KR20140120446A - Pharmaceutical composition for prevention and treatment for Hepatitis C containing MAPKAPK3 expression or activity inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing and treating hepatitis C, and more specifically, to a composition containing a mitogenactivated protein kinase-activated protein kinase 3 (MAPKAPK3) expression or activity inhibitor. The MAPKAPK3 expression inhibitor is one selected from a group of antisense nucleotide, shRNA, and siRNA complementarily combined to mRNA.

Description

[0001] The present invention relates to a pharmaceutical composition for the prevention and treatment of hepatitis C comprising a MAPKAPK3 expression or an activity inhibitor,

The present invention relates to a pharmaceutical composition for the prevention and treatment of hepatitis C, and more particularly to a composition comprising a mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) expression or activity inhibitor.

Hepatitis C virus (HCV) is a transmembrane virus consisting of positive-sense, single-stranded RNA genomes. HCV causes an acute or chronic infection and often causes cirrhosis and hepatocellular carcinoma (HCC) (Saito I. et al., 1990. Proc. Natl. Acad Sci. US 405 A. 87: 6547-6549) . HCV belongs to the genus Flaviviridae and Hepacivirus (Giannini C, Brechot C. 2003. Hepatitis C virus biology. Cell Death Differ. Suppl 1: S27-38). The HCV genome consists of 9,600 bases and encodes 3010 amino acids from a single open reading frame. The polyprotein is translated and processed post-translationally by virus and host cell proteases into 10 functional proteins, including structural proteins (core, E1 and E2) and non-structural proteins (p7, NS2 to NS5B) (Lindenbach BD et al, 2005. Nature 436: 933-938). Structural proteins constitute the structure of viral particles, while nonstructural proteins are involved in the replication of viral genomes.

The HCV core protein is a nucleocapsid protein that envelops the viral RNA genome. The amino acid sequence of the core is highly conserved in the six HCV genotypes. HCV cores can form multimers and self-assemble to form nucleocapsid-like particles. The core interacts with many cellular proteins and regulates transcription, signal transduction, cell cycle, steatosis and tumorigenesis. Interaction between core and apolipoprotein AII induces binding of the core to lipid droplets and fat accumulation in hepatocytes (Barba G. et al., 1997. Proc. Natl. Acad. 412 Sci. USA 175: 740-744, Shi ST et al., 2002. Virology 292: 198-210). In addition, PPAR alpha activation is essential for HCV core protein-induced hepatitis and liver cancer in mice (Tanaka N. et al., 2008. J. Clin. Invest. 118: 683-694). Indeed, the HCV core is known to be a multifunctional protein that affects liver disease and liver cancer.

Map-kinase (MAPK) signaling pathways regulate a variety of cellular responses including gene expression, proliferation, differentiation and immune responses (Dong C, Davis RJ, Flavell RA 2002. Annu. Rev. 420 Immunol. -72). The MAPK-activated protein kinase (MAPKAPK) family, which is activated by MAPKs such as MAPKAPK2 / MK2 and MAPKAPK3 / MK3, is one of the downstream targets of the MAPKs cascade (Gaestel M. 2006. Nat. Mol., 422 Cell Biol., 7: 120-130). The MAPKAPK3 protein belongs to the serine / threonine MAPKAPK family. MAPKAPK3 is closely related to MAPKAPK2, which shares 72% base and 75% amino acid (Sithanandam G. et al., 1996. Mol. Cell. Biol. 16: 868-876). MAPKAPK3 has been defined as MAP kinase-activated protein kinase present in the small cell lung cancer tumor suppressor gene region (Sithanandam G. et al., 1996. Mol. Cell. Biol. 16: 868-876). Until now, the function of MAPKAPK3 has been largely unknown. MAPKAPK3 has been reported to be activated by influenza virus infection (Luig C. et al., 2010. FASEB J. 24: 4068-4077). In addition, Tsukada et al. Recently found that two single-nucleotide polymorphisms associated with interferon therapy were present in MAPKAPK3 in patients infected with HCV genotype 1b (Tsukada H. et al., 2009. Gastroenterology. : 1796-433 1805.e6). However, the function of MAPKAPK3 in HCV-infected cells is not yet clear.

Patent Publication No. 10-2011-52642 discloses a method for detecting cancer by detecting a MAPKAPK3 protein or a polynucleotide in a subject. However, this invention is not related to hepatitis C virus.

The present invention aims at solving the problem that there is no effective therapeutic agent for hepatitis C and providing an effective preventive and therapeutic agent for hepatitis C infection.

The inventors used functional proteomics to identify cellular proteins that interact with the HCV core protein. Protein microarray technology is a powerful tool for analyzing protein-protein, protein-phospholipid, protein-nucleic acid and protein-small molecule interactions (Satoh J. et al., 2006. J. Neurosci Methods. 152: 278-288, Tripathi LP et al., 2010. Mol. Biosyst. 6: 2539-2553). Protein microarray analysis is a fast, systematic, economical, and productive screening method that is important for biological function and drug discovery research (Fenner BJ et al., 2010. Expanding the substantial interactome of NEMO using protein microarrays. 5: e8799, Hallda et al., 2007. Mech., Aging Dev. 442 128: 161-167). We have identified about 100 HCV core proteins that interact with host cell proteins using protein microarray analysis. Of these core proteins, MAPKAPK3 was selected and characterized. Binding of the HCV core to MAPKAPK3 was confirmed using both in vitro pull down and common immunoprecipitation assays. MAPKAPK3 expression silencing resulted in lower levels of protein, extracellular HCV RNA and HCV infection. These data suggest that MAPKAPK3 is involved in HCV proliferation.

The present invention relates to a pharmaceutical composition for the prophylaxis and treatment of hepatitis C comprising a mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) expression or activity inhibitor.

The present invention also provides a method for producing a C-type MAPKAPK3 gene, wherein the MAPKAPK3 expression inhibitor is any one selected from the group consisting of an antisense nucleotide complementary to the mRNA of the MAPKAPK3 gene, short RNA (shRNA) and siRNA (short interfering RNA) To a pharmaceutical composition for preventing and treating hepatitis.

The present invention also relates to a pharmaceutical composition for the prevention and treatment of hepatitis C, wherein the siRNA is SEQ ID NO: 1 or 2. However, since it is easy for a person having ordinary skill in the art to produce siRNA targeting MAPKAPK3 in addition to SEQ ID NOS: 1 and 2, it is not limited to SEQ ID NOS: 1 and 2, Do.

