KR20060099558A - Peptide for regulating the function of hcv ns5b protein and the use thereof - Google Patents

Abstract

The present invention provides a method for regulating RNA replication of hepatitis C virus using a peptide that specifically modulates the function of NS5B protein, HCV RNA polymerase, and an intracellular protein that specifically binds the peptide as a target protein. It is. According to the present invention, it is possible to inhibit HCV RNA replication by inhibiting phosphorylation of NS5B protein using siRNA that inhibits the expression of intracellular target protein.

HCV, NS5B, PRK2

Description

Peptides for regulating the function of HCV NS5B protein and the use

Figures 1a and 1b is a view showing the expression and purification results of NS5B protein HCV RNA polymerase used in the present invention.

Figure 2 shows the results of analyzing the peptide sequence (P1 to P8) that specifically binds to the NS5B protein screened through biological panning through DNA sequencing.

3A shows that the P1 peptide sequence has a very high homology with amino acids 518-525 residues of the intracellular PRK2 protein.

3B shows that the P1 peptide sequence has specific homology with PRK2.

4 shows the specific interaction of the P1 peptide with the NS5B protein.

5 shows the results of confirming the extracellular interaction of NS5B protein and PRK2 protein.

6 shows the results of confirming intracellular interaction of NS5B protein and PRK2 protein.

Figure 7 shows the results confirmed the intracellular fusion of NS5B protein and PRK2 protein.

8A shows the results of confirming phosphorylation of NS5B protein by PRK2 protein.

8B shows the results of confirming phosphorylation of NS5B protein by PKC-α, β and γ proteins.

9 is a result confirming that PRK2 protein is specifically involved in the extracellular phosphorylation of NS5B protein.

10A shows a schematic of making a recombinant NS5B mutation to identify amino acid residues in the NS5B protein that bind to PRK2 protein.

10B shows the results of coimmunoprecipitation using the recombinant NS5B mutant of 10a and anti-PRK2 antibody of PRK2 protein.

Figure 11 shows the results confirming the amino acid residues in the NS5B protein phosphorylation is induced by PRK2 protein.

12 shows the results of confirming intracellular phosphorylation of NS5B protein by PRK2 protein.

Figure 13a shows that the expression of PRK2 protein is inhibited in Huh7TR-NS, a liver cancer cell transfected with siRNA that inhibits intracellular expression of PRK2.

FIG. 13B shows that phosphorylation of NS5B protein was inhibited in Huh7TR-NS, a liver cancer cell transfected with siRNA that inhibits the expression of PRK2 protein.

14 shows the results of identifying amino acid residues that cause phosphorylation of the NS5B protein by immunoprecipitation using anti-phosphorylated-serine, anti-phosphorylated-threonine and anti-phosphorylated-tyrosine antibodies, respectively.

Figure 15 shows the results of transfection of siRNA that inhibits the expression of PRK2 protein in R-1 cell line and the phosphorylation of NS5B protein.

Figure 16a shows the results of quantifying HCV RNA replication regulation according to the change in the concentration of siRNA transfected in the R-1 cell line by real-time PCR method.

Figure 16b shows the results of confirming the expression of PRK2 protein under the same conditions as in Figure 16a.

17 shows the results of observing phosphorylation of NS5B protein after transfection of plasmid (pcDNA3.1-PRK2) in R-1 cell line.

18a shows the results of quantifying HCV RNA replication by real-time PCR method with increasing concentration of plasmid (pcDNA3.1-PRK2) in R-1 cell line.

Figure 18b shows the results of observing the expression of PRK2 protein under the same conditions as 18a.

19 shows the result of quantifying the replication degree of HCV RNA by real-time PCR method when HA1077, an inhibitor of PRK2 activity, is treated in R-1 cell line.

Technical Field

The present invention relates to a peptide that specifically binds to RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) and to controlling HCV RNA replication using the same.

Prior art

Hepatitis C virus is the virus that is the leading cause of non-A and non-B hepatitis. It is known that 10-20% of infected people develop acute hepatitis and 80-90% of those infected develop chronic infection states (Alter HJ et al., N. Eng.). J. Med., 1989, 321: 1494-1500). In addition, a 2003 report by the Centers for Disease Control and Prevention found that by 2030, the number of new patients infected with HCV is reduced to less than 1%, while the number of patients with severe liver disease, including chronic hepatitis, is increasing. It is expected to increase. An estimated 170 million people worldwide are infected, especially in Africa, Japan and Korea, where 1.5 to 2.0% of the population is chronically infected, and the number of newly infected HCV patients is about 1 million every year. It is estimated to reach. The main feature that distinguishes hepatitis from HCV infection from hepatitis B virus (HBV) infection is that the likelihood of chronic hepatitis is much higher than that from HBV infection. Anti-HCV antibody formation rates in patients not infected with HBV and with cirrhosis or liver cancer for other causes have been reported to be significantly higher than in controls (Ikeda et al., Hepatology, 1993, 18: 47-53). 75% of HCV-infected patients develop liver cancer after 15 years. This is much higher compared to a 27% chance of developing liver cancer during HBV infection, indicating a stronger correlation with the onset of cancer than any carcinogen known to date.

Ribavirin and α-interferon have been used together to effectively treat chronic hepatitis of HCV. However, only about 20% of hepatitis patients receiving α-interferon have been reported to have therapeutic effects (Hoofnagal JH., And. Intern. Med., 1994, 39: 241-275). As described above, α-interferon is the most widely used type of HCV therapeutic agent in the world, but the HCV-1b type, which has a high rate of liver cirrhosis or liver cancer, has a low effect and has a problem in treating hepatitis caused by HCV infection. . It has also been reported to have no effect on HCV with an interferon resistance sequence. Therefore, there is a continuing need for the development of new therapeutic agents or treatments for hepatitis caused by HCV infection.

HCV belongs to the flavivirus family and consists of positive single stranded RNA (HC) with a genome of about 9.5 kb. The RNA is a polyprotein consisting of one open reading frame (ORF) and about 3,011 amino acids, and consists of sequences of about 10 different proteins. In particular, about 340 nucleotides (nt) important for protein synthesis are 5'UTR (untraslated region) and 3'UTR consisting of 98 nt as a short variable region-polypyrimidine tract. It includes. The 98 nt 3'UTR is known to affect the activity of NS5B protein, which is a highly conserved sequence in various HCV and HCV RNA polymerase.

The polyprotein is composed of NH ₃ -C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH. That is, at the amino terminus of the polyprotein, there are structural proteins, C (core), E1 and E2, which are components of the nucleocapside and envelope of the virus, and HCV is present at the carboxyl terminus. Non-structural proteins essential for proliferation are located, namely NS2, NS3, NS4A, NS4B, NS5A and NS5B. When the signal polytease in the host cell and the proteolytic enzymes NS2-3 and NS3 act on the precursor polyprotein, specific sites of the polyprotein are cleaved to form ten mature viral proteins. Specifically, cleavage between NH ₃ -C-E1-E2-p7-NS2 is cleaved by a host cell protease, and between NS2-NS3 is a metalloprotease consisting of the amino terminal 1/3 of NS2 and NS3 By autocleavage, and the rest is cleaved by NS3 serine protease. Of these, the NS5B protein has a conservative GDD motif of RNA-dependent RNA polymerase (RdRp) of RNA viruses, which is used to remove minus strands from positive strands. It is a very important component for viral replication that synthesizes and synthesizes the viral genome using this negative strand as a template. The NS5B protein has no specificity for the HCV RNA genome and is reported to be capable of performing primer-dependent or primer-independent RNA synthesis using monopolymeric RNA as a template. (Behren SE et al., EMBO J., 1996, 15: 12-22; Luo G et al., J. Virol., 2000, 74: 851-863).

Research on NS5B protein has been focused on in vitro studies on its activity as an RNA polymerase mainly using recombinant NS5B protein, but recently, a system using human-derived cells capable of HCV RNA replication ( Construction of the HCV replicon cell line and the crystal structure (Lesburg CA et al., Nat. Struct. Biol., 1999, 6: 937-943) revealed that the NS5B protein has been studied as an important protein in regulating HCV RNA replication. have. In addition, screening for anti-HCV targeting the NS5B protein and functional studies at the cellular level to maintain conditions in vitro as well as in vivo are being actively conducted. Various anti-HCVs targeting the NS5B protein have been screened and reported, including non-nucleoside inhibitors (Beaulieu PL et al., Bioog. Med. Chem. Lett., 2004, 14: 119-124; Chan L et al., Bioog. Med. Chem. Lett., 2004, 14: 793-796; Chan L et al., Bioog. Med. Chem. Lett., 2004, 14: 797-800; Beaulieu PL et al. , Bioog.Med. Chem. Lett., 2004, 14: 967-971; Gopalsamy A et al., Bioog.Med.Chem. Lett., 2004, 14: 4221-4224), chemical combination inhibitors (Mckercher G et. al., Nucleic Acid Res., 2004, 32: 422-431; Lou H et al., Virology, 2003, 317: 65-72), nucleotides including DNA and RNA (Bellecave P et al., Oligonucletides, 2003) 13: 455-463; Vo NV et al., Virology, 2003, 307: 301-316) and small molecular inhibitors analyzed by high-throughput screening through the crystal structure of HCV RNA polymerase , Love RA et al., J. Virol., 2003, 77: 7575-7581), and in vitro studies In addition, research has been conducted at the human cell level.

In addition, the recent research on NS5B proteins focuses on the ability to interact with other cellular proteins in the replication of HCV RNA in the cell, thus screening for protein binding and analyzing its function. It's going on. P68 cellular RNA helicase (Goh PY et al., J. Virol., 2004, 78: 5288-5298), alpha-actin as an intracellular protein that binds NS5B protein intracellularly to date Nin (α-actinin, Lan S et al., FEBS Lett., 2003, 554: 289-294), nucleolin (nucleolin, Hirano M et al., J. Biol. Chem., 2003, 278: 5109- 5115), and eIF4AII (Biochem. Biophys. Res. Commum., Kyono K et al., 2002, 292: 659-666). However, in these studies, it was difficult to understand the function of the intracellular NS5B protein because it did not specifically describe how the NS5B protein binds to these intracellular proteins and affects HCV RNA replication.

