was confirmed by immunoblotting with anti-Flag or anti-Myc monoclonal antibody (right panel). (C) Myc-NS5B and the mutation. (D) CycA2 interacts with the palm domain of NS5B. HEK293T cells were co-transfected with the mutants of Flag-CycA2 and Myc-NS5B expression vectors. After 48 hours, the cell lysate was immunoprecipitated with anti-Flag monoclonal antibody, and the bound protein was immunoblotted with anti-Myc monoclonal antibody.
Figure 6 shows that the RxL / HxL motif of the palm domain of NS5B mediates protein interaction with CycA2. (A) Represents wild type and mutant type of RxL motif of NS5B (genotype 1b). (B) HEK293T cells were transfected with the wild-type or mutant form of the RxL motif of Myc-NS5B. After 48 hours, total cell lysates were collected and incubated with GST or GST-CycAs. The complexes were precipitated with glutathione beads and the bound proteins were analyzed by immunoblotting using anti-Myc antibodies (top panel). Expression of wild-type or mutant forms of Myc-NS5B was confirmed with anti-Myc antibodies (middle panel). Expression of GST and GST-CycA2 proteins was confirmed by immunoblotting with anti-GST antibody (panel below). (C) HEK293T cells were co-transfected with Flag-CycA2 and either wild-type or RxL mutants. After 48 hours, the cell lysate was immunoprecipitated with anti-Flag monoclonal antibody, and the bound protein was immunoblotted with anti-Myc monoclonal antibody (upper panel). Protein expression of Flag-CycA2 in the same cell broth was also confirmed by immunoblot analysis (bottom panel). (D) NS5B (Genotype Type 2a) Wild type and mutant type of HxL motif. (E) HEK293T cells were co-transfected with either wild-type or RxL mutants in Flag-CycA2 and Myc-NS5B. After 48 hours, the cell lysate was immunoprecipitated with anti-Flag monoclonal antibody, and the bound protein was immunoblotted with anti-Myc monoclonal antibody (upper panel). Flag-CycA2 protein expression in the same cell lysate was confirmed by immunoblot analysis (lower panel).
Figure 7 shows the role of CycA2-binding motif in HCV proliferation. (A) Huh7.5 cells were transfected with 10 [mu] g of wild-type or RxL mutant RNA of Jc1. After 4 days, total cell lysate was collected and immunoblotted with the indicated antibody. (B) Cultured supernatants of transfected cells were collected and extracellular HCV RNA was quantified by qPCR. (C) Naive Huh7.5 cells were mock-infected or infected with wild type or mutant forms of Jc1 collected in (B) above. After 2 days, the collected cell lysate was immunoblotted with the indicated antibody. (D) Culture supernatant was collected and extracellular HCV RNA was quantified by qPCR. (E) NS5B wild type and mutant type polymerase activity. Briefly, HEK293T cells were cotransfected with IFNb-luc, Flag-RIG-I, beta-galactosidase and various NS5B mutant plasmids. After 24 hours, the cells were lysed and luciferase activity was measured and normalized to? -Galactosidase activity. The data are mean values obtained from three independent experiments, which are repeated twice, and the bars represent the standard deviation of the mean. The asterisk indicates a significant difference (p <0.05).
Figure 8 is data showing that the NS5B RxL motif of Genotype 1b is required for CycA2 interaction and RNA replication. (A) Wild type and mutant amino acid sequence of NS5B RxL motif of genotype 1b. (B) HEK293T cells were cotransfected with wild-type or mutant forms of Flag-CycA2 and NS5B expression vectors. Total cell lysate was collected after 48 hours and immunoprecipitated with anti-Myc monoclonal antibody, and the bound protein was immunoblotted with anti-Flag monoclonal antibody (upper panel) . (C) NS5B wild type and mutant type polymerase activity. The data are the mean of the results obtained in two independent trials twice, and the bars represent the standard deviation of the mean. (D) a schematic diagram of wild type (RSL in NS5B) and mutant structure (ASL in NS5B) of genotype 1b. Both plasmids were constructed by replacing neojin with the firefly luciferase gene from pFK-neo / NS3-3 '(Lohmann et al . J. Virol. 2001; 75: 1437-1449). (E) Huh7.5 cells were electroporated with 10 μg of wild-type or R254A mutant-luc RNA. Cells were collected and luciferase activity was measured. The activity is normalized and expressed in relative light units (RLU). Data are the mean values of the results obtained in two independent experiments in duplicate.