In addition, the present invention relates to a pharmaceutical composition for the prevention and treatment of hepatitis C, wherein the MAPKAPK3 activity inhibitor is an anti-MAPKAPK3 antibody binding to MAPKAPK3 protein.

The composition for the prevention and treatment of hepatitis C infection comprising the inhibitor of the expression or activity of the Pin1 protein of the present invention as an active ingredient contains 0.0001 to 50% by weight of the active ingredient, based on the total weight of the composition.

The composition of the present invention may contain at least one active ingredient which exhibits the same or similar function to the MAPKAPK3 protein expression or activity inhibitor. 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.

In the present invention, the inhibitor for MAPKAPK3 may be an antisense oligonucleotide for the mRNA of the gene.

Antisense oligonucleotides have been used successfully to achieve gene-specific inhibition both in vivo as well as in vitro. Antisense nucleotides are short length DNA synthesis strands (or DNA analogs) that are antisense (or complementary) to a particular DNA or RNA target. Antisense oligonucleotides have been proposed to inhibit the expression of proteins encoded by DNA or RNA targets by binding to the target and stopping expression at the transcription, translation or splicing step. Antisense oligonucleotides have also been successfully used in animal models of cell culture and disease (Hogrefe, 1999). Other variations of antisense oligonucleotides to make the oligonucleotides more stable and resistant to degradation are known and understood by those skilled in the art. The antisense oligonucleotides used herein include double stranded or single stranded DNA, double stranded or single stranded RNA, DNA / RNA hybrids, DNA and RNA analogs and oligonucleotides with base, sugar or backbone modifications. Oligonucleotides are modified by methods known in the art to increase stability and increase resistance to nuclease degradation. These modifications include, but are not limited to, modifications of oligonucleotide backbones known in the art, modifications of sugar moieties, or modifications of bases.

In addition, the inhibitor for MAPKAPK3 may be siRNA (Small Interfering RNA) of the gene.

That is, the nucleotide sequence of the siRNA may be a sequence of 19 to 23 contiguous nucleotides of the nucleotide sequence of the MAPKAPK3 gene (mRNA).

The sequence of the siRNA represents a sense strand for convenience, and constitutes a double ribonucleic acid chain together with an antisense strand indicating an actual gene suppression effect.

siRNA has the potential to become a very potent drug against the inhibition of the expression of specific genes in vivo in terms of long-lasting effects in cell culture and in vivo, the ability to transfect cells in vivo, and resistance to serum degradation (Bertrand et al., 2002). Expression constructs / vectors including siRNA delivery and siRNA are known to those skilled in the art. For example, U.S. Patent Application Nos. 2004/106567 and 2004/0086884 disclose delivery mechanisms including viral vectors, non-viral vectors, liposome delivery vehicles, plasmid injection systems, artificial viral envelopes and polylysine conjugates, / Vector.

siRNAs have been proposed as an alternative to antisense oligonucleotides because they can achieve equivalent or even higher effects than those obtained by antisense oligonucleotides at relatively low concentrations (Thompson, 2002). The use of siRNAs has been shown to inhibit gene expression in animal models of disease. Those skilled in the art can synthesize and modify the antisense oligonucleotides and siRNAs as desired using methods known in the art (Andreas Henschel, Frank Buchholzl and Bianca Habermann (2004) DEQOR: a web-based tool for the design and quality control of siRNAs. Nucleic Acids Research 32 (Web Server Issue): W113-W120 Reference). Also, those skilled in the art are well aware of regulatory sequences (e.g., constitutive promoters, inducible promoters, tissue-specific promoters, or combinations thereof) useful in expression constructs / vectors with antisense oligonucleotides or siRNA.

The antisense oligonucleotides or siRNA of the present invention used for the treatment of hepatitis C can be administered in the form of a composition further comprising a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrin, glycerol, ethanol as well as combinations thereof. Such compositions may be formulated to provide a rapid release, sustained or delayed release of the active ingredient after administration.

The inhibitor of the MAPKAPK3 gene may be any substance that inhibits the expression of the gene in addition to the antisense oligonucleotide or siRNA. Therefore, compounds known as inhibitors of the genes in the art are also available.

The present invention also provides a composition for treating hepatitis C comprising an inhibitor against a protein expressed from MAPKAPK3 gene. When the gene of the present invention is inhibited, expression of the protein is inhibited and HCV proliferation is inhibited. Therefore, inhibition of the protein of the gene can inhibit HCV proliferation.

Accordingly, the present invention provides a therapeutic use of an inhibitor of the above-mentioned protein for hepatitis C virus. In the present invention, treatment of hepatitis C includes prevention and inhibition of hepatitis C virus.

The inhibitor for the protein may be an antibody to a protein expressed from the gene of the present invention. The monoclonal antibody against the protein may be produced by using a monoclonal antibody production method customary in the art, or may be commercially available. In addition, a polyclonal antibody recognizing the protein may be used in place of the monoclonal antibody, or it may be produced and used through an antiserum preparation method customary in the art.

The composition for treating hepatitis C of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. When the inhibitor for the protein of the present invention is an antibody, the pharmaceutically acceptable carrier may be composed of a minimum amount of auxiliary substance such as a wetting agent or an emulsifying agent, an antiseptic or a buffer to increase the shelf life or effectiveness of the binding protein.

In addition, the composition for treating hepatitis C of the present invention can be used together with one or more therapeutic agents for hepatitis C virus. The composition for treating hepatitis C of the present invention may further comprise a chemotherapeutic agent well known to those skilled in the art.

The composition for treating hepatitis C of the present invention may include pharmaceutically acceptable and physiologically acceptable adjuvants in addition to the above-mentioned effective components. Examples of such adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, swelling agents, lubricants , A lubricant or a solubilizing agent.

In addition, the composition of the present invention may be formulated into a pharmaceutical composition containing at least one pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration.

Examples of the pharmaceutical carrier which is acceptable for the composition to be formulated into a liquid solution include sterilized and sterile water, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, And other conventional additives such as an antioxidant, a buffer, and a bacteriostatic agent may be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

The pharmaceutical preparation form of the hepatitis C therapeutic composition of the present invention may be in the form of granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, And the like.

The composition for treating hepatitis C of the present invention may be administered orally or parenterally in the form of intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, ≪ / RTI > can be administered in a conventional manner.