In addition, a study was conducted to select peptides that specifically bind to NS5B protein using phage peptide libraries extracellularly targeting the NS5B protein (Amin A et al., Virology, 2003, 313: 158-169). Peptides selected from were found to affect the activity of the NS5B protein, but no functional studies have been found in the cells.

Therefore, the inventors screened (biopanning) a peptide that specifically binds to the recombinant HCV NS5B protein (recombinant NS5B protein) using a phage random peptide library. In addition, the present inventors confirmed that the PRK2 protein in the cell has a very high homology with the selected peptide sequence, and confirmed that the intracellular protein phosphorylates NS5B protein not only in the cell but also in the cell.

In addition, it was confirmed that the phosphorylation of the NS5B protein is also regulated by regulating the intracellular proteins, and the phosphorylation residues in the NS5B protein are also revealed, thereby providing a method of selectively regulating the phosphorylation of the NS5B protein. As such, the present inventors have completed the present invention by providing a method for regulating HCV RNA replication by intracellular PRK2 protein by revealing that HCV RNA replication is specifically regulated through phosphorylation of NS5B.

An object of the present invention is to provide a peptide that specifically binds to NS5B protein which is HCV RNA polymerase.

Another object of the present invention is to provide an intracellular target protein for regulating HCV RNA replication using a peptide that specifically binds to NS5B protein.

The present invention provides a method of regulating HCV RNA replication by regulating phosphorylation of NS5B protein using intracellular PRK2 protein. It is also an object of the present invention to provide a technique utilizing the function of targeting the NS5B protein in regulating HCV RNA replication.

In order to achieve the above object, the present invention was selected by sequencing the peptide specifically binding to NS5B protein HCV RNA polymerase. A search was performed for intracellular proteins having high homology with each of these peptides and confirmed whether they were involved in phosphorylation of HCV NS5B protein.

Specifically, the present invention uses a peptide library displayed on phage to select peptides that specifically bind to NS5B protein, a non-structural protein that functions as an RNA-dependent RNA polymerase in the replication of hepatitis C virus. Panning was performed. Fusion of M13 phage coat protein III using a recombinant NS5B protein expressed in E. coli BL21 (Novagen) and purified through an affinity column and an ion-exchange column. 12 peptide libraries that are expressed and expressed, and select the bound phage only to select peptides that specifically bind to the NS5B protein through amplification and sequencing, respectively, P1 (SEQ ID NO: 1) and P2 (SEQ ID NO: : 2), P3 (SEQ ID NO: 3), P4 (SEQ ID NO: 4), P5 (SEQ ID NO: 5), P6 (SEQ ID NO: 6), P7 (SEQ ID NO: 7), and P8 (SEQ ID NO: 8 ). In Figure 2, the second peptide of the P1 sequence, the 12th of the P4 sequence, the first of the P5 sequence, the fourth and fifth of the P6 sequence, the sixth and ninth of the P7 sequence, and the fifth glutamine of the P8 sequence (Q ) Was identified as "TAG" sequence by sequence search. This codon is generally suppressed by glutamine (Q) in E. coli SOLR (supE), an amber stop codon, or a strain used for library production, and is therefore treated as a glutamine codon, and thus is also translated into glutamine in the present invention.

Among the peptides that specifically bind to the NS5B protein, the P1 peptide (N-TSTAGRIVRRAI-C) (SEQ ID NO: 1), which shows the highest binding frequency, is a homologous PRK2 (protein kinase C-related kinase 2) protein in human cells. And showed the highest homology. Intracellular PRK2 protein was confirmed to bind to NS5B protein in vitro and in vivo , thereby confirming the function of NS5B protein by human intracellular kinase that binds to NS5B protein and intracellularly. That is, the specific phosphorylation of NS5B protein in vitro by intracellular PRK2 protein was observed by inhibitors specific to PRK2 (HA1077) and non-specific inhibitors (PI3 kinase inhibitors LY294002 and Wortmannin, p38MAP kinase inhibitors SB203580 and PD98059, PKC). Assay was performed using the inhibitor GF109203X).

As such, it was confirmed that the phosphorylation of the NS5B protein by the PRK2 protein in vitro , and then phosphorylation in vivo was tested. To this end, first, a Huh7TR-NS cell line in which human liver cancer cell line Huh7 expressing HCV nonstructural protein is expressed by tetracycline was prepared, and the phosphorylation of NS5B protein in vivo was verified using this. Cover the cell line was modified with ortho ^[32 P] phosphate (ortho ^[32 P] phosphate) and then, via immunoprecipitation with anti -NS5B antibody was confirmed that the NS5B protein phosphorylation in the in vivo. Next, siRNA was prepared to suppress the expression of intracellular PRK2 protein in order to confirm whether the phosphorylation of the intracellular NS5B protein is specific by PRK2 protein. siRNA inhibited the expression of PRK2 protein in the cell, and thus phosphorylation of NS5B protein was inhibited.

In addition, the present invention specifically prepared a NS5B mutant to specifically identify the phosphorylation site of the NS5B protein induced by PRK2 protein. PET28a (+) / NS5B (1-371) and pET28a (+) / NS5B (187-) expressing proteins corresponding to amino acids 1 to 371, 187 to 591 and 372 to 591, respectively, of the NS5B protein (SEQ ID NO: 13) 591) and pET28a (+) / NS5B (372-591) were prepared to identify sites in NS5B protein that bind to and phosphorylate PRK2 protein by immunoprecipitation and in vitro kinase assay. As a result, it was confirmed that the amino acid 1-187 site corresponding to the finger portion of the NS5B protein binds to and phosphorylates PRK2.

In addition, the phosphorylation residues of NS5B protein phosphorylated intracellularly were analyzed. To this end, immunoprecipitation with anti-phosphorylated-serine, anti-phosphorylated-threonine and anti-phosphorylated-tyrosine antibodies was performed and the phosphorylation residues in the NS5B protein were analyzed. As a result, it was confirmed that phosphorylation was induced at the serine residue in the NS5B protein. As such, it was confirmed that the NS5B protein is phosphorylated in vitro and in vivo by the PRK2 protein, and the residue from which phosphorylation is induced is a serine residue of amino acids 1 to 187, which is a finger portion of the NS5B protein.

The mechanism of HCV RNA replication regulated by NS5B protein phosphorylated by PRK2 protein was studied. To this end, after inhibiting or overexpressing the intracellular protein of PRK2 in R-1, a HCV replicon cell line in which HCV RNA is stably replicated, the regulation of HCV RNA replication is performed in real time using primers specific for HCV 5'-UTR. Analysis was by real-time PCR (PCR). Through the above analysis, it was confirmed that HCV RNA replication was inhibited when PRK2 protein expression in R-1 cell line was inhibited by siRNA.In contrast, pcDNA3.1-PRK2 transfected with PRK2 protein expression in R-1 cell line. Overexpression has been shown to increase HCV RNA replication. In other words, treatment of siRNA that inhibits PRK2 protein expression in R-1 cell line inhibits phosphorylation of HCV NS5B protein, whereas treatment of pcDNA3.1-PRK2 that overexpresses PRK2 protein expression in R-1 cell line results in HCV NS5B protein. Phosphorylation of was found to increase. This demonstrates that the expression of PRK2 protein in cells is associated with phosphorylation of NS5B protein and thus HCV RNA replication.

Thus, the present invention suggests that PRK2 protein can be used as a target protein for the regulation of HCV RNA replication.

In the present invention, a "fragment" or "derivative" of a peptide / protein is defined as comprising part or all of the amino acid sequence of the peptide / protein of the invention and sharing the specific binding properties possessed by the peptide / protein of the invention. .

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are only for illustrating the present invention, but the scope of the present invention is not limited by these examples. References cited in the present invention are incorporated as part of the present invention.

Example

Example 1: Expression and Purification of Recombinant HCV NS5B Protein

For expression of the NS5B protein (SEQ ID NO: 13), a hepatitis C virus (HCV) RNA polymerase, the gene was cloned into the pTrcHisB vector (Invitrogen) using 5'-NheI and 3'-EcoRI restriction enzymes. E. coli BL21 (Novagen) was transformed and expressed the recombinant protein fused with 6x histidine. Selected E. coli transformants were incubated at 37 ° C. in LB medium (luria-bertani medium, added 100 μg / ml ampicillin), and the absorbance at 600 nm was 0.8 mM, 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside). After inducing and inducing protein expression for 6 to 12 hours while incubating at 25 ℃, cells were recovered from the culture. Cells obtained by centrifugation were washed once with PBS (phosphate buffered saline), followed by lysis buffer (50 mM Na-phosphate buffer [pH 8.0], 1% nonidet P-40, 10 mM mercaptoethanol, 10% glycerol, Cells were disrupted by sonication by suspension in 10 mM imidazole) and centrifuged (12,000 rpm, 30 minutes, 4 ° C.) to remove precipitated cell residue and supernatant was taken. The obtained supernatant was adsorbed on an affinity column, Ni ²⁺ -NTA column (nitrilotriacetic acid, Qiagen), eluted with 50 to 450 mM imidazole, and buffer A (50 mM Tris-HCl). Heparine-agarose column, Amesham Biosiciences, an ion-exchanger column treated with [pH 8.0], 50 mM sodium chloride, 5 mM magnesium chloride, 1 mM DTT) and treated with the same buffer. G) and eluted with Buffer A containing 0.1-1.0 M sodium chloride. The obtained fraction was purified using an ion exchange column, Sepharose column (sp-sepharose column, Amersham Biosciences). The crude extract, precipitate, supernatant and purified protein were stained with coomassie blue after electrophoresis (10% SDS-PAGE) to confirm the expression of the protein. Next, the protein separated on the 10% SDS-PAGE gel was transferred to a nitrocellulose membrane (Amersham Biosciences) using a protein electroblotter (Hoefer), and the histidine residues attached to the N-terminus were recognized. Using an anti-6 × His antibody (Qiagen) as a primary antibody and peroxidase-labeled anti-mouse antibody as a secondary antibody (peroxidase-conjugated anti-mouse IgG antibody, Sigma) Finally, Western blotting was performed using ECL substrate (Amersham Biosciences). As confirmed in Figures 1a and 1b, it was confirmed that the high concentration in the purified NS5B protein fraction.