Figure 9 shows that protein interaction between CycA2 and NS5B does not alter cell cycle in HCV infected cells. Huh7.5 cells were infected with the GFP-labeled Jc1 virus. After 48 hours, the cells were fixed with 775% ethanol for 2 hours, treated with RNase (2 mg / ml), and stained with 50 쨉 g / ml propidium iodide. Samples were filtered with nylon mesotubes (BD Biosciences) to remove cell aggregates and analyzed by FACSCalibur flow cytometer 29 (BD biosciences). (A) GFP-negative or positive cells were divided into gating based on GFP signal. Each cell was analyzed for cell cycle using FlowJo software (TreeStar, San Carlos, CA). The cell cycle distribution is the same as in the data (left panel), and the mean value of each cell cycle step was obtained from two independent experiments and is shown on the right panel. (B) GFP-negative cells were gated (three panels on the left) using the additivity group as a control in each sample to detect the number of replication-competent cells (GFP-positive cells). The wild-type and H254A mutant type replication competent cells are shown on the right panel. The data are mean values from two independent experiments twice, and the asterisks show significant differences (Student's t test; _** , P <0.01).
Figure 10 shows that tilophorin inhibits HCV replication in replicon cells. (A) Huh 7 cells containing subgenomic replicon (genotype 1b) were treated with tilapolin for 48 hours and 72 hours, respectively. Protein expression of HCV and CycA2 was analyzed by immunoblotting. Cell viability of (B) replicon cells was determined by MTT analysis after 72 hours of treatment with tilapolin.
Fig. 11 shows that tilaphorin inhibits HCV proliferation in a dose-dependent manner. (A) Huh7.5 cells were treated with various concentrations of tilapolin for 48 h after addition or addition of Jc1 for 4 h. Intracellular RNA was isolated from the cells and analyzed by qPCR. (B) Total cell lysate was collected and immunoblotted with the antibody. (C) Culture supernatant was treated with A and extracellular HCV RNA was quantitated with qPCR. (D) Huh7.5 cells infected or infected with Jc1 were treated for 48 hours with an increase in the amount of thylol. Cells were fixed and incubated with rabbit anti-NS5A polyclonal antibody. Washed with PBS and incubated with FITC-conjugated goat anti-rabbit IgG. Cells were stained with DAPI to label the nuclei. Samples were analyzed with a Zeiss LSM 700 laser confocal microscope. (E) Huh7.5 cells were transfected with vector or Flag labeled CycA2. After 24 hours, the cells were infected with Jcl for four hours and cultured in the absence of DMSO or in the presence of tilophorin (0.06 [mu] M). After 24 hours, the cell lysate was collected and immunoblotted with the antibody. (F) Huh7.5 cells were transfected with 10 [mu] g of JFH1-luc RNA for 24 hours and further transfected with vector or CycA2 for 24 hours. Cells were treated with DMSO 9 carrier) or with tilapolin for 24 hours and luciferase activity was measured. Data are the mean values of two independent experiments. The asterisk indicates a significant difference (p <0.05).
FIG. 12 shows that inhibition of HCV replication and CycA2 expression by tilapolin occurs prior to cell growth arrest. (A) Diagram showing the time for treating tilolol with two different concentrations. (B) Huh7.5 cells were treated at the times indicated with DMSO or Tylophorin. Cell proliferation was determined by MTT assay. (C) Huh7.5 cells were transfected with 10 μg of JFH1-luc RNA for 15 hours and then treated at the times indicated with DMSO or tilophorin and luciferase activity was measured. Data are the mean of two independent experiments (left panel) ( _* , P <0.05; _** , P <0.01). Huh7.5 cells treated as described in the left panel were immunoblotted to determine CycA2 expression levels (right panel).
Figure 13 shows that inhibition of cell growth in HCV replication is not a mechanism based on the effect of tilapolin. The diagram (A) shows a time chart of the treatment with tilolol. Huh7.5 cells were infected with Jc1 for four hours and the dose was incubated for 24 hours in the presence of other thylophorin, followed by removal of the thiophylline and further incubation for 24 hours (T1). Huh7.5 cells infected with Jc1 were cultured for 48 hours in the presence of other thylophorin doses (T2). (B) Cell proliferation at 0.12 mM (left panel) or 0.25 mM (right panel) of the indicated time points was detected by MTT assay. The data are the mean of three independent experiments twice each, and the bars represent the standard deviation. (C) Huh7.5 cells were infected with Jc1 for four hours and treated as in (A). The cell lysate was immunoblotted with the indicated antibody.
Figure 14 shows that HCV proliferation is impaired by gene silencing only CycA2 while leaving other cyclins or CDKs intact. Huh7.5 cells were transfected with 20 nM indicated siRNAs for 48 h and then infected with HCV Jc1 for four hours. Two days after infection, culture supernatant was collected and extracellular HCV RNA was quantified by qPCR.