In the treatment method of the present invention, "effective amount" means an amount required for achieving the effect of inhibiting hepatitis C infection. Thus, the "effective amount" of the active ingredient of the present invention will vary depending upon factors such as the type of disease, the severity of the disease, the type and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the age, weight, The dosage, the time of administration, the route of administration and the rate of administration of the composition, the duration of the treatment, the drugs used concurrently, and the like. In the case of an adult, 0.01 ng / kg to 10 mg / kg in the case of siRNA and 0.01 ng / kg to 10 mg / kg in the case of antisense oligonucleotides to mRNA of the gene when the gene or protein inhibitor is administered once to several times a day, Kg to 10 mg / kg in the case of the compound, and 0.1 ng / kg to 10 mg / kg in the case of the monoclonal antibody against the protein.

In the present invention, the MAPKAPK3 gene may be a nucleotide sequence in which at least one base of a known nucleotide sequence is deleted, substituted or inserted.

It is apparent to those skilled in the art that the siRNA sequence of the MAPKAPK3 gene is not limited thereto. Sequences having substantial sequence identity or substantial sequence homology to the sequences are also within the scope of the present invention. As used herein, the term "substantial sequence identity" or "substantial sequence identity" is used to denote that the sequence represents substantial structural or functional identity with another sequence. These differences are due, for example, to inherent variations in the codon usage between different species. If there is a significant amount of sequence redundancy or similarity between two or more different sequences, the structural differences will be negligible if they have similar physical properties even if the length or structure of these sequences is different.

(2001)) and Frederick et al. (Frederick et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (Ausubel et al., Current Protocols in Molecular Biology Volume 1, 2, 3, John Wiley & Sons, Inc. (1994)).

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.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. The present invention may, however, be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Is provided to fully convey the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

According to the present invention, MAPKAPK3 interacts with the HCV core from both genotypes 1b and 2a. In addition, the HCV core interacts with MAPKAPK3 through the N-terminal half of the kinase domain of MAPKAPK3 and the 41-75 amino acid residues of the core.

Also, according to the present invention, HCV increases the level of MAPKAPK3 expression, and when the MAPKAPK3 is knocked down in HCV subgenomic replicon cells, the level of HCV protein expression is suppressed.

Further, according to the present invention, MAPKAPK3 knockdown inhibits HCV proliferation.

In addition, protein interactions between the HCV core protein and MAPKAPK3 are essential for HCV IRES-mediated translation.

Therefore, a substance that inhibits the activity or expression of mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) of the present invention is useful as a prophylactic and therapeutic agent for hepatitis C by inhibiting replication of HCV.