Example 2: Screening of Peptides That Binding Specificly to NS5B Proteins: Biological Panning Using Phage Peptide Libraries

In order to select peptides that specifically bind to NS5B protein, biological panning using phage peptide libraries was performed on the fractions purified according to Example 1. The phage peptide library contains (NNK) ₁₂ (N = A, C, G, or T; K = G, T) random nucleotides with a pCANTAB5E vector (Amersham Biosciences) using 5'-SfiI and 3'-NotI restriction enzymes. ) By cloning. The random sequence is a library fused to the N-terminus of coat protein III of M13 bacteriophage to express 12 random peptides. The PBS solution containing the recombinant NS5B protein (800 ng) purified in Example 1 was coated on a Ni-NTA HisSorb plate (Qiagen), and then reacted at 4 ° C. for 12 hours. After the reaction was completed, 0.1% BSA for the first and third biological panning and 5% for the second biological panning, respectively, to remove the remaining PBS solution on the plate and to disrupt non-specific binding between the NS5B protein and the phage peptide library. PBS solution containing skim milk was dispensed to disturb the plate and allowed to react at room temperature for 2 hours. After completion of the reaction, phage peptide libraries (1.08 × 10 ¹² cfu phage / well) were dispensed on the plates, then reacted for 12 hours at 4 ° C. and non-specific phages were removed using PBS solution at room temperature. Next, 0.1 M glycine [pH2.2] solution was dispensed on the plate for 20 minutes to elute specifically bound phages, and immediately neutralized by addition of 1 M Tris-HCCL [pH8.8] solution. . Some of the phages were infected with E. coli XL1-Blue (Stratagene) incubated in LB liquid medium (125 μg / ml tetracycline, 100 μg / ml ampicillin) at 37 ° C. for 15 minutes and the LB solid medium (125 μg / ml Incubated in ml tetracycline, 100 μg / ml ampicillin), the eluted phages were confirmed by treating colonies on solid medium with BamHI and EcoRI restriction enzymes and confirming the inserts by DNA electrophoresis. The eluted phages were infected with Escherichia coli SOLR (Stratagene) incubated in LB liquid medium (250 μg / ml kanamycin, 100 μg / ml ampicillin), and the absorbance at 600 nM when incubated at 37 ° C. reached M13- Phages were amplified with VCS helper phage (2.46 × 10 ¹¹ pfu phage / ml). The supernatant obtained by centrifugation was concentrated using 30% PEG8000 / 2.5 M sodium chloride, dissolved in TE solution (10 mM Tris-HC, 1 mM EDTA [pH 8.0]) and used for the next biological panning. . After tertiary biological panning, the phages were infected with E. coli SOLR (Stratagene) incubated in LB liquid medium (250 μg / ml kanamycin, 100 μg / ml ampicillin). Next, DNA sequencing was performed by randomly selecting colonies grown in LB solid medium (250 μg / ml kanamycin, 100 μg / ml ampicillin). As a result, eight kinds of peptide sequences were identified and named as P1 to P8 (SEQ ID NO: 1 to SEQ ID NO: 8, respectively). Among them, the P1 peptide sequence was analyzed with the highest frequency. The base sequence encoding the P1 peptide is shown in SEQ ID NO: 9.

Example 3: Homology Analysis of P1 Peptide and PRK2 Protein

Proteins with the highest homology in human cells were analyzed using the P1 peptide sequence with the highest binding frequency among peptides that specifically bind to NS5B protein through biological panning. Human intracellular protein having high homology with P1 peptide was analyzed through protein BLAST provided by NCBI (national center for biotechnology information). As a result, as shown in FIG. 3A, the N-GRLVRRAI-C amino acid sequence of the P1 peptide has a proline-rich region in the PRK2 protein (protein kinase C-related kinase 2, GenBank accession number NM_006256) (SEQ ID NO: 10). proline rich region) showed very high homology with amino acids sequence 518-525 of 87.5%. In addition, as shown in FIG. 3B, the P1 peptide sequence was analyzed by comparison with all isotypes of protein kinase C (PKC), and the sequence was found to have specific homology with the PRK2 protein.

Example 4: Specific Binding of P1 Peptide to NS5B Protein

In order to analyze specific binding of NS5B protein to P1 peptide having the highest frequency among peptides selected through biological panning as binding to NS5B protein, P1 peptide synthesized with phage expressing P1 peptide to NS5B protein ( Competitive ELISA between SP1) was performed. Synthetic P1 peptide (SP1) was produced by Peptron Co., Korea. Colonies of E. coli SOLR cells transformed with phagemid expressing P1 peptides were inoculated in LB medium (250 μg / ml kanamycin, 100 μg / ml ampicillin) and incubated at 37 ° C. at 600 nm. When the absorbance was 1.0, M13-VCS helper phage (Helper phage) was added to amplify phage expressing the P1 peptide. The supernatant obtained by centrifugation was then concentrated using 30% PEG8000 / 2.5 M sodium chloride (NaCl), and the concentrated phages were washed with TE solution (10 mM Tris-HCl, 1 mM EDTA [pH8.0]). Melted. To perform competitive ELISA, purified 800 ng of NS5B protein was coated on a Ni-NTA HisSorb plate at 4 ° C. for 12 hours, followed by 2% PBS solution containing 1% BSA to prevent non-specific binding. The reaction was carried out at room temperature for hours. After washing the plate three times with PBS solution, a mixture of phage expressing P1 peptide (1.08 × 10 ¹⁰ cfu phage / well) and synthetic P1 peptide (0.001 to 10 pmol) of various concentrations were mixed for 12 hours at 4 ° C. Reacted for a while. After completion of the reaction, the plate was washed 10 times and 20 times with PBS solution and PBS solution containing 0.1% Tween20, respectively, and P1 peptide was prepared using anti-M13 antibody (Amersham Biosciences) as the primary antibody. Reacted with expressing phage. Next, alkaline phosphatase-conjugated goat anti-rat IgG (Alkaline phosphatase-conjugated goat anti-mouse IgG, Sigma) was used as a secondary antibody, and finally, p-nitrophenylphosphate (Sigma) was used. 4) was used as a substrate and the absorbance was measured at 405 nm on an ELISA reader (Molecular Devices). As shown in FIG. 4, binding of the phage expressing the selected P1 peptide to the NS5B protein was found to significantly decrease the binding capacity due to a competitive relationship as the amount of the synthetic P1 peptide (SC1) increased. . Thus, the selected P1 peptide was identified as a peptide that is very specific to the NS5B protein and has a very high binding capacity.

Example 5: In vitro Binding of NS5B protein and PRK2 protein in

In vitro , specific interaction between NS5B protein and PRK2 protein was confirmed. Coimmunoprecipitation was performed using HEK293T endogenous PRK2 protein and recombinant NS5B protein. 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptoto HEK293T cells (ATCC) were cultured in animal cell medium DMEM additionally added to mycin (streptomycin) and 10% fetal bovine serum (FBS), and the cells were recovered by centrifugation (21,000 × g, 4 ° C., 20 minutes). . After washing three times with PBS solution, a cell disruption solution [20 mM Tris-HCl [pH7.5], 1 mM EDTA, 1 mM DTT, to which an additional protease inhibitor (EDTA free-protease inhibitor cocktail, Roche) was added 50 mM sodium fluoride (NaF), 1 mM sodium vanadate (Na ₃ VO ₄ ), 0.5% Triton X-100, and 100 mM sodium chloride (NaCl)] to disrupt cells, anti-PRK2 antibody, recombinant NS5B The PRK2-NS5B complex was isolated by co-immunoprecipitation with the addition of protein and protein A-Sepharose. The PRK2-NS5B complex was added to a 6 × SDS solution, heat treated at 95 ° C., and the proteins isolated on 8% SDS-PAGE gels. The isolated protein was blotted onto a nitrocellulose membrane, and 1 hour in TBST solution [20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween20] to which 5% BSA was added. After interfering with non-specific binding, anti-PRK2 antibody (Cellsignaling), anti-NS5B antibody (SantaCruz), anti-pan-PKC antibody (Zymed Laboratory) or anti-histidine antibody (Qiagen), respectively. Was diluted as a primary antibody in a 1: 1 ratio of TBST solution and reacted at room temperature for 1 hour. After completion of the reaction, the membranes were washed three times with TBST solution for 10 minutes each, using anti-rabbit IgG, anti-goat IgG and anti-mouse IgG bound to horse radish peroxidase (HRP), respectively, as a secondary antibody. The reaction was carried out for 1 hour at room temperature diluted with TBST solution in proportion. After completion of the reaction, the membrane was washed three times with TBST solution and finally the protein was reactive with antibody using ECL as substrate. As a result, as shown in Figure 5, the NS5B protein was directly cross-linked with the PRK2 protein, but not with the isomers of PKC-α, β, γ. This demonstrates that the interaction of PRK2 and NS5B proteins is specific and direct.

Example 6: In vivo Binding of NS5B protein and PRK2 protein in

6.1 Preparation of HCV Replicon Cell Line

According to the prior art, pZS2 (Institute for Cancer Center, Fox Chase Cancer Center, Philadelphia, USA) derived from HCV Con-1 Riplicon I ₃₇₇ / NS3-3 (GenBank accession number AJ242652) was single-stranded with ScaI restriction enzyme. And used for in vitro transcription (Oh JW et al., 1999, J. Virol., 73: 7694-7702). The produced HCV RNA was transfected into Huh7 (ATCC), a human liver cancer cell line using a Gene Pulser system (BioRad), and selected using 1 mg / ml G418 (Gibco-BRL) (Guo JT et al. , 2001, J. Virol., 75: 8516-8523, Lohmann V et al., 2001, J. Virol., 75: 1437-1449). The obtained HCV replicon cell line was named R-1.