Hereinafter, the configuration of the present invention will be described in more detail with reference to specific embodiments. However, it is apparent to those skilled in the art that the scope of the present invention is not limited to the scope of the embodiments.

Plasmid construction

Full-length CycA2 was amplified by PCR using cDNA prepared from HeLa cells and cloned into the Hin dIII / Eco RI site of p3XFLAG-CMV10 (Sigma Aldrich). GST-CycA2 was subcloned into pGEX-4T-1 vector (Amersham Biosciences AB, Uppsala). CycA2 and NS5B mutations were prepared by PCR and subcloned into p3XFLAG-CMV10 and pEF6B / His-Myc expression vectors, respectively. Point mutants of NS5B and Jc1 were constructed using the QuikChange II XL site directed mutagenesis kit (Stratagene). pEF-BOS-Flag-RIG- Takashi Fujita (Kyoto University, Japan).

Cell culture and DNA transfection

All cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal calf serum and 100 units / ml penicillin-streptomycin. HCV subgenomic replicon (genotype 1b) cells and IFN treated cells were cultured according to the methods described in the prior art (Park CY et al., J. Hepatol. 2009; 51: 853-864]. For DNA transfection ~5 x ¹⁰ cells were cultured in 60-mm dishes and transfected with expression plasmids using polyethyleneimine (Sigma-Aldrich) [Park CY et al., J. Hepatol. 2009; 51: 853-864].

GST pulldown analysis

The glutathione S-transferase (GST) -CycA2 fusion protein expressed in E. coli BL21 (DE) (Novagen) was purified with glutathione-sepharose 4B beads and incubated with Myc-NS5A or Myc-NS5B expressed in HEK293T cells And subjected to GST pulldown analysis [Park CY et al., J. Hepatol. 2009; 51: 853-864].

Lucifer Raise Replication Analysis

For dual Luciferase reporter assays, Huh7.5 cells were transfected with 20 nM indicated siRNAs for 24 h and then transfected together with pRL-HL plasmid and [beta] -galactosidase plasmid [Kang SM et al ., FEBS Lett. 2011; 585: 3236-3244]. For HCV reporter assays, Huh7.5 cells were electroporated with 10 [mu] g of in vitro transcribed JFH-Luc RNA and treated with the indicated timoletolol. Luciferase assay was performed as in the prior art [Lim YS et al., J. Virol. 2011; 85: 8777-8788]. Cell-based RNA-dependent RNA polymerase analysis of HCV NS5B using pEF-BOS-Flag-RIG-I was performed [Ranjith-Kumar CT et al., PloS One 2011; 6: e22575]. Briefly, 2.5x10 ⁵ HEK293T cells were seeded in 6-well plates and transfected with the indicated plasmids. Tylophorin was added 4 hours after plasmid transfection. Luciferase activity was measured 24 hours after plasmid transfection.

Viral infectivity assay

The infectious virus titer released from the cells was measured [Lim YS et al., J. Virol. 2011; 85: 8777-8788]. Huh7.5 cells were infected with HCV and then monitored for viral infection. Virus Stain Focus per ml And calculated as focus-forming units (FFU).

MTT analysis

Approximately 1.5 x 10 ⁴ cells were seeded into 24-well plates and transfected with the corresponding siRNAs. Host cell viability was measured using MTT {3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide} (Sigma).