Figure 1 is a probe of a human protein microarray with HCV core protein. (A, B) HCV core protein was expressed in E. coli and purified by single step Ni-NTA agarose affinity chromatography. Purified proteins at various concentrations were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (A) or immunoblotting (B) with the indicated antibody. Blood serum albumin was run as a reference. (C) The frequency histogram of the core-probe protein array shows the obtained Z-score range. Protein interactors with a Z-score of 3 or more (Z? 3) were considered important. Scores were calculated using Invitrogen Protoarray version 5.2 software. (D) MAPKAPK3 was identified in protein microarrays (left panel). Positive and negative controls were also shown (right panel).
Figure 2 shows the ontology analysis results of the HCV core interactors. The gene ontology was determined for each core interactor, and the results for each of the three standard ontology categories were expressed as a percentage.
Figure 3 shows the interaction of the HCV core protein with MAPKAPK3. (A) HEK293T cells were transiently transfected with vector or Flag-labeled MAPKAPK3. Total cell lysate was collected and incubated with His-labeled core protein purified in E. coli 36 hours after transfection. Bound proteins were precipitated with Ni-NTA beads and detected by anti-Flag monoclonal antibody immunoblotting (IB) (top). The core protein was identified as an anti-core antibody (bottom). (B) HEK293T cells were cotransfected with Flag-tagged MAPKAPK3 and myc-tagged core expression plasmids from genotype 1b or 2a. After 48 hours, the cell lysate was immunoprecipitated (IP) with anti-Myc monoclonal antibody and the bound protein was detected by immunoblotting (IB) analysis with anti-Flag monoclonal antibody (top). Protein expression of Flag-labeled AMPKAPK3 and Myc-labeled core was confirmed by immunoblotting with anti-Flag monoclonal antibody and anti-core polyclonal antibody, respectively (bottom). The asterisk denotes the IgG heavy chain. (C) The HCV core protein interacts with endogenous MAPKAPK3 in HCV infected cells. Huh7.5 cells were electroporated with 10 [mu] g of Jc1 RNA. Four days after electroporation, the collected cells were immunoprecipitated with anti-MAPKAPK3 antibody or control IgG. Bound proteins were immunoblotted with MAPKAPK3 antibody (top, lanes 3 and 4) or anti-core antibody (bottom, lanes 3 and 4). The asterisk denotes the heavy chain. MK3 represents MAPKAPK3. (D) Huh7.5 cells were infected for four hours with an additive salt or Jc1. After 48 hours, cells were fixed with 4% paraformaldehyde and immunofluorescent staining was performed using anti-MAPKAPK3 monoclonal antibody and FITC (fluorescein isothiocyanate) -based goat anti-mouse IgG to detect MAPKAPK3 (green) , And immunofluorescent staining was performed with rabbit anti-core antibody and TRITC-conjugated donkey anti-rabbit IgG to detect core (red). Double staining showed the common location of MAPKAPK3 and core in yellow fluorescence in the combined images. Cells were stained with DAPI (4 ', 6-diamidino-2-phenylindole) to label the nucleus (blue). (E) HEK293T cells were cotransfected with Myc-labeled core and Flag-labeled MAPKAPK3 or Flag-labeled MAPKAPK2. 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 (top). Protein expression of Myc-labeled core was confirmed by immunoblotting with anti-core polyclonal antibody (bottom). The asterisk denotes the IgG heavy chain.
Figure 4 shows that the HCV core protein interacts with MAPKAPK3 through the 41-75 amino acid residues of the core and the N-terminal half of the MAPKAPK3 kinase domain. (A) HCV core expression plasmid wild type and variants thereof. aa is an amino acid. (B) MAPKAPK3 interacts with 41-75 amino acid residues of the core. HEK293T cells were cotransfected with Flag-labeled MAPKAPK3 and Myc-labeled core expression plasmids. After 48 hours, the cell lysate was immunoprecipitated with anti-Myc monoclonal antibody, and the bound proteins were immunoblotted with anti-Flag monoclonal antibody. Protein expression of Myc-labeled core and Flag-labeled MAPKAPK3 was confirmed by immunoblotting with anti-Myc or anti-Flag monoclonal antibodies using the same cell lysate. (C) MAPKAPK3 expression plasmid wild type and variant. (D) The HCV core interacts with the N-terminal part of the kinase domain of MAPKAPK3. HEK293T cells were cotransfected with the indicated combination of expression plasmids. After 48 hours, the cell lysate was immunoprecipitated with anti-Myc monoclonal antibody, and the bound protein was immunoblotted with anti-Flag monoclonal antibody (right panel). Arrows point to joined proteins. The asterisk indicates the IgG heavy chain and light chain. Expression of Myc-tagged core and Flag-tagged MAPKAPK3 protein was confirmed by immunoblotting with rabbit anti-core antibody or anti-Flag monoclonal antibody using the same cell culture medium
(Left panel).
Figure 5 shows that HCV increases MAPKAPK3 expression levels. (A) MAPKAPK3 mRNA isolated from interferon-treated cells or subgenomic replicon cells was quantitated by qPCR. The asterisk indicates a significant difference (*, p <0.05) from the control value. The error bars represent the standard deviation. (B) Total cell lysates collected from interferon-treated cells or subgenomic replicon cells were immunoblotted with the indicated antibodies. (C) Huh7.5 cells were infected with either additive or HCV Jcl for four hours. After 96 hours, MAPKAPK3 mRNA was analyzed by qPCR. Data from two independent experiments were quantified. (D) Total cell lysate collected 96 hours after infection was immunoblotted with the indicated antibody. (E) Huh7.5 cells were infected for four hours with either additive or HCV JFH1. After 96 hours, MAPKAPK3 mRNA was analyzed by qPCR. Data from two independent experiments were quantified. Asterisks indicate significant differences (**, p <.0.01). The error bars represent the standard deviation. (F) After 96 hours of infection, total cell lysate was collected and immunoblotted with the indicated antibody. (G) Huh7.5 cells were transiently infected with vector or Myc-labeled core, NS3, NS4B, NS5A and NS5B expression plasmids, respectively. After 72 hours total cells
The bacterial suspension was immunoblotted with the indicated antibody.
Figure 6 shows that MAPKAPK3 knockdown decreases HCV protein expression levels in HCV subgenomic replicon cells. (A) Huh7.5 cells containing the HCV subgenomic replicon were transfected with the indicated siRNA constructs. After 72 hours, the total cell lysate was immunoblotted with the indicated antibody. Strip intensity was quantified using ImageJ software. (B, C) Total RNA was extracted from cells 72 hours after siRNA transfection, and MAPKAP2 / 3 mRNA (B) or intracellular HCV RNA (C) was quantified by qPCR. "Negative": universal negative control siRNA; "positive": HCV-specific siRNA; MK3, MAPKAPK3-specific siRNA; MK2, MAPKAPK2-specific siRNA. The asterisk indicates a significant difference (**, p <0.01) from the negative control value. The experiment was performed twice. The error bars represent the standard deviation.
Figure 7 shows that MAPKAPK3 knockdown inhibits HCV proliferation. (A) Huh7.5 cells were transfected with the indicated siRNA. After 2 days, the cells were infected with Jc1 for 4 hours, and after 48 hours, the cells were collected and immunoblotted with the indicated antibody. The band intensity was quantified with ImageJ software. (B) Intracellular HCV RNA was isolated 48 hours after infection and analyzed by qPCR. (C) Extracellular RNA was isolated from these cells and quantified by qPCR. (D) Huh7.5 cells were transfected with the indicated siRNA. After 2 days, the cells were infected with JFH1 for 4 hours. After 48 hours, the cell lysate was collected and immunoblotted with the indicated antibody. (E) Total RNA was extracted from Huh7.5 cells after 48 hours. Intracellular and extracellular HCV RNA was quantified by qPCR. The asterisk indicates a significant difference (*, p <0.05; **, p <.0.01). The experiment was carried out three times. The error bars represent the standard deviation. MK3, MAPKAPK3; MK2, MAPKAPK2. (F) The culture supernatants collected in (C) were used for Huh7.5 cell infections that had never been exposed to the antigen. The virus reversal was determined by a focus-forming assay. The asterisk indicates a significant difference (*, p <0.05; **, p <0.01) from the negative control value. (G) Huh7.5 cells were transfected with 50 nM of indicated siRNAs. After 4 days, cell viability was determined by MTT assay. "Negative": universal negative control siRNA; "positive": HCV-specific siRNA; MK3, MAPKAPK3; MK2, MAPKAPK2. (H) Huh7.5 cells were transfected with the indicated siRNA. After 48 hours, the cells were infected with Jc1 for four hours and transfected with a combination of indicated expression plasmids. After 48 hours, the cell lysate was immunoblotted using the indicated antibody. MAPKAPK3-WT, Flag-labeled wild-type MAPKAPK3; MAPKAPK3-SR, Flag-tagged siRNA-resistant variant MAPKAPK3; MAPKAPK3-KA, Flag-tagged siRNA-resistant ATP binding defect variant MAPKAPK3. The experiment was performed twice.
Figure 8 shows that MAPKAPK3 increases HCV IRES translation. (A) pRL-HL plasmid. (B) (left panel) Huh7.5 cells were transfected with vectors, Flag-tagged wild-type MAPKAPK3, Flag-tagged mutant MAPKAPK3-KA, respectively, with MyL-tagged plasmids pRL-HL and? -Galactosidase expression plasmids The amount of core was increasingly increased, or co-transfected without a Myc-labeled core. Luciferase activity was determined after 48 hours of transfection and normalized to? -Galactosidase. HCV IRES activity was expressed as the ratio of FLuc / RLuc (left, top panel). MAPKAPK3, MAPKAPK3-KA, core and actin protein expression were identified using the same cell lysate as directed antibody (left, bottom panel). The asterisk indicates a significant difference (*, p <0.05; **, p <0.01). The experiment was performed twice. The error bars represent the standard deviation. (Right panel) Huh7.5 cells were cotransfected with vector or Myc-labeled core with or without Flag-labeled wild-type MAPKAPK3 with pRL-HL and beta-galactosidase expression plasmids. Luciferase activity was determined after 48 hours of transfection and normalized to? -Galactosidase. HCV IRES activity was expressed as the ratio of FLuc / RLuc (upper right panel). The asterisk indicates a significant difference (*, p <0.05; **, p <0.01) from the vector control group. The experiment was carried out twice, and the error bars indicate the standard deviation. The expression of core, MAPKAPK3-WT, MAPKAPK3-KA and actin protein was confirmed using the same cell lysate as the indicated antibody (right panel, bottom panel). (C) HEK293T cells were cotransfected with the indicated expression plasmid combination. 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. Protein expression of Flag-MAPKAPK3, Flag-MAPKAPK3-SR, Flag-MAPKAPK3-KA, Myc-core and actin in the same cell lysate was measured by using anti-Flag monoclonal antibody, anti-core polyclonal antibody and anti- And confirmed by immunoblot analysis. Asterisks indicate IgG heavy and light chains. (D) (left panel) Huh7.5 cells were transfected with 50 nM of indicated siRNA. After 24 hours, the cells were transfected with vectors (white bars) or different amounts of Myc-Glu, with or without the siRNA-resistant variant MAPKAPK3 (Flag-MAPKAPK3-SR) And transfected with labeled core (black bars). Luciferase activity was determined after 48 hours of transfection and normalized to? -Galactosidase. HCV IRES activity was expressed as the FLuc / RLuc ratio (top panel). The experiment was performed twice. The asterisk indicates a significant difference (*, p <0.05; **, p <0.01). The error bars represent the standard deviation. MAPKAPK3, MAPKAPK3-SR, core and actin protein expression were identified using the same cell lysate as the indicated antibody (panel below). (Right panel) Huh7.5 cells were cotransfected with Flag-labeled MAPKAPK3-WT and pcDNA3 cores with pRL-HL and beta-galactosidase expression plasmids. After 24 hours, the cells were transfected with vector or Myc-labeled core variants as directed. Luciferase activity was determined after 36 hours of transfection and normalized to? -Galactosidase. HCV IRES activity was expressed as FLuc / RLuc ratio (right, top panel). The experiment was performed twice. The asterisk indicates a significant difference (*, p <0.05; **, p <0.01). The error bars represent the standard deviation. ns, and p values were not significant. MAPKAPK3-WT, pCDNA3-core, Myc-tagged core variants and actin protein expression were identified using the same cell lysate as directed antibody (right and bottom panels).