6.2 Specific Binding of NS5B Protein and PRK2 Protein in HCV Replicon Cell Line

In vivo , immunoprecipitation using R-1 cell line was performed to confirm specific interaction between NS5B protein and PRK2 protein. 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10 The R-1 cell line was cultured in animal cell medium DMEM added with% FBS, and the cultured cells were recovered by centrifugation (21,000 × g, 4 ° C., 20 minutes). After washing three times with PBS solution, a cell disruption solution [20 mM HEPES [pH7.5], 1 mM EDTA, 10% glycerol, to which an additional protease inhibitor (EDTA free-protease inhibitor cocktail, Roche) was added, 100 mM sodium fluoride (NaF), 10 mM tetrasodium pyruphosphate, 17.5 mM β-glycerophosphate, 1% triton X-100, 150 mM sodium chloride (NaCl)], and disrupted the cells, followed by anti-PRK2. The PRK2 complex was isolated by immunoprecipitation with the addition of antibody and protein A-Sepharose. The isolated PRK2 complex was added to a 6 × SDS solution and heat treated at 95 ° C., and the proteins were separated on 8% SDS-PAGE gels. The isolated protein was blotted onto nitrocellulose membrane and non-specific binding was inhibited for 1 hour in TBST solution [20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween20] with 5% BSA. After interruption, anti-PRK2 antibody, anti-NS5B antibody or anti-NS5A antibody (Biodesign International Co., Ltd.) were used as primary antibodies and diluted in TBST solution in a ratio of 1: 1000 and reacted at room temperature for 1 hour. After completion of the reaction, the membranes were washed three times with TBST solution for 10 minutes each, using anti-rabbit IgG, anti-goat IgG and anti-mouse IgG bound to horse radish peroxidase (HRP), respectively, as a secondary antibody. Diluted in the TBST solution in a ratio and reacted for 1 hour at room temperature. After the reaction, the membrane was washed three times with TBST solution, and finally, ECL was used as a substrate to analyze proteins reactive with the antibody. As a result, as shown in Figure 6, the PRK2 protein was interconnected with the NS5B protein, but not with the NS5A protein. This demonstrates that PRK2 protein specifically interacts with NS5B protein.

Example 7: I n vivo Fusion of NS5B Protein and PRK2 Protein in

7.1 Preparation of Huh7TR-NS Cell Line

Huh7TR-NS cell line was prepared in which HCV nonstructural protein (NS3-NS5B) was expression induced by tetracycline. First, the human liver cancer cell line Huh7 (ATCC) was transfected with pcDNA6 / TR (Invitrogen), and then screened with 10 μg / ml of blasticidin S (blasticidin S, Invitrogen) to obtain a Huh7TR cell line. . Selected cell lines have a blastetidine selection marker (Marker), and has a gene encoding a tetracycline operon repressor protein (tetracycline) is characterized by controlling the expression of the protein with tetracycline. Next, Huh7TR-NS cell line was prepared. First, primers HCV-NS3F (5'-ATAAGAATGCGGCCGCACCATGGCGCCCATCACGGCCTACTCC-3 ') (SEQ ID NO: 11) and NS5B-BIII (5'-GCTCTAGATCGGTTGGGGAGCAGGTAAATG-3') (sequence) with pCV-J4L6S (NIH, USA) as a template Polymerase chain reaction (PCR) was used to amplify the gene encoding the HCV nonstructural protein, and the PCR product was prepared using pcINA4.0 / TO / myc-hisB vector (NotI and XbaI restriction enzymes). Invitrogen) was cloned. The cloned pcDNA4.0-NS plasmid was transfected into a Huh7TR cell line, screened with 10 μg / ml of blasticidin S and 100 μg / ml of geocin (Zeocin, Invitrogen) and finally Huh7TR- NS cell lines were prepared. This cell line was used to confirm intracellular fusion of NS5B protein and PRK2 protein.

7.2 Fusion of NS5B and PRK2 Proteins in Huh7TR-NS Cell Lines Using Immunofluorescence Microscopy

In order to confirm the fusion of NS5B protein and PRK2 protein in vivo , the huh7TR-NS cell line prepared in 7.1 was used. 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10 Incubate Huh7TR-NS cell line to about 60% in 8-well chamber (Nunc) using animal cell medium DMEM with% FBS, and induce HCV nonstructural protein expression with 1 μg / ml tetracycline. It was. After 48 hours, cells were washed with PBS solution and fixed with PBS solution with 4% paraformaldehyde for 30 minutes at -20 ° C. The fixed cells were permeated with PBS solution with 0.2% Triton X-100 for 30 minutes at room temperature. After washing 5 times with PBS solution, the cells were reacted for 30 minutes at room temperature with a blocking solution to which 1% BSA, 0.1% gelatin and 5% goat serum were added, and as anti-PRK2 antibody and anti-NS5B as primary antibody, respectively. After reacting at 4 ° C. for 12 hours using an antibody, the resultant was washed five times using a PBS solution containing 1% BSA and 0.1% gelatin. Next, the cells were reacted for 2 hours with goat anti-rabbit IgG antibody (Vector Laboratory) bound to FITC and horse anti-mouse IgG antibody (Vector Laboratory) bound to TEXAS-red, respectively, as secondary antibodies. Finally, after washing five times with PBS solution, the nuclei were stained with 1 μM 4,6-diimidino-2-phenylindole (DAPI) and BIO-RAD Radiance 2000 multi-photon laser confocal microscope PRK2 protein and NS5B protein images were confirmed. As a result, as shown in FIG. 7, it was confirmed that the green PRK2 protein and the red NS5B protein were fused (yellow) around the nucleus (blue) in the cell.

Example 8: In vitro Phosphorylation of NS5B Protein by PRK2 Protein in

To confirm the phosphorylation of NS5B protein by PRK2 protein from In vitro, the cell was performed Endogenous PRK2 of recombinant protein and NS5B protein, and [γ- ³² P] in vitro kinase assay using isotope (In vitro kinase assay). HEK293T cells (ATCC) in DMEM animal cell medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10% FBS ) Were cultured and centrifuged (21,000 × g, 4 ° C., 20 minutes) to collect the cells. After washing three times with PBS solution, the cell disruption solution [20 mM Tris-HCl [pH7.5], 1 mM EDTA, 1 mM DTT, further added with a protease inhibitor (EDTA free-protease inhibitor cocktail, Roche) 50 mM sodium fluoride (NaF), 1 mM sodium vanadate (Na ₃ VO ₄ ), 0.5% Triton X-100, and 100 mM sodium chloride (NaCl)] to disrupt cells and anti-PRK2 antibody and protein A -PRK2 complexes were isolated by immunoprecipitation with the addition of Sepharose. Twice the PRK2 complex into a cell disruption solution, and 1 × phosphorylation solution [20 mM HEPES [pH7.5], 50 mM sodium chloride, 5 mM magnesium chloride (MgCl ₂ ), 5 mM manganese chloride (MnCl ₂ ), 1 mM DTT] once, followed by in vitro kinase reaction (30 ° C., 30 minutes) by adding 1 μg of NS5B protein, 100 μM ATP and 10 μCi [γ- ³² P] isotope. The reaction was added to a 6 × SDS solution and heat treated at 95 ° C., the protein was separated on an 8% SDS-PAGE gel, and the gel was dried on 3M paper to analyze the phosphorylation of the protein on the film. As a result, the phosphorylation of NS5B protein was induced by PRK2 protein (FIG. 8A), but phosphorylation by isomers of PKC-α, β, and γ was not induced (8b). This demonstrates that PRK2 protein is specific in vitro and directly phosphorylates the NS5B protein.

Example 9: In vitro Specificity of Phosphorylation of NS5B Protein by PRK2 Protein in

In vitro kinase using endogenous PRK2 protein and recombinant NS5B protein, HA1077 (PRK2 inhibitor, Calbiochem) and [γ- ³² P] isotope to confirm specific phosphorylation of NS5B protein by PRK2 protein in vitro Assay was performed. HEK293T cells (ATCC) in DMEM animal cell medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10% FBS ) Were cultured and centrifuged (21,000 × g, 4 ° C., 20 minutes) to collect the cells. After washing three times with PBS solution, the cell disruption solution [20 mM Tris-HCl [pH7.5], 1 mM EDTA, 1 mM DTT, further added with a protease inhibitor (EDTA free-protease inhibitor cocktail, Roche) 50 mM sodium fluoride (NaF), 1 mM sodium vanadate (Na ₃ VO ₄ ), 0.5% Triton X-100 and 100 mM sodium chloride (NaCl)] to disrupt the cells, and anti-PRK2 antibody and protein A -PRK2 complexes were isolated by immunoprecipitation with the addition of Sepharose. The separated PRK2 complex was purified twice with a cell disruption solution and 1 × phosphorylated solution [20 mM HEPES [pH7.5], 50 mM sodium chloride, 5 mM magnesium chloride (MgCl ₂ ), 5 mM manganese chloride (MnCl ₂ ) and 1 mM DTT], followed by in vitro kinase reaction (30 ° C., 30 minutes) by adding 20 μM of HA1077, 1 μg of NS5B protein, 100 μM ATP and 10 μCi of [γ- ³² P] isotope. ) The reaction was added to a 6 × SDS solution, heat treated at 95 ° C., the protein was separated on an 8% SDS-PAGE gel, and the gel was dried on 3M paper to analyze protein phosphorylation on film. As a result, as shown in Figure 9, phosphorylation of NS5B protein by PRK2 protein was significantly inhibited by PRK2 specific inhibitor HA1077, but specific inhibitor of PI3K protein [50 μM LY294002 (Calbiochem), 1 μM Wortmannin (Sigma)], NS5B protein by a specific inhibitor of p38 MAPK protein [10 μM of SB203580 (Calbiochem), 50 μM of PD98059 (Calbiochem)] and a specific inhibitor of PKC protein [10 μM of GF109203X (Calbiochem)] Phosphorylation inhibition was not induced. Phosphorylation of NS5B protein by PRK2 protein was confirmed to be specifically induced by PRK2 protein.