RNA transcripts and infectious HCV production

HCV RNA transcripts were prepared according to the prior art [Lim YS et al., J. Virol. 2011; 85: 8777-8788]. Briefly, the HCV pFK-Jc1 and pFK-Jc1-GFP plasmids were digested with Mlu I followed by phenol-chloroform extraction. In vitro transcription was performed using the T7 RiboMAX ^TM expression system (Promega). RNA transcripts were purified with TRIzol ^® LS (Invitrogen). 10 μg of genomic Jc1 RNA transcribed in vitro was inoculated into 400 μl of a solution containing 120 mM KCl, 0.15 mM CaCl ₂ , 10 mM CaCl ₂ , 25 mM HEPES, 2 mM EDTA, 5 mM MgCl ₂ , 2 mM ATP and 4 mM GSH The cells were mixed with 7.5 x 10 ⁶ Huh7.5 cells resuspended in cytomix buffer (pH 7.6) and 0.4-cm cuvette. The mixture was electroporated at 270 V and 950 μF using a Bio-Rad GenePulser II electric perforator. Cells are carefully seeded in complete medium (low glucose DMEM containing 10% FBS, 100 units / ml penicillin, 100 ug / ml streptomycin, 2 mM L-glutamine, 1 mM nonessential amino acid [NEAA] and 10 mM HEPES) And then placed in a 150-mm dish. After 24 hours, the cells were changed to fresh medium and the apoptotic cells were removed. Four days after electroporation, the culture medium was collected, filtered through a 0.45-mm syringe-top filter unit (Milipore) and stored. A Jc1 / GNN mutant containing an NS5B active site mutation that disrupts RNA-dependent RNA polymerase activity was prepared in parallel. The culture supernatants collected from cells transfected with Jc1 / GNN mutant RNA were prepared as described and used for mock infection.

Immunoprecipitation

HEK293T cells were transfected with 2 [mu] g Flag-CycA2, 2 [mu] g Myc-NS5A and 8 [mu] g Myc-NS5B plasmid, respectively. The total DNA amount was adjusted to be constant by adding an empty vector. After 48 hours of transfection, the cells were collected and lysed in lysis buffer (50 mmol / l HEPES (pH 7.6), 250 mmol / l NaCl, 5 mmol / l EGTA, 0.2% NP-40, 1 mmol / l b- glycerophosphate, mmol / l NaF, 1 mmol / l Na ₃ VO ₄ and 1 mmol / l phenylmethyl-sulfonyl fluoride]. The cell lysate was pulverized by passing through a 25-gauge needle ten times on ice and centrifuged at 13,000 rpm for 10 minutes. The supernatants were over-night incubated at 4 ° C with anti-Flag antibody or anti-Myc antibody. The samples were further incubated for 1.5 hours with 30 μl of protein A beads (Zymed Laboratories Inc.). The beads were washed four times with cell lysate ELB buffer. The bound proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and detected by immunoblot analysis with secondary antibody.

Immunoblot analysis

Equal amounts of protein were electrophoresed on SDS-PAGE and nitrocellulose membranes. The membrane was blocked with TBST buffer containing 5% skim milk powder {Tris-buffered saline-Tween: 20 mmol / liter Tris-HCl [pH 7.6], 500 mmol / liter NaCl and 0.25% Tween 20) for 1 hour, Containing TBST buffer at 4 [deg.] C. Rabbit anti-rabbit antibody or goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) Combined with wasabi peroxidase in TBST containing 1% defatted milk powder after three washes with TBST buffer The membranes were incubated at room temperature for a period of time. Proteins were detected with ECL kit (Amersham Biosciences).

Chemicals and Antibodies

Antibodies to CycA2, GAPDH and Myc were purchased from Santa Cruz, GST antibodies were purchased from Cell signaling, and Flag antibodies were purchased from Sigma Aldrich.

Statistical analysis

Data were expressed as mean ± standard deviation of at least two independent experiments. Student's t test was used for statistical analysis using Microsoft Excel (Microsoft Corporation, USA). A P value of <0.05 is considered statistically significant.

<Result>

Result 1: CycA2 is essential for HCV replication.