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

Plasmid construction

The cDNA of HCV core protein was amplified by PCR using genotype 1b derived cDNA. PCR products were obtained between the Bam HI site and the Hin dIII site of the pET21b vector (Invitrogen), between the Hin dIII site and the Bam HI site of the pCDNA3 (Invitrogen) vector, and between the Bam HI site and the Xba I site of the pcDNA4 vector (Invitrogen) Respectively. HCV core mutants were constructed using full length cores as templates. Total RNA extracted from Huh7.5 cells for MAPKAPK3 cloning was used for RT-PCR with the primers of Table 1. The PCR product was sandwiched between the Xba I site and the Bam HI site of the plasmid p3XFlag-CMV10 (Sigma Aldrich). MAPKAPK3 deletion mutants were generated by site-specific mutagenesis (Enzynomics) using the primers in Table 1. The siRNA-resistant mutant MAPKAPK3 contains four silent mutations at the siRNA binding site. siRNA-resistant inactive mutants (ATP-binding defect mutations) MAPKAPK3 (Ludwig S. et al. 1996, Mol. Cell. Biol. 16: 6687-6697), a K73A substitution mutation was introduced into the siRNA-resistant mutant MAPKAPK3. The Myc-labeled NS3, NS4B, NS5A and NS5B plasmids were prepared according to the prior art (Lim YS et al., 2011. Peptidyl-prolyl isomerase Pin1 is a cellular 447 factor required for hepatitis C virus propagation. : 8777-8788). Flag-labeled MAPKAPK2 was provided by Jun-ichi Abe (University of Rochester).

Antibody

Antibodies were purchased from each of the following sites: MAPKAPK3 antibody, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) antibody and Myc antibody in Santa Cruz; Flag antibody and actin antibody were purchased from Sigma-Aldrich; The HCV core antibody, the NS3 antibody and the NS5A antibody are the same as described in the paper (Lim YS et al., 2011. Peptidyl-prolyl isomerase Pin1 is a cellular 447 factor required for hepatitis C virus propagation J. Virol. 85: 8777-8788 ).

Microarray Probe Manufacturing

The HCV core protein expressed in E. coli was purified using Invitrogen Ni-NTA (nitrilotriacetic acid) -agarose beads. The protein was restored to its folded state and stored at -70 ° C.

Protein Array Screening

Protein microarray screening was performed using version 5.0 of Invitrogen Protoarray. The arrays were incubated with blocking buffer (50 mM HEPES pH 7.5, 25% glycerol, 0.08% triton X-100, 200 mM NaCl, 20 mM reduced glutathione and 0.1 mM DTT) at 4 ° C for one hour with gentle shaking, After the addition of 120 μl of the probe buffer (PBS containing 0.1% Tween 20), 6 μg of the purified HCV core protein was diluted to the array. After incubation at 4 ° C for 1.5 hours, the array was washed five times with cold buffer and then treated with anti-V5-AlexaFluor 647 antibody (Invitrogen) for 1.5 hours at 4 ° C. After drying, the array was analyzed using a Perkin Elmer Scanarray Ex-pressHT system and Invitrogen Prospector version 5.2 software. Significant interactions were identified based on a Z-score cutoff value of 3.0.

Cell culture

All cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal calf serum and 100 units / ml penicillin / streptomycin at 37 ° C under 5% CO ₂ . Interferon-treated and HCV subgenomic replicon cells were cultured (Lim YS et al., 2011. Peptidyl-prolyl isomerase Pin1 is a cellular factor required for hepatitis C virus propagation J. Virol. 85: 8777-8788).

Immune precipitation

HEK293T cells were cotransfected with 4 [mu] g of Myc-labeled core and 2 [mu] g of Flag-labeled MAPKAPK3 or MAPKAPK2. The total amount of DNA was adjusted by adding an empty vector. After 48 hours of transfection, cells were collected and analyzed for immunoprecipitation (Lim YS et al., 2011. Peptidyl-prolyl isomerase Pin1 is a cellular factor required for hepatitis C virus propagation J. Virol. 85: 8777-8788) .

Invitro pull-down analysis

About 1 μg of the purified core protein was incubated with 30 μl of Ni-NTA agarose beads at 4 ° C for 1 hour with gentle agitation. The beads were washed three times with buffer (50 mM Na ₂ HPO ₄ (pH 8.0), 100 mM NaCl, 1 mM PMSF, 1% protein inhibitor cocktail) and incubated for 2 hours at 4 ° C with cells expressing Flag-labeled MAPKAPK3 And cultured with the bacterial solution. The cells were washed five times with lysis buffer and the bound proteins were then immunoblot analyzed using anti-Flag monoclonal antibody.