Example 10 Identification of PRK2 Protein and Cross-Binding Site in NS5B Protein

10.1 Preparation of Recombinant NS5B Mutants

Recombinant NS5B mutants were prepared in order to identify sites that interact with PRK2 protein in the NS5B protein. To prepare a recombinant NS5B mutant, PCR was performed using the pThNS5B plasmid used in Example 1 as a template, and the PCR preparation was prepared using pET28a (+) using 5'-NheI and 3'-EcoRI restriction enzymes, respectively (Novagen). PET28a (+) / NS5B (372-591), which is a plasmid which is cloned into a) and corresponds to the NS5B protein (SEQ ID NO: 13) amino acids (372-591); PET28a (+) / NS5B (1-371), which is a plasmid corresponding to amino acids (1-371); And pET28a (+) / NS5B (188-591), which are plasmids corresponding to amino acids (188-591), respectively. As a result, as shown in FIG. 10A, a mutant capable of identifying a site in the NS5B protein that mutually binds to PRK2 protein was obtained.

10.2 Expression and Purification of Recombinant NS5B Mutant Proteins

Each of the NS5B mutants prepared in 10.1 was transformed into Escherichia coli BL21 (DE3) (Novagen), and the selected E. coli colonies were absorbed at 600 nm while incubated at 37 ° C. in LB medium (250 μg / ml kanamycin added). At 0.8, 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was added and incubated at 25 ° C. for 6-12 hours to induce protein expression, and then cells were recovered from the culture. Cells obtained by centrifugation were washed once with PBS solution, followed by lysis buffer (50 mM Na-phosphate buffer [pH 8.0], 1% Nonidet P-40, 10 mM mercaptoethanol, 10% glycerol and 10 mM imidazole). Cells were disrupted by sonication, suspended, and centrifuged (12,000 rpm, 30 minutes, 4 ° C.) to remove precipitated cell residue and supernatant was taken. The supernatant obtained therefrom was adsorbed onto an affinity column, Ni ²⁺ -NTA column (nitrilotriacetic acid, Qiagen), eluted with 50 to 450 mM imidazole, and buffer A (50 mM). Final purification was performed by dialysis in tris-hydrochloric acid [pH 8.0], 50 mM sodium chloride, 5 mM magnesium chloride, 1 mM DTT). As a result, as shown in the third panel of FIG. 10B, each NS5B mutant was identified by Western blotting analysis using an anti-histidine antibody. The purified NS5B mutant was used to identify sites in the NS5B protein that interact with the PRK2 protein.

10.3 Identification of Sites Interacting with PRK2 Protein in NS5B Protein

In vitro , coimmunoprecipitation was performed using a recombinant NS5B mutant purified from 10.2 with HEK293T endogenous PRK2 protein in order to identify a site within the NS5B protein that interacts with PRK2 protein. 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml strepto HEK293T cells were cultured in animal cell medium DMEM additionally added to mycin (streptomycin) and 10% fetal bovine serum (FBS), and the cultured cells were recovered by centrifugation (21,000 x g, 4 ° C, 20 minutes). Cells were washed three times with PBS solution, followed by additional cell disruption solution containing a protease inhibitor (EDTA free-protease inhibitor cocktail, Roche) [20 mM Tris-HCl [pH7.5], 1 mM EDTA, 1 mM DTT, 50 mM sodium fluoride (NaF), 1 mM sodium vanadate (Na ₃ VO ₄ ), 0.5% Triton X-100 and 100 mM sodium chloride (NaCl)], and the anti-PRK2 antibody, recombinant The PRK2-NS5B mutant complex was isolated by co-immunoprecipitation with the addition of the NS5B mutant and protein A-Sepharose. The isolated PRK2-NS5B mutant complex was added to a 6 × SDS solution, heat treated at 95 ° C., and the protein was separated on an 8% SDS-PAGE gel. The isolated protein was blotted onto a nitrocellulose membrane, and 1 hour in TBST solution [20 mM Tris-HCl, 150 mM sodium chloride and 0.1% Tween20] to which 5% BSA was added. After interfering with non-specific binding, anti-PRK2 antibody or anti-histidine antibody, respectively, was used as a primary antibody and diluted in a TBST solution in a ratio of 1: 1000 and reacted at room temperature for 1 hour. After completion of the reaction, the membrane was washed three times with TBST solution for 10 minutes. Anti-rabbit IgG or anti-rat IgG conjugated with HRP (horse radish peroxidase), respectively, was used as a secondary antibody, diluted in TBST solution at a ratio of 1: 5000, and reacted at room temperature for 1 hour. After completion of the reaction, the membranes were washed three times with TBST solution and the proteins were reactive with the antibody using ECL as substrate. As a result, as shown in FIG. 10B, the NS5B (amino acids 1-371) mutant directly interlinked with the PRK2 protein, while the NS5B (amino acids 372-591) mutant and NS5B (amino acids 188-591) mutant were PRK2 protein. It did not crosslink with. Thus, it can be seen that the site in the NS5B protein that binds to the PRK2 protein is amino acid (1-187), which is the site corresponding to the finger domain of the NS5B protein.

Example 11: Amino Acid Sites of NS5B Protein Phosphorylated by PRK2 Protein

To determine the NS5B protein region that is phosphorylated by protein in PRK2 In vitro, the cell was performed Endogenous PRK2 of recombinant NS5B protein and mutants and [γ- ³² P] in vitro kinase assay using the isotope. Incubating HEK293T cells in DMEM animal cell medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10% FBS Cells were collected by centrifugation (21,000 × g, 4 ° C., 20 minutes). After washing three times with PBS solution, a cell disruption solution [20 mM Tris-HCl [pH7.5], 1 mM EDTA, 1 mM DTT, 50 was added with a protease inhibitor [EDTA free-protease inhibitor cocktail, Roche]. mM sodium fluoride (NaF), 1 mM sodium vanadate (Na ₃ VO ₄ ), 0.5% Triton X-100 and 100 mM sodium chloride (NaCl)], and disrupted the cells, followed by anti-PRK2 antibody and protein A-. The PRK2 complex was isolated by immunoprecipitation with the addition of Sepharose. The PRK2 complex was twice with a cell disruption solution, 1 × phosphorylation solution (20 mM HEPES [pH7.5], 50 mM sodium chloride, 5 mM magnesium chloride (MgCl ₂ ), 5 mM manganese chloride (MnCl ₂ ), 1 mM DTT ) Was washed once, followed by an in vitro kinase reaction (30 ° C., 30 min) with addition of 1 μg NS5B mutant, 100 μM ATP and 10 μCi [γ- ³² P] isotope. The reaction product was added to a 6 × SDS solution, heat treated at 95 ° C., the protein was separated on an 8% SDS-PAGE gel, and the gel was dried on 3M paper to analyze the phosphorylation of the protein on a film. As a result, as shown in FIG. 11, the NS5B (amino acids 1-371) mutant was phosphorylated by PRK2 protein, but the NS5B (amino acids 372-591) mutant and NS5B (amino acids 188-591) mutant were By phosphorylation. These results suggest that the site in the NS5B protein phosphorylated by PRK2 protein is amino acid (1-187), which is the site corresponding to the finger domain of the NS5B protein.

Example 12: In vivo Phosphorylation of NS5B Protein by PRK2 Protein in

In order to analyze the phosphorylation of NS5B protein in vivo , immunoprecipitation was performed by metabolic cell labeling using the Huh7TR-NS cell line prepared in Example 7. Huh7TR- in DMEM (Phosphate-free DMEM) without animal cell media phosphate added with 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 10% dialyzed FBS NS cell lines were cultured and cells were labeled for 4 hours using 80 μCi of ortho [ ³² P] phosphate isotope. The cultured cells were washed three times with cold PBS solution, and then the cells were collected by centrifugation (21,000 × g, 4 ° C., 20 minutes). The harvested cells were lysed (100 mM Tris-HCl [pH 8.0], 10 mM sodium fluoride (NaF), 1 mM sodium oral) with the addition of an EDTA free-protease inhibitor cocktail (Roche). Sobadate, 10 mM tetrasodium pyruphosphate, 17.5 mM β-glycerophosphate, 1% Triton X-100, 150 mM sodium chloride), disrupted, anti-NS5B antibody and protein A-Sepharose NS5B complex was isolated by immunoprecipitation method. The NS5B complex was added to a 6 × SDS solution, heat treated at 95 ° C., the protein was separated on an 8% SDS-PAGE gel, and the gel was dried on 3M paper to analyze the phosphorylation of the protein on the film. As a result, as shown in FIG. 12, NS5B protein was phosphorylated in vivo .

Example 13: In vivo Phosphorylation of NS5B Protein by PRK2 Protein in Rats

13.1 Preparation of siRNA that Inhibits Expression of Intracellular PRK2 Protein

To prepare siRNAs that inhibit the expression of intracellular PRK2 protein, the nucleotide sequence (1,055-1,073) in the PRK2 protein (GenBank accession number NM_006256) was targeted. For the preparation of the target sequence, primers 5'-GATCCCCCATCAGTTGCACTGCCTGGTTCAAGAGACCAGGCAGTGCAACTGATGTTTTTGGAAA-3 '(SEQ ID NO .: 14) and 5'-AGCTTTTCCAAAAACATCAGTTGCACTGCCTGGTCTCTTGAACCAGGCAGTGCAACTGATGGGG-3' (SEQ ID NO: 9,2,3,5,3,3,3,3,3,3,3,3,3,3,3,3,3, etc.) It was cloned into pSUPER vector (Oligoengine) and named pSUPER-PRK2 as siRNA which suppresses expression of intracellular PRK2 protein. In order to confirm the expression inhibitory effect of the intracellular PRK2 protein by the obtained siRNA, pSUPER-PRK2 plasmid was transfected into the Huh7TR-NS cell line and immunobrotting was performed. Incubate the Huh7TR-NS cell line in animal cell medium DMEM with additional 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin and 10% FBS, and 6 μg pSUPER- PRK2 plasmids were transfected to inhibit intracellular PRK2 protein expression. The cultured cells were washed three times with cold PBS solution and then centrifuged (21,000 × g, 4 ° C., 20 minutes) to recover the cells. The recovered cells were added with a protease inhibitor cocktail (EDTA free-protease inhibitor cocktail, Roche) [100 mM Tris-HCl [pH 8.0], 10 mM sodium fluoride (NaF), 1 mM sodium olovana Date, 10 mM tetrasodium pyriphosphate, 17.5 mM β-glycerophosphate, 1% Triton X-100 and 150 mM sodium chloride], and the 6 × SDS solution was added to the crushed solution and heat-treated at 95 ° C. Afterwards, proteins were separated on 8% SDS-PAGE gels. The isolated protein was blotted onto the nitrocellulose membrane and non-specific binding was inhibited for 1 hour in a TBST solution [20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween20] to which 5% BSA was added. After interfering, the anti-PRK2 antibody or the anti-NS5B antibody, respectively, was used as the primary antibody and diluted in a TBST solution in a ratio of 1: 1000 and reacted at room temperature for 1 hour. After completion of the reaction, the membrane was washed three times with TBST solution for 10 minutes. Anti-rabbit IgG or anti-goat IgG bound to horse radish peroxidase (HRP), respectively, was used as a secondary antibody and diluted with TBST solution at a ratio of 1: 5000, and then reacted at room temperature for 1 hour. After completion of the reaction, the membranes were washed three times with TBST solution and the proteins were reactive with the antibody using ECL as substrate. As a result, as shown in FIG. 13A, intracellular PRK2 protein expression was significantly inhibited by siRNA, but no inhibition was observed in relation to NS5B protein and alpha-cubulin protein expression. This demonstrates that pSUPER-PRK2 specifically inhibits only PRK2 protein expression in cells.