First, 11 candidates were screened by screening an siRNA library targeting 131 genes that regulate cell cycle in HCVcc infected cells [Lim YS et al., J. Virol. 2011; 85: 8777-8788]. Among these genes, genes coding for CycA2 have been selected and identified because CycA2 modulated mitigation has been implicated in myeloma mediated carcinogenesis and malignant transformation [Yam CH et al., Cell Mol Life Sci 2002; 59 : 1317-1326, Wang HC Hepatology 2005; 41: 761-770, Wang j. et al. Nature 1990; 343: 555-557). Inhibition of CycA2 expression does not induce cytotoxicity (FIG. 1B), and HCV infectivity is markedly impaired (FIG. 1A). Figure 1C shows that CycA2 mRNA levels were strongly inhibited by siRNA treatment. As a result, intracellular HCV RNA levels were also significantly decreased in CycA2 knockdown cells. To further confirm the effect of CycA2 on HCV replication, we examined the level of viral protein expression in CycA2 knockdown cells. Similarly, silencing CycA2 expression impaired HCV expression (Fig. 1D, lane 2). To further test HCV production in CycA2 knockdown cells, extracellular HCV RNA was quantitated by qPCR. As shown in FIG. 1E, silencing CycA2 expression strongly inhibits HCV production. The role of CycA2 in HCV replication was tested in Huh7 cells containing the HCV subgenomic replicon. We have shown that CycA2 knockdown in HCV replicon cells significantly inhibited viral replication (Fig. 1F, lane 2). Collectively, these data suggest that CycA2 is essential for HCV replication.

Result 2: CycA2 interacts with HCV NS5B protein in Invitro and Invivo.

Because HCV requires CycA2 for proliferation, we hypothesized that CycA2 would upregulate HCV proliferation through interaction with viral proteins. Since NS5A and NS5B are essential components of the viral replication complex, we first investigated possible protein interactions by in vitro GST pulldown analysis. FIG. 2A shows that GST or NS5A does not interact with CycA2, but selectively binds NS5B. A common immunoprecipitation assay was performed to further confirm the results with Invit. Indeed, only NS5B, not NS5A, specifically interacted with CycA2 in the invivo (Fig. 2B, lane 5). In addition, it was confirmed that both NS5B derived from genotype 1b and 2a interacted with CycA2 (Fig. 2C, lanes 4 and 5). Next, we investigated whether NS5B could interact with endogenous CycA2 using common immunoprecipitation assays. As expected, the NS5B protein interacted with endogenous CycA2 (Fig. 2D, lane 4 on the top panel). We have shown that NS5B (FIG. 2E, lane 4 of the top panel), not NS5A (FIG. 2E, middle panel lane 4) interacts with endogenous CycA2 in HCV replicon cells. These results suggest that NS5B is located in the same location as CycA2. To confirm this possibility, immunofluorescence analysis was performed using Naive Huh7 and HCV replicon cells. Figure 2F shows that NS5B is predominantly in the cytoplasm, whereas CycA2 is present in both the cytoplasm and nucleus. Double staining appears as yellow fluorescence in the overlapping image, showing that CycA2 and NS5B are located at the same position in the cytoplasm. To confirm this result, the coexistence of CycA2 and NS5B was determined by the Manders overlap coefficient [Lim YS et al., J. Virol. 2011; 85: 8777-8788]. The overlap factor of NS5B labeled NS5B with TRITC and FITC was ~ 0.95 (Figure 2G, bar # 1). The overlap coefficient of CycA2 was also determined because CycA2 is mainly present in the nucleus and in some cytoplasm. The double staining data show that the overlap coefficient of CycA2 in the nucleus is 0.96 (Fig. 2G, bar 2), while the CycA2 overlap coefficient of cytoplasm is 0.91 (Fig. 2G, bar 3). The overlap coefficient of CycA2 / NS5B was 0.78, indicating that these proteins coexist in the cytoplasm (Fig. 2G, bar 4). In addition, we have shown that CycA2 and NS5B interact through the cyclin box of CycA2 and the palm domain of NS5B (FIG. 5). Taken together, these data show that CycA2 specifically interacts with NS5B in vitro and in vivo.

Result 3: Protein interaction between CycA2 and NS5B is essential for HCV replication.

Many cell and viral proteins interacting with CycA2 commonly have RxL motifs (where x is a variable) [Chen J. et al., Mol Cell Bio 1996; 16: 4673-4682, Adams P.D. et al., Mol Cell Biol 1999; 19: 1068-1080, Ma T. et al., Proc Natl Acad Sci USA 1999; 96: 382-387). To test the functional validity of the interaction between NS5B and CycA2 in HCV replication, we examined what amino acids are needed for the RxL motif in the palm domain for interaction with CycA2. Using the GST pull-down analysis and common immunoprecipitation analysis, we have shown that both the R / HxL motifs of the NS5B farm domain from genotype 1b (RxL) and genotype 2a (HxL) are responsible for binding to CycA2 (Figure 6).