Immunoblot analysis

The same amount of protein was loaded on SDS-PAGE and electrophoresed on a nitrocellulose membrane. The membrane was blocked with TBS / Tween {20 mM Tris-HCl (pH 7.6), 500 mM NaCl and 0.25% Tween 20) buffer containing 5% skimmed milk for one hour and then blocked with TBS / Tween overnight at &lt; RTI ID = 0.0 &gt; 4 C. &lt; / RTI &gt; After three washes with TBS / Tween, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody or goat anti-mouse antibody (Jackson ImmunoResearch Laboratories, West Grove, Pa.) In TBS / Lt; / RTI &gt; Proteins were detected using ECL kit (Amersham Biosciences).

Luciferase reporter gene analysis

Huh7.5 cells were cotransfected with pRL-HL plasmid with? -Galactosidase and the indicated plasmid. After 48 hours, the cells were collected and analyzed for luciferase (Lim YS, Tran HT, Yim SA, Hwang SB, 2011. Peptidyl-prolyl isomerase Pin1 is a cellular factor required for hepatitis C virus propagation J. Virol. 85: 8777-8788 ).

RNA interference

(Sense 5'-GGAUCAACCAAUCGAUGGU-3 '; antisense 5'-ACCAUCGAUUGGUUGAUCC-3') targeting MAPKAPK3, siRNA (sense 5'-ACGAGCAGAUCAAGAUAAA-3 '; antisense 5'-UUUAUCUUGAUCUGCUCCU- ) And a universal negative control were purchased from Bioneer (Korea). SiRNA (5'-CCUCAAAGAAAAACCAAACUU-3 ') targeting 5'NTR of Jc1 was used as a positive control (Lim YS, Tran HT, Yim SA, Hwang SB 2011. Peptidyl-prolyl isomerase Pin1 is a cellular factor required for hepatitis C virus propagation. J. Virol. 85: 8777-8788). SiRNA transfection was performed using Lipofectamine RNAiMax reagent (Invitrogen, Carlsbad, Calif.).

Focus-forming assay

A Huh7.5 cells were seeded to 2 x 10 ⁴ cells in a 4-well culture chamber slide (Millipore). After incubation for 24 hours, cells were inoculated with serial dilutions of cell culture medium collected from HCVcc-infected cells. Two days after inoculation, indirect immunofluorescence was performed to confirm the presence of intracellular core antibodies to determine the number of focus-forming units (FFU) per ml (Lim YS et al., 2011. Peptidyl- prolyl isomerase Pin1 is a cellular factor required for hepatitis C virus propagation. J. Virol. 85: 8777-8788).

Quantification of MAPKAPK and HCV RNA

Total RNA was isolated from HCVcc-infected cells, cell culture medium or replicon cells using RiboEx LS reagent (Geneall Biotechnology) and reverse transcribed using ReverTra Ace kit (Toyobo). Quantitative real-time PCR (qRT-PCR) experiments were performed using the iQ5 multicolor Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) (Lim YS et al., 2011. Peptidyl-prolyl isomerase Pin1 is a cellular factor required for hepatitis C virus propagation. J. Virol. 85: 8777-8788).

MTT analysis

Huh7.5 cells were transfected with the indicated siRNA and seeded in ⁴ x 10 4 cells in a 24-well plate. After 4 days, MTT {3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide} reagent (Sigma) was added to the cells and incubated at 37 ° C for 2 hours. Cell viability was assessed according to conventional methods (Lim YS, Tran HT, Yim SA, Hwang SB, 2011. Peptidyl-prolyl isomerase Pin1 is a cellular 447 factor required for hepatitis C virus propagation J. Virol. 85: 8777-8788 )

Immunofluorescence analysis

Huh7.5 cells cultured on glass slides were fixed with PBS containing 4% paraformaldehyde for 12 minutes at room temperature. After washing twice with PBS, the immobilized cells were treated with 0.1% Triton X-100 for 15 minutes to increase the permeability, followed by incubation with 0.5% bovine serum albumin for 2 hours. Cells were over-incubated with mouse anti-MAPKAPK3 antibody and rabbit anti-core antibody, respectively. After washing three times with PBS, cells were incubated with FITC-conjugated goat anti-mouse IgG or TRITC-conjugated donkey anti-rabbit IgG for two hours at room temperature. After washing three times with PBS, the cells were analyzed with a Zeiss LSM 700 laser confocal microscope system (Carl Zeiss, Inc., Thornwood, NY).

Result 1: Identification of cellular proteins interacting with HCV core using protein arrays

Core proteins were purified to identify cellular proteins that interact with the HCV core protein (Figure 1A). The purified core protein was verified using anti-core, anti-V5 and anti-His antibodies (FIG. 1B) and applied to a spot on a human protein microarray kit (Invitrogen) containing about 9,000 human proteins. Statistically significant interactor candidates were determined by measuring the average spot intensity at a Z-score of at least 3 as a boundary value (Figure 1C). Scanned array data analysis shows that approximately 100 cellular proteins have been identified as HCV core interactors (Tables 2 through 5). MAPKAPK3 was identified as one of the candidate hits, and positive control hits and negative control hits are shown in Figure 1D. The ontology data revealed that half of the estimated HCV core interactors were proteins and nucleic acid binding proteins involved in gene regulation, cell cycle, proliferation and development (FIG. 2).

Result 2: MAPKAPK3 interacts with HCV cores derived from both genotypes 1b and 2a.