13.2 Demonstration of Specificity of NS5B Protein Phosphorylation in Huh7TR-NS Cell Lines Transfected with siRNA Inhibiting Intracellular PRK2 Protein Expression

To demonstrate the specificity of NS5B protein phosphorylation by PRK2 protein in vivo , the Huh7TR-NS cell line prepared in Example 7 was transfected with the pSUPER-PRK2 plasmid prepared in Example and subjected to metabolic cell labeling. Coimmunoprecipitation was performed. Huh7TR-NS cell line in phosphate-free DMEM (Phosphate-free DMEM) with additional addition of 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin and 10% dialyzed FBS After incubation and transfection of the pSUPER-PRK2 plasmid to inhibit intracellular PRK2 protein expression, cells were labeled for 4 hours using 80 μCi of ortho [ ³² P] phosphate isotopes. The cultured cells were washed three times with cold PBS solution and then centrifuged (21,000 × g, 4 ° C., 20 minutes) to recover the cells. Cell disruption solution further added with a protease inhibitor cocktail (EDTA free-protease inhibitor cocktail, Roche), 100 mM Tris-HCl [pH 8.0], 10 mM sodium fluoride (NaF), 1 mM sodium orthovanadate, 10 mM tetrasodium pyruphosphate, 17.5 mM β-glycerophosphate, 1% Triton X-100 and 150 mM sodium chloride], and the cells were crushed and immunoprecipitated with anti-NS5B antibody and protein A-Sepharose. NS5B complex was separated. The isolated NS5B complex was added to a 6 × SDS solution, heat treated at 95 ° C., and the protein was separated on an 8% SDS-PAGE gel. Gels were dried on 3M paper to analyze the phosphorylation of proteins on film. As a result, as shown in FIG. 13B, phosphorylation of NS5B protein was induced in vivo. As such, the fact that the phosphorylation of NS5B protein is inhibited as the expression of PRK2 protein in cells is inhibited demonstrates that phosphorylation of NS5B protein in human cells is caused by PRK2 protein.

Example 14: In vivo Phosphorylation sites within NS5B protein

To confirm the phosphorylation site in the NS5B protein in vivo , immunoprecipitation using R-1 cell line was performed. 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10 The R-1 cell line was cultured in animal cell medium DMEM additionally added with% FBS, and the cells were recovered by centrifugation (21,000 × g, 4 ° C., 20 minutes). After washing three times with PBS solution, a cell disruption solution [20 mM HEPES [pH7.5], 1 mM EDTA, 10% glycerol, to which an additional protease inhibitor (EDTA free-protease inhibitor cocktail, Roche) was added, 100 mM sodium fluoride (NaF), 10 mM tetrasodium pyruphosphate, 17.5 mM β-glycerophosphate, 1% tritonX-100 and 150 mM sodium chloride] and disrupted the cells, respectively, and anti-p-serine Intracellular phosphorylated proteins were isolated by immunoprecipitation with the addition of antibodies, anti-p-threonine antibodies, anti-p-tyrosine antibodies and protein A-sepharose. Phosphorylated protein was added to 6 × SDS solution and heat treated at 95 ° C., and the protein was separated on 8% SDS-PAGE gel. The isolated protein was blotted onto the nitrocellulose membrane and non-specific binding was inhibited for 1 hour in a TBST solution [20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween20) to which 5% BSA was added. After interfering, using anti-p85 PI3K antibody (SantaCruz Biotechnology), anti-NS5B antibody or anti-Akt antibody (Cellsignaling) as primary antibody, diluted 1: 1000 ratio in TBST solution for 1 hour at room temperature, respectively. Reacted for a while. After completion of the reaction, the membrane was washed three times with TBST solution for 10 minutes. Next, anti-rabbit IgG, anti-goat IgG, and anti-rabbit IgG, each of which were bound with HRP (horse radish peroxidase), were used as secondary antibodies, diluted with TBST solution at a ratio of 1: 5000, and reacted at room temperature for 1 hour. I was. After completion of the reaction, the membranes were washed three times with TBST solution and the proteins were reactive with the antibody using ECL as substrate. As a result, as shown in Figure 14, NS5B protein was analyzed in the protein immunoprecipitated with anti-p-serine (serine). However, NS5B protein could not be detected in proteins immunoprecipitated with anti-p-Threonine and anti-p-Tyrosine. This demonstrates that the NS5B protein is phosphorylated at the serine site.

Example 15 Analysis of Regulation of NS5B Protein Phosphorylation by siRNA Inhibiting Intracellular PRK2 Protein Expression

In order to analyze whether NS5B protein phosphorylation is regulated by siRNA that inhibits PRK2 protein expression in vivo, immunoprecipitation using R-1 cell line was performed. 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10 The R-1 cell line was cultured in animal cell medium DMEM to which% FBS was additionally added, and 6 µg of pSUPER-PRK2 plasmid was transfected. After 48 hours, the cultured cells were centrifuged (21,000 × g, 4 ° C., 20 minutes) and the cells collected. After washing three times with PBS solution, a cell disruption solution added with a protease inhibitor (EDTA free-protease inhibitor cocktail, Roche) [20 mM HEPES [pH7.5], 1 mM EDTA, 10% glycerol, 100 mM sodium fluoride (NaF), 10 mM tetrasodium pyruphosphate, 17.5 mM β-glycerophosphate and 1% Triton X-100, 150 mM sodium chloride] to disrupt cells and anti-p-serine Intracellular phosphorylated protein was isolated by immunoprecipitation method with antibody and protein A-Sepharose. Phosphorylated protein was added to 6 × SDS solution and heat treated at 95 ° C., and the protein was separated on 8% SDS-PAGE gel. The isolated protein was blotted onto a Nitrocellulose membrane and non-specific for 1 hour in TBST solution [20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween20] with 5% BSA. After interfering with the specific binding, the anti-NS5B antibody was used as the primary antibody, diluted in the TBST solution at a ratio of 1: 1000, and reacted at room temperature for 1 hour. After completion of the reaction, the membrane was washed three times with TBST solution for 10 minutes. Next, HRP (horse radish peroxidase) conjugated anti-goat IgG was used as a secondary antibody and diluted in a TBST solution at a ratio of 1: 5000, and reacted at room temperature for 1 hour. After completion of the reaction, the membrane was washed three times with TBST solution and ECL was used as a substrate to analyze proteins reactive with the antibody. As a result, as shown in FIG. 15, NS5B protein was analyzed in the protein immunoprecipitated with anti-p-serine, and in particular, it was confirmed that NS5B protein phosphorylation was significantly suppressed in the cell line transfected with pSUPER-PRK2. This demonstrates that the expression of NS5B protein is inhibited following the inhibition of PRK2 protein expression.

Example 16: Analysis of HCV RNA Replication Regulation by siRNA Inhibiting Intracellular PRK2 Protein Expression

To analyze whether HCV RNA replication is regulated by siRNA that inhibits PRK2 protein expression in vivo, a real-time PCR (RPC) technique using an R-1 cell line was performed. 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10 The R-1 cell line was cultured in animal cell medium DMEM to which% FBS was additionally added, and 6 µg of pSUPER-PRK2 plasmid was transfected. After 48 hours, the cultured cells were centrifuged (21,000 × g, 4 ° C., 20 minutes) to recover the cells. After washing the cells three times with BS solution, RNA was extracted and purified using Trizol reagent (Invitrogen). After quantifying the extracted RNA, amplified using the Taqman EZ RT-PCR kit (Applied Biosystems), and 5'-UTR-F, 5'-GCGTCTAGCCATGGCCTTAGTATGAGTGTC-3 '(sense primer specific for HCV 5'-UTR) SEQ ID NO: 16) and antisense primers 5'-UTR-R, 5'-ACCACAAGGCCTTTCGCGACCCAACACTAC-3 '(SEQ ID NO: 17), and dual fluorophore-labeled probe, 5'-FAM Reverse transcription (2 minutes at 50 ° C., 30 minutes at 65 ° C.) was performed using (6-carboxy-fluorine) -CTGCGGAACCGGTGAGTACAC-TAMRA (6-carboxy-tetramethyl-rhodamine) -3 ′. RNA was then quantified with the ABI PRISM 7700 Sequence Search Kit by real-time PCR (20 seconds at 95 ° C., 1 minute at 62 ° C., 40 cycles). All experiments were performed three times independently (FIG. 16). As a result, the amount of HCV RNA in the cell line was significantly suppressed by increasing the transfection concentration of pSUPER-PRK2. This demonstrates that HCV RNA replication is inhibited by inhibition of PRK2 protein expression. 16B is a result of Western blot analysis that the pSUPER-PRK2 plasmid was transfected into the R-1 cell line to inhibit the expression of the PRK2 protein and the phosphorylated PRK2 protein in the cell.