The interaction between NS5B and CycA2 increases the likelihood that HCV will abduct CycA2 through NS5B for its replication. To test this hypothesis, alanine-scanning mutations were introduced into the CycA2 binding motif of NS5B in the background of the Jc1 genome. As a result, the H254R mutant produced similar levels of viral proteins and extracellular RNA as compared to the wild type, but the RSL / AAA mutation showed defects in proliferation and thus could not detect viral protein and RNA production (Figures 7A-D ). However, the RSL / AAA mutation had severe defects in RNA synthesis due to the loss of luciferase activity (Fig. 7E). A series of mutants were constructed in the RSL motif to exclude the negative effects of alanine mutations in RSL / AAA (Figure 3A). Immunoprecipitation data confirm that substitution of the RSL motif impairs the binding suitability of CycA2 (FIG. 3B). Interestingly, although the level of expression was significantly reduced compared to the wild type, the virus propagation integrity of the H254A mutant was still maintained (Fig. 3C-F). Because the H254A mutation induces structural changes, the folding structure of the NS5B protein may collapse and the polymerase activity may be destroyed. To exclude this possibility, the polymerase activity of the H254A mutation was analyzed using pEF-BOS-Flag-RIG-I [Ranjith-Kumar C. T. et al., PloS One 2011; 6: e22575]. As in Figure 3G, the polymerase activity of NS5B was not compromised by the H254A mutation. This suggests that protein interaction between NS5B and CycA2 creates a cellular environment to increase HCV replication. To further confirm the functional significance of the NS5B R / HxL motif, we constructed the R254A mutant in the genotype 1b replicon conformation and studied the effect of this mutation on CycA2 interaction and RNA replication. As shown in Fig. 8, this mutant lost the binding fitness but retained the polymerase activity. Nonetheless, the replication fitness of this mutant was dramatically reduced, suggesting that protein interactions between NS5B and CycA2 play a central role in HCV genotype 1b replication. It is noteworthy that the L256 mutation not only compromised the binding suitability with CycA2, but also impaired polymerase activity, so that this HCV mutant could no longer proliferate. Overall, these results suggest that protein interactions between CycA2 and NS5B play a crucial role in HCV proliferation. Next, we asked if HCV infection could rely on protein interactions between CycA2 and NS5B to alter cell cycle progression. Cell cycle distributions were analyzed for Huh7.5 cells infected with GFP-labeled wildtype or GFP-labeled H254A mutants. As shown in Figure 9, there was no difference in the cell cycle distribution determined by the number of G1, S and G2 / M stages of cells between the wild type and cells infected with the H245A mutant HCV (not interacting with CycA2). In addition, as already reported [Kannan R.P. et al., J. Birol. 2011; 85: 7989-8001], the cell accumulation of the G2 / M stage occurred in both wild-type and mutant virus-infected cells, suggesting that the change in cell cycle progression is a major effect of protein interactions between NS5B and CycA2 in HCV replication Tell me or not.

Result 4: Tilophorin inhibits HCV proliferation.