Because MAPK is involved in the regulation of proinflammatory cytokines in influenza virus infected cells (Luig C, Kother K. et al., 2010. FASEB J. 24: 4068-4077), MAPKAPK3 Respectively. To demonstrate protein array data, we performed in vitro binding assays using His-tagged core proteins purified in E. coli and cell lysates expressing Flag-labeled MAPKAPK3. Figure 3 shows that MAPKAPK3 selectively interacts with the HCV core protein. Common immunoprecipitation data confirmed that MAPKAPK3 specifically interacted with cores derived from genotypes 1b and 2a (FIG. 3B). Next, the present inventors investigated whether the HCV core protein interacts with endogenous MAPKAPK3 in Huh7.5 cells electroporated with Jc1 RNA. The cell lysate obtained 4 days after electroporation was immunoprecipitated with anti-MAPKAPK3 antibody or control IgG, and the bound proteins were analyzed by immunoblotting with anti-core antibody. Indeed, the HCV core interacted with the endogenous MAPKAPK3 protein (Figure 3C). These data suggest that MAPKAPK3 may be in the same position as the core in HCV-infected cells. To investigate this possibility, Huh7.5 cells infected with mock-infected or Jc1 were analyzed by immunofluorescence analysis. As expected, MAPKAPK3 was widely expressed in both nucleus and cytoplasm of infected cells (Figure 3D). It is noteworthy that MAPKAPK3 accumulates mainly in the cytoplasm in HCV infected cells. Figure 3C shows that both MAPKAPK3 and HCV core are common to cytoplasm that appears to be yellow fluorescence. Since MAPKAPK3 shares 72% nucleic acid identity and 75% amino acid identity with MAPKAPK2 and is closely related to MAPKAPK2 (Sithanandam G. et al., 1996. 3 pK, a new mitogen-activated protein kinase-activated protein kinase 16: 868-876), the present inventors have questioned whether the core can also interact with MAPKAPK2. For this purpose, HEK293T cells co-transfected with Myc-labeled core and Flag-labeled MAPKAPK3 / MAPKAPK2 were immunoprecipitated with anti-Myc antibodies and the bound proteins were immunoblotted with anti-Flag antibody. Figure 3E shows that only MAPKAPK3, not MAPKAPK2, interacts with the HCV core. In fact, MAPKAPK2 has not been identified as a candidate for core interactors in protein microarrays (Fig. 1D). When these data are combined, it can be seen that MAPKAPK3 specifically interacts with the HCV core in both Invitro and Invivo.

Result 3: The HCV core interacts with MAPKAPK3 through the N-terminal half of the kinase domain of MAPKAPK3 and the 41-75 amino acid residues of the core.

The interaction between the various deletion mutants of MAPKAPK3 and the core was determined by a transfection-based common immunoprecipitation assay (Fig. 4A) to identify the core regions involved in MAPKAPK3 binding. As shown in Figure 4B, MAPKAPK3 interacted with aa mutation containing aa M41-173 but did not interact with aa M76-173 core mutation. This result suggests that aa 41-75 of the core plays a role in binding to MAPKAPK3. Next, the present inventors constructed various domain variants of MAPKAPK3 to determine the site of MAPKAPK3 involved in core binding (Fig. 4C) and determined the binding domain in the same manner as described above. Figure 4D shows that the core interacts with a mutation in which the kinase domain is intact but does not interact with the C-terminal half-arm mutation of the kinase domain of MAPKAPK3, and the core is a portion containing the N- terminal half of the kinase domain To interact with MAPKAPK3.

Result 4: HCV increases MAPKAPK3 expression level.

MAPKAPK3 mRNA and protein levels in replicon cells were examined to determine whether MAPKAPK3 expression levels were regulated by HCV. As shown in FIG. 5A, MAPKAPK3 mRNA levels in replicon cells were significantly increased compared with cells treated with interferon. Figure 5B shows that MAPKAPK3 protein levels were significantly increased in HCV replicon cells. We found that MAPKAPK3 mRNA and protein levels were significantly increased in both Jc1-infected cells (FIG. 5C, 5D) and JFH1-infected cells (FIG. 5E, 5F) as compared to virally infected cells. These data suggest that HCV nonstructural proteins play a role in upregulating MAPKAPK3 expression. For this purpose, Huh7.5 cells were transiently transfected with vector or Myc-labeled core, NS3, NS4B, NS5A and NS5B, respectively. After 72 hours of transfection, total cell lysate was immunoblotted with the indicated antibody. As shown in FIG. 5G, except for the other HCV proteins, only NS5A increased the level of MAPKAPK3 protein at a dose-dependent concentration. Collectively, these data clearly indicate that HCV increases MAPKAPK3 expression levels.

Result 5: MAPKAPK3 knockdown suppresses HCV protein expression level in HCV subgenomic replicon cells.

To investigate the effect of MAPKAPK3 on HCV replication, HCV subgenomic replicon cells were transfected with siRNA targeting MAPKAPK3 or indicated control siRNA. Figure 6A shows that silencing MAPKAPK3 in replicon cells dramatically reduces NS3 and NS5A protein levels. Notably, the inhibitory activity of MAPKAPK3 siRNA was as effective as positive control siRNA (Fig. 6A, lane 2 vs. lane 3). MAPKAPK3 mRNA knockdown was confirmed by qPCR (Fig. 6B). Interestingly, intracellular HCV RNA levels did not change to MAPKAPK3 knockdown (FIG. 6C). These data suggest that MAPKAPK3 is involved in the translation phase of the HCV life cycle.

Result 6: MAPKAPK3 knockdown inhibits HCV proliferation.

To further investigate the effect of MAPKAPK3 on HCV proliferation, Huh7.5 cells transfected with siRNA targeting MAPKAPK3 or with indicated control siRNA were infected with Jc1. After 48 hours of infection, protein and RNA levels were determined. MAPKAPK3 silencing significantly inhibited both NS3 and core protein levels (Figure 7A). It is notable that the inhibitory activity of MAPKAPK3 siRNA is similar to that of positive control siRNA (Fig. 7A, lane 2 vs. lane 3). Furthermore, MAPKAPK3 alone, rather than MAPKAPK2, specifically inhibited HCV protein expression. As in the replicon cells, intracellular HCV RNA levels were not affected by MAPKAPK3 knockdown in Jc1-infected cells (Fig. 7B). MAPKAPK3 expression silencing induced a strong inhibition of extracellular HCV RNA levels (Figure 7C). We also found that MAPKAPK3 knockdown significantly reduced both HCV protein and extracellular HCV RNA levels in JFH1-infected cells (FIGS. 7D and 7E). To confirm this result, Huh7.5 cells were infected with Jc1 collected in Figure 7C and the degree of HCV infection was determined. As shown in FIG. 7F, the degree of HCV infection in MAPKAPK3 knockdown cells was significantly inhibited. Since the siRNA transfection did not induce cytotoxicity (Fig. 7E), the silencing effect in HCV-infected cells was specific for MAPKAPK3. To exclude the off-target effect of MAPKAPK3 siRNA, a MAPKAPK3 mutation lacking siRNA-resistant MAPKAPK3 mutation and siRNA-resistant ATP-binding was prepared (Ludwig S. et al., 1996. 3pK, a (MAP) kinase-activated protein kinase, is targeted by three MAP kinase pathways, Mol. Cell. Biol. 16: 6687-6697). As in Figure 7F, exogenous expression of the siRNA-resistant MAPKAPK3 mutation relieved HCV protein expression (lane 4), whereas wild-type MAPKAPK3 or mutant MAPKAPK3 deficient in ATP-binding was not. Collectively, these data suggest that MAPKAPK3 is required for HCV proliferation.