Example 17 Analysis of Regulation of NS5B Protein Phosphorylation by Intracellular PRK2 Protein Overexpression

In order to analyze whether NS5B protein phosphorylation is regulated by overexpression of PRK2 protein in vivo, immunoprecipitation using R-1 cell line was performed. 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10 The R-1 cell line was cultured in animal cell medium DMEM to which% FBS was added, and 6 µg of pcDNA3.1-PRK2 plasmid was transfected. After 48 hours, the cultured cells were centrifuged (21,000 × g, 4 ° C., 20 minutes) to recover the cells. After washing three times with PBS solution, a cell disruption solution [20 mM HEPES [pH7.5], 1 mM EDTA, 10% glycerol, to which an additional protease inhibitor (EDTA free-protease inhibitor cocktail, Roche) was added, 100 mM sodium fluoride (NaF), 10 mM tetrasodium pyruphosphate, 17.5 mM β-glycerophosphate and 1% Triton X-100, 150 mM sodium chloride] to disrupt cells and anti-p-serine antibody Intracellular phosphorylated protein was isolated by immunoprecipitation method with and protein A-Sepharose. Phosphorylated protein was then added to 6 × SDS solution and heat treated at 95 ° C., and the protein was separated on 8% SDS-PAGE gel. The isolated protein was blotted onto the nitrocellulose membrane and blocked for non-specific binding for 1 hour in TBST solution [20 mM Tris-HCl, 150 mM sodium chloride, 0.1% Tween20] with 5% BSA added. Thereafter, the anti-NS5B antibody was used as the primary antibody, diluted in a TBST solution in a 1: 1000 ratio, and reacted at room temperature for 1 hour. After completion of the reaction, the membrane was washed three times with TBST solution for 10 minutes. Next, HRP-bound anti-goat IgG was used as a secondary antibody, diluted in a TBST solution at a ratio of 1: 5000, and reacted at room temperature for 1 hour. After completion of the reaction, the membranes were washed three times with TBST solution and the proteins were reactive with the antibody using ECL as substrate. As a result, as shown in Figure 17, NS5B protein was analyzed in the protein immunoprecipitated with anti-p-serine, especially in the cell line transfected with pcDNA3.1-PRK2 significantly increased the phosphorylation of NS5B protein. This demonstrates that phosphorylation of NS5B protein is increased concurrently with overexpression of PRK2 protein.

Example 18 Analysis of HCV RNA Replication Regulation by Overexpression of Intracellular PRK2 Protein

To analyze HCV RNA replication regulation by overexpression of PRK2 protein in vivo, real-time PCR was performed using R-1 cell line. 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10 The R-1 cell line was cultured in animal cell medium DMEM to which% FBS was added, and 6 µg of pcDNA3.1-PRK2 plasmid was transfected. After 48 hours, the cultured cells were centrifuged (21,000 × g, 4 ° C., 20 minutes) to collect the cells. After washing three times with PBS solution, RNA was extracted and purified using Trizol (Trizol reagent, Invitrogen). After quantifying the extracted RNA, amplified using the Taqman EZ RT-PCR kit (Applied Biosystems), and 5'-UTR-F, 5'-GCGTCTAGCCATGGCCTTAGTATGAGTGTC-3 '(sense primer specific for HCV 5'-UTR) SEQ ID NO: 16) and antisense primers 5'-UTR-R, 5'-ACCACAAGGCCTTTCGCGACCCAACACTAC-3 '(SEQ ID NO: 17) and dual fluorophore-labeled probe, 5'-FAM ( Reverse transcription was performed using 6-carboxy-fluorine) -CTGCGGAACCGGTGAGTACAC-TAMRA (6-carboxy-tetramethyl-rhodamine) -3 '(Reverse transcription, 2 minutes at 50 ° C, 30 minutes at 65 ° C). RNA was then quantified with the ABI PRISM 7700 sequencing kit using the Real-Time-PCR (20 sec at 95 ° C., 1 min at 62 ° C., 40 cycles) technique. All experiments were performed three times independently. 18A shows the results of HCV RNA quantitation by real-time PCR. As the transfection concentration of pcDNA3.1-PRK2 was increased, the amount of HCV RNA in the cell line increased significantly. This demonstrates that HCV RNA replication is increased by overexpression of PRK2 protein. FIG. 18B is a result of confirming that the pcDNA3.1-PRK2 plasmid was transfected into the R-1 cell line to increase the expression of the PRK2 protein and the phosphorylated PRK2 protein in the cells.

Example 19 Analysis of HCV RNA Replication Inhibition Effect by HA1077 that Inhibit Intracellular PRK2 Protein Activity

To analyze whether HCV RNA replication is regulated by HA1077 (Calbiochem), which specifically inhibits the activity of PRK2 protein in vivo, real-time reverse transcription PCR (Real- time RT-PCR) was used to quantify HCV gene copy number. 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acid, 100 U / ml penicillin, 100 μg / ml streptomycin and 10 R-1 cell lines were cultured in animal cell medium DMEM added with% FBS and treated with HA1077 (0, 10, 20 and 50 μM, respectively) that specifically inhibited the activity of PRK2 protein. After 48 hours, the cultured cells were collected by centrifugation (21,000 × g, 4 ° C., 20 minutes). After washing three times with PBS solution, RNA was extracted and purified using Trizol (Trizol reagent, Invitrogen). After quantifying the extracted RNA, amplified using Taqman EZ RT-PCR kit (Applied Biosystems), 5'-UTR-F, 5'-UTR-F, 5'-GCGTCTAGCCATGGCCTTAGTATGAGTGTC-3 ', which is a specific sense primer for HCV 5'-UTR. (SEQ ID NO: 16) and antisense primers 5'-UTR-R, 5'-ACCACAAGGCCTTTCGCGACCCAACACTAC-3 '(SEQ ID NO: 17), and dual fluorophore-labeled probe, 5'- Reverse transcription was performed using FAM (6-carboxy-fluorine) -CTGCGGAACCGGTGAGTACAC-TAMRA (6-carboxy-tetramethyl-rhodamine) -3 '(Reverse transcription, 2 minutes at 50 ° C, 30 minutes at 65 ° C). . The HCV gene copy number was then analyzed using an ABI PRISM 7700 sequence detection instrument using real-time PCR (20 seconds at 95 ° C., 1 minute at 62 ° C., 40 cycles). All experiments were performed three times independently. As a result, as shown in FIG. 19, HCV RNA was quantitatively analyzed by real-time PCR, and it was confirmed that the amount of HCV RNA in the R-1 cell line was significantly suppressed as the concentration of HA1077 was gradually increased. This demonstrates that HCV RNA replication is inhibited by inhibiting the activity of PRK2 protein.

Example 20 Regulation of Expression of PRK2 Protein Using Fusion Molecules of siRNA and Protein Transduction Domain (PTD)

First, a fusion molecule of siRNA (pSUPER-PRK2) prepared in Example 13 and Mph1 (available from Forhumantech, WO03 / 059941A1) was prepared according to the method described in WO03 / 059941A1 of Forhumantech. The resulting fusion molecules were delivered to the HuH7TR-NS cell line of Example 7 and the expression of PRK2 protein was observed in the same manner as described in Example 13. As a result, it was confirmed that expression of PRK2 protein was significantly suppressed by the introduction of the fusion (the result is not shown).

As such, the present invention includes an anti-HCV viral agent that regulates HCV RNA replication by specifically regulating the function of HCV RNA polymerase using peptides and intracellular proteins identified in the present invention, for example, PRK2 protein. It can be useful for the development of HCV inhibitors.

<110> OH, JONG-WON <120> Peptide for regulating the function of HCV NS5B protein and the          use <130> IPM-26205 <160> 17 <170> KopatentIn 1.71 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> P1 Peptide <400> 1 Thr Ser Thr Ala Gly Arg Ile Val Arg Arg Ala   1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> P2 Peptide <400> 2 Cys Gln Leu Leu Val Arg Trp Cys Gly Ile Gly Cys   1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> P3 Peptide <400> 3 Asp Glu Leu Glu Ala Ser Leu Phe Gly Phe Ala Gly   1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> P4 Peptide <400> 4 Ile Arg Trp Ala Asp Phe Arg Ile Cys Val Gly Gln   1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> P5 Peptide <400> 5 Gln Arg Cys Gly Pro Leu Gln Val Val Ala Tyr Ser   1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> P6 Peptide <400> 6 Ala Ala Phe Gln Gln Arg Gln Gly Leu Cys Gly Leu   1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> P7 Peptide <400> 7 Leu Leu Ser Arg Gln Gln Leu Trp Gln Val Pro Val   1 5 10 <210> 8 <211> 12 <212> PRT <213> Artificial Sequence <220> P223 Peptide <400> 8 Gln Asp Phe Phe Gln Phe Ser Ser Leu Ile Val Thr   1 5 10 <210> 9 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide encoding P1 Peptide <400> 9 acttcgacgg cgggtcggat tgttcgtcgt gctatt 36 <210> 10 <211> 984 <212> PRT <213> Homo sapiens <400> 10 Met Ala Ser Asn Pro Glu Arg Gly Glu Ile Leu Leu Thr Glu Leu Gln   1 5 10 15 Gly Asp Ser Arg Ser Leu Pro Phe Ser Glu Asn Val Ser Ala Val Gln              20 25 30 Lys Leu Asp Phe Ser Asp Thr Met Val Gln Gln Lys Leu Asp Asp Ile          35 40 45 Lys Asp Arg Ile Lys Arg Glu Ile Arg Lys Glu Leu Lys Ile Lys Glu      50 55 60 Gly Ala Glu Asn Leu Arg Lys Val Thr Thr Asp Lys Lys Ser Leu Ala  65 70 75 80 Tyr Val Asp Asn Ile Leu Lys Lys Ser Asn Lys Lys Leu Glu Glu Leu                  85 90 95 His His Lys Leu Gln Glu Leu Asn Ala His Ile Val Val Ser Asp Pro             100 105 110 Glu Asp Ile Thr Asp Cys Pro Arg Thr Pro Asp Thr Pro Asn Asn Asp         115 120 125 Pro Arg Cys Ser Thr Ser Asn Asn Arg Leu Lys Ala Leu Gln Lys Gln     130 135 140 Leu Asp Ile Glu Leu Lys Val Lys Gln Gly Ala Glu Asn Met Ile Gln 145 150 155 160 Met Tyr Ser Asn Gly Ser Ser Lys Asp Arg Lys Leu His Gly Thr Ala                 165 170 175 Gln Gln Leu Leu Gln Asp Ser Lys Thr Lys Ile Glu Val Ile Arg Met             180 185 190 Gln Ile Leu Gln Ala Val Gln Thr Asn Glu Leu Ala Phe Asp Asn Ala         195 200 205 Lys Pro Val Ile Ser Pro Leu Glu Leu Arg Met Glu Glu Leu Arg His     210 215 220 His Phe Arg Ile Glu Phe Ala Val Ala Glu Gly Ala Lys Asn Val Met 225 230 235 240 Lys Leu Leu Gly Ser Gly Lys Val Thr Asp Arg Lys Ala Leu Ser Glu                 245 250 255 Ala Gln Ala Arg Phe Asn Glu Ser Ser Gln Lys Leu Asp Leu Leu Lys             260 265 270 Tyr Ser Leu Glu Gln Arg Leu Asn Glu Val Pro Lys Asn His Pro Lys         275 280 285 Ser Arg Ile Ile Ile Glu Glu Leu Ser Leu Val Ala Ala Ser Pro Thr     290 295 300 Leu Ser Pro Arg Gln Ser Met Ile Ser Thr Gln Asn Gln Tyr Ser Thr 305 310 315 320 Leu Ser Lys Pro Ala Ala Leu Thr Gly Thr Leu Glu Val Arg Leu Met                 325 330 335 Gly Cys Gln Asp Ile Leu Glu Asn Val Pro Gly Arg Ser Lys Ala Thr             340 345 350 Ser Val Ala Leu Pro Gly Trp Ser Pro Ser Glu Thr Arg Ser Ser Phe         355 360 365 Met Ser Arg Thr Ser Lys Ser Lys Ser Gly Ser Ser Arg Asn Leu Leu     370 375 380 Lys Thr Asp Asp Leu Ser Asn Asp Val Cys Ala Val Leu Lys Leu Asp 385 390 395 400 Asn Thr Val Val Gly Gln Thr Ser Trp Lys Pro Ile Ser Asn Gln Ser                 405 410 415 Trp Asp Gln Lys Phe Thr Leu Glu Leu Asp Arg Ser Arg Glu Leu Glu             420 425 430 Ile Ser Val Tyr Trp Arg Asp Trp Arg Ser Leu Cys Ala Val Lys Phe         435 440 445 Leu Arg Leu Glu Asp Phe Leu Asp Asn Gln Arg His Gly Met Cys Leu     450 455 460 Tyr Leu Glu Pro Gln Gly Thr Leu Phe Ala Glu Val Thr Phe Phe Asn 465 470 475 480 Pro Val Ile Glu Arg Arg Pro Lys Leu Gln Arg Gln Lys Lys Ile Phe                 485 490 495 Ser Lys Gln Gln Gly Lys Thr Phe Leu Arg Ala Pro Gln Met Asn Ile             500 505 510 Asn Ile Ala Thr Trp Gly Arg Leu Val Arg Arg Ala Ile Pro Thr Val         515 520 525 Asn His Ser Gly Thr Phe Ser Pro Gln Ala Pro Val Pro Thr Thr Val     530 535 540 Pro Val Val Asp Val Arg Ile Pro Gln Leu Ala Pro Pro Ala Ser Asp 545 550 555 560 Ser Thr Val Thr Lys Leu Asp Phe Asp Leu Glu Pro Glu Pro Pro Pro                 565 570 575 Ala Pro Pro Arg Ala Ser Ser Leu Gly Glu Ile Asp Glu Ser Ser Glu             580 585 590 Leu Arg Val Leu Asp Ile Pro Gly Gln Asp Ser Glu Thr Val Phe Asp         595 600 605 Ile Gln Asn Asp Arg Asn Ser Ile Leu Pro Lys Ser Gln Ser Glu Tyr     610 615 620 Lys Pro Asp Thr Pro Gln Ser Gly Leu Glu Tyr Ser Gly Ile Gln Glu 625 630 635 640 Leu Glu Asp Arg Arg Ser Gln Gln Arg Phe Gln Phe Asn Leu Gln Asp                 645 650 655 Phe Arg Cys Cys Ala Val Leu Gly Arg Gly His Phe Gly Lys Val Leu             660 665 670 Leu Ala Glu Tyr Lys Asn Thr Asn Glu Met Phe Ala Ile Lys Ala Leu         675 680 685 Lys Lys Gly Asp Ile Val Ala Arg Asp Glu Val Asp Ser Leu Met Cys     690 695 700 Glu Lys Arg Ile Phe Glu Thr Val Asn Ser Val Arg His Pro Phe Leu 705 710 715 720 Val Asn Leu Phe Ala Cys Phe Gln Thr Lys Glu His Val Cys Phe Val                 725 730 735 Met Glu Tyr Ala Ala Gly Gly Asp Leu Met Met His Ile His Thr Asp             740 745 750 Val Phe Ser Glu Pro Arg Ala Val Phe Tyr Ala Ala Cys Val Val Leu         755 760 765 Gly Leu Gln Tyr Leu His Glu His Lys Ile Val Tyr Arg Asp Leu Lys     770 775 780 Leu Asp Asn Leu Leu Leu Asp Thr Glu Gly Phe Val Lys Ile Ala Asp 785 790 795 800 Phe Gly Leu Cys Lys Glu Gly Met Gly Tyr Gly Asp Arg Thr Ser Thr                 805 810 815 Phe Cys Gly Thr Pro Glu Phe Leu Ala Pro Glu Val Leu Thr Glu Thr             820 825 830 Ser Tyr Thr Arg Ala Val Asp Trp Trp Gly Leu Gly Val Leu Ile Tyr         835 840 845 Glu Met Leu Val Gly Glu Ser Pro Phe Pro Gly Asp Asp Glu Glu Glu     850 855 860 Val Phe Asp Ser Ile Val Asn Asp Glu Val Arg Tyr Pro Arg Phe Leu 865 870 875 880 Ser Thr Glu Ala Ile Ser Ile Met Arg Arg Leu Leu Arg Arg Asn Pro                 885 890 895 Glu Arg Arg Leu Gly Ala Ser Glu Lys Asp Ala Glu Asp Val Lys Lys             900 905 910 His Pro Phe Phe Arg Leu Ile Asp Trp Ser Ala Leu Met Asp Lys Lys         915 920 925 Val Lys Pro Pro Phe Ile Pro Thr Ile Arg Gly Arg Glu Asp Val Ser     930 935 940 Asn Phe Asp Asp Glu Phe Thr Ser Glu Ala Pro Ile Leu Thr Pro Pro 945 950 955 960 Arg Glu Pro Arg Ile Leu Ser Glu Glu Glu Gln Glu Met Phe Arg Asp                 965 970 975 Phe Asp Tyr Ile Ala Asp Trp Cys             980 <210> 11 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> HCV-NS3F Primer <400> 11 ataagaatgc ggccgcacca tggcgcccat cacggcctac tcc 43 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> NS5B-BIII Primer <400> 12 gctctagatc ggttggggag caggtaaatg 30 <210> 13 <211> 591 <212> PRT <213> Hepatitis C virus <400> 13 Ser Met Ser Tyr Thr Trp Thr Gly Ala Leu Ile Thr Pro Cys Ala Ala   1 5 10 15 Glu Glu Ser Lys Leu Pro Ile Asn Pro Leu Ser Asn Ser Leu Leu Arg              20 25 30 His His Asn Met Val Tyr Ala Thr Thr Ser Arg Ser Ala Ser Leu Arg          35 40 45 Gln Lys Lys Val Thr Phe Asp Arg Leu Gln Val Leu Asp Asp His Tyr      50 55 60 Arg Asp Val Leu Lys Glu Met Lys Ala Lys Ala Ser Thr Val Lys Ala  65 70 75 80 Lys Leu Leu Ser Ile Glu Glu Ala Cys Lys Leu Thr Pro Pro His Ser                  85 90 95 Ala Lys Ser Lys Phe Gly Tyr Gly Ala Lys Asp Val Arg Asn Leu Ser             100 105 110 Ser Arg Ala Val Asn His Ile Arg Ser Val Trp Glu Asp Leu Leu Glu         115 120 125 Asp Thr Glu Thr Pro Ile Asp Thr Thr Ile Met Ala Lys Ser Glu Val     130 135 140 Phe Cys Val Gln Pro Glu Lys Gly Gly Arg Lys Pro Ala Arg Leu Ile 145 150 155 160 Val Phe Pro Asp Leu Gly Val Arg Val Cys Glu Lys Met Ala Leu Tyr                 165 170 175 Asp Val Val Ser Thr Leu Pro Gln Ala Val Met Gly Ser Ser Tyr Gly             180 185 190 Phe Gln Tyr Ser Pro Lys Gln Arg Val Glu Phe Leu Val Asn Thr Trp         195 200 205 Lys Ser Lys Lys Cys Pro Met Gly Phe Ser Tyr Asp Thr Arg Cys Phe     210 215 220 Asp Ser Thr Val Thr Glu Ser Asp Ile Arg Val Glu Glu Ser Ile Tyr 225 230 235 240 Gln Cys Cys Asp Leu Ala Pro Glu Ala Arg Gln Ala Ile Arg Ser Leu                 245 250 255 Thr Glu Arg Leu Tyr Ile Gly Gly Pro Leu Thr Asn Ser Lys Gly Gln             260 265 270 Asn Cys Gly Tyr Arg Arg Cys Arg Ala Ser Gly Val Leu Thr Thr Ser         275 280 285 Cys Gly Asn Thr Leu Thr Cys Tyr Leu