The fact that CycA2 is required for HCV proliferation has raised questions about how CycA2 inhibitors will affect HCV proliferation. Recently, Wu et al . Reported that a natural compound derived from a plant named Tylophora indica specifically lowers CycA2 expression but does not degrade other cyclins and CDKs (Wu CM et al., Biochem. Biophys. Res. Commun. 2009; 386: 140-145). In order to investigate the effect of thyllofurin on the proliferation of HCV, HCV replicon cells were treated with different doses for 48 hours and 72 hours, respectively. As shown in FIG. 10A, intracellular HCV protein and CycA2 levels decreased in a dose-dependent manner. In addition, thiamine did not induce cytotoxicity in Huh7 cells, as measured by MTT assay (Fig. 10b). Cells infected with Jc1 were treated with various concentrations of thiroline to examine the effect of thirolone on HCV proliferation. 0.06 μM of tilaphorin effectively inhibited HCV proliferation at the levels of intracellular HCV RNA (FIG. 11A), viral proteins (FIG. 11B), and extracellular HCV RNA (FIG. 11C), and this inhibition was dose dependent. The inhibitory effect of the thyrofoline on HCV proliferation was further confirmed by confocal microscopy (Fig. 11d). Previous studies have reported that overexpression of CycA2 can relieve inhibition of cell cycle progression induced by tilapolin in the G1 phase (Wu CM et al., Biochem. Biophys. Res. Commun. 2009; 386: 140-145). Thus, the present inventors have tested whether overexpression of CycA2 can restore inhibition of tyrolein-mediated HCV replication. Indeed, CycA2 overexpression markedly restored HCV replication (Fig. 11e, lanes 3 and 4), suggesting that the inhibitory effect of tilaphorin on HCV replication is mediated by CycA2. Showed about 85% inhibition compared to the control cell treated with HCV-replete gatifloiene using the JFH1-luc reporter system (Fig. 11 (f)). However, HCV replication was significantly restored (50% vs. 85%) in cells externally expressing CycA2. This data confirms that tytilin inhibits HCV replication by down-regulating CycA2, which may be a new mechanism of inhibition of HCV-mediated by tyrophagurin. Drug ablation experiments were performed to further investigate whether the inhibitory effect of thyrofoline on HCV replication is due to the inhibition of growth inhibitory mediator cells [Wu CM et al., Biochem. Biophys. Res. Commun. 2009; 386: 140-145). As shown in Fig. 12, the damage of HCV replication and CycA2 expression by tyrophagurin occurred before the cell growth inhibition. In addition, the inhibitory effect of thyrofoline on HCV replication was more pronounced than the inhibitory effect on cell growth (FIG. 13). These results suggest that the inhibitory effect of thyllofurin on HCV replication is not due to the mechanism of cell growth inhibition, and it also suggests that tiloporpholine is a possible therapeutic agent for HCV.

Result 5: CycA2 is involved in the genomic replication phase of the HCV life cycle.

Since CycA2 knockdown significantly inhibited HCV proliferation, the present inventors sought to determine at which stage of the HCV life cycle CycA2 was involved. We used two reporter constructs as in Figure 4a. Huh7.5 cells transfected with SiRNA were transfected with pRL-HL [Kang S.M. et al., FEBS Lett. 2011; 585: 3236-3244] and β-galactosidase plasmid, and then luciferase activity was measured. We have demonstrated that the knockdown of CycA2 has no effect on HCV IRES-dependent translation (Figure 4b). Likewise, it has been shown that treatment with tilolol does not affect HCV IRES-dependent translation (Fig. 4C). These results suggest that CycA2 may be involved in HCV genomic amplification. To demonstrate this, we used a JFH1-luc reporter plasmid in which gene expression was induced by HCV IRES-dependent translation. This structure allows genomic translation to be monitored prior to genomic RNA replication. As shown in FIG. 4d, the initial translation of the injected HCV genomic RNA reached a peak after five hours of electroporation and decreased when RNA depletion began (left panel). Luciferase activity was not different between the control (DMSO) and thiroline treated cells, suggesting that the CycA2 downregulator had no effect on the HCV IRES dependent translation. The longer treatment of DMSO or tilophorin demonstrated a significant increase in luciferase activity in DMSO treated cells (48 hours after electroporation). However, as in the cells transfected with the GNN mutant defective in replication, the luciferase activity was continuously lost in the cells treated with tilapolin (Fig. 4D, right panel). This result showed that HCV RNA replication was dramatically inhibited by tilapolin. Overall, these results indicate that CycA2 is involved in the genomic cloning stage, not the translation stage, in the HCV life cycle.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for prevention and treatment for          hepatitis c containing cyclin A2 expression or activity inhibitor <130> hallym-Ilsong-SBHwang-CYCA2 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA of cycA2 <400> 1 ggaaauggag guuaaaugu 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA of cycA2 <400> 2 uagcagaguu uguguacau 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA of cycA2 <400> 3 augaggauau ucacacaua 19 <210> 4 <211> 18 <212> RNA <213> Artificial Sequence <220> <223> siRNA of cycA2 <400> 4 ugauagaugc ugacccau 18

Claims (6)

A pharmaceutical composition for the prevention and treatment of hepatitis C comprising an antibody against tilapolin or cyclin A2 as a cyclin A2 protein activity inhibitor.
A pharmaceutical composition for the prevention and treatment of hepatitis C virus comprising siRNA (short interfering RNA) complementarily binding to the mRNA of the cyclin A2 gene selected from the group consisting of SEQ ID NOS: 1 to 4. delete delete delete delete

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