Result 7: Protein interaction between core and MAPKAPK3 is essential for HCV IRES-mediated translation.

HCV RNA translation is primarily mediated by the internal ribosome entry site (IRES) located within the viral RNA 5 ' UTR (Ji H. et al., 2004. Proc. Natl. Acad. Sci. USA 101: 16990-16995, Spahn CM et al., 2001. Science 291: 1959-1962, Wang L. et al., 2011. J. Virol. 85: 7954-64). Since MAPKAPK3 silencing has no effect on intracellular HCV RNA levels, we investigated the effect of MAPKAPK3 on the translation step of the HCV life cycle. For this purpose we used the pRL-HL structure (Figure 8A) (Honda M. et al., 2000. Gastroenterology 118: 152-162). Indeed, MAPKAPK3 overexpression significantly increased HCV IRES activity (Figure 8B, left panel). We have observed that MAPKAPK3-dependent HCV IRES-mediated translation is dose dependent increase by core protein. Reporter assays were performed using the MAPKAPK3 mutation (MK3-KA) defective in ATP-binding to confirm the effect of MAPKAPK3 on HCV IRES-mediated translation. As shown in Figure 8B (left panel), the inactive MAPKAPK3 mutation could not augment the HCV IRES-mediated translocation. These data suggest that MAPKAPK3 is involved in HCV IRES-mediated translation. It has been previously reported that the HCV core stimulates HCV IRES activity (Boni S. et al., 2005. J. Biol. Chem. 465 280: 17737-17748, Lourenco S. et al., 2008. FEBS J. 275: 4179-4197). Indeed, core protein overexpression increased HCV IRES activity by 1.5-fold compared to vector control and core-mediated IRES activity was further increased in a dose-dependent manner by MAPKAPK3 (Figure 8B, right panel). We further found that ATP-binding defect mutations of MAPKAPK3 can not bind HCV core protein (Fig. 8C), which suggests that protein interaction is important for HCV protein expression. Next, we studied the functional relevance of protein interaction between core and MAPKAPK3 in HCV IRES-mediated translation. Huh7.5 cells were transfected with negative siRNA or MAPKAPK3 siRNA and transfected with empty vector or core plasmid with or without siRNA resistance mutation MAPKAPK3 with reporter plasmid. As shown in Figure 8D, HCV IRES activity increased in a dose dependent manner with only core protein (left panel). MAPKAPK3 knockdown impaired HCV IRES-mediated translation activity. However, the expression of the siRNA-resistant MAPKAPK3 mutation restored IRES activity, which was dose-dependently increased by the core protein (Fig. 8D, left panel). Thus, the effect of the core on HCV IRES activity was MAPKAPK3-dependent. Furthermore, we assessed the functional significance of protein interactions between the core and MAPKAPK3 for HCV IRES-mediated translation using defective core mutations in binding potency. For this purpose, Huh7.5 cells were co-transfected with wild-type MAPKAPK3 and full-length cores with reporter plasmids. The cells were transfected again with various mutant structures of the core plasmid. As in Figure 8D (right panel, lane 2), MAPKAPK3-dependent HCV IRES activity was significantly increased by full-length core proteins. In the competition assay method, all core mutants except M76-173 effectively distorted protein interactions between wild type core and MAPKAPK3, thus impairing HCV IRES activity (Fig. 8D, right panel). Since M76-173 is a mutant defective in the binding ability of the core (Fig. 4B), these data suggest that protein interaction between core and MAPKAPK3 plays a crucial role in HCV proliferation.

<110> Industry Academic Cooperation Foundation, Hallym University <120> Pharmaceutical composition for prevention and treatment for          hepatitis C containing MAPKAPK3 expression or activity inhibitor <130> Hallym-SBhwang-MAPKAPK3 <160> 21 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA targetting MAPKAPK3 <400> 1 ggaucaacca aucgauggu 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA targetting MAPKAPK3 <400> 2 accaucgauu gguugaucc 19 <210> 3 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA targetting MAPKAPK2 <400> 3 acgagcagau caagauaaa 19 <210> 4 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siRNA targetting MAPKAPK2 <400> 4 uuuaucuuga ucugcucgu 19 <210> 5 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siRNA targetting 5'NTR of Jc1 <400> 5 ccucaaagaa aaaccaaacu u 21 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atcaactagt gatgagcacg aatcct 26 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 attatctaga gcaaccgggc aagtt 25 <210> 8 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gcaagcttat gagcacgaat cctaaacc 28 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cgggatcctc aagagcaacc gggcaagttc 30 <210> 10 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gcggatccga acaaaaactc atctcagaag aggatctgat gagcacgaat cctaaacc 58 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cgtctagaag agcaaccggg caagttc 27 <210> 12 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 cgctctagaa tggatggtga aacagcag 28 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 atggatccct actggttgtt gcagccc 27 <210> 14 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 accacccctg gatcaaccag agtatggtag tgccacagac cc 42 <210> 15 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gggtctgtgg cactaccata ctctggttga tccaggggtg gt 42 <210> 16 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gacagaagtg tgccctggcg ctcctgtatg acagcc 36 <210> 17 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ggctgtcata caggagcgcc agggcacact tctgtc 36 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tgagtgtcgt acagcctcca 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 acgctactcg gctagcagtc 20 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 tgacagcagt cggttggagc g 21 <210> 21 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 gacttcctgt aacaacgcat ctcata 26

Claims (4)

A pharmaceutical composition for the prevention and treatment of hepatitis C virus comprising MAPKAPK3 (mitogen-activated protein kinase-activated protein kinase 3) expression or activity inhibitor.
The method according to claim 1,
Wherein the MAPKAPK3 expression inhibitor is any one selected from the group consisting of an antisense nucleotide complementary to mRNA of MAPKAPK3 gene, short hairpin RNA (shRNA), and siRNA (short interfering RNA).
3. The method of claim 2,
Wherein said siRNA is SEQ ID NOS: 1-2.
The method according to claim 1,
Wherein the MAPKAPK3 activity inhibitor is an anti-MAPKAPK3 antibody binding to MAPKAPK3 protein.

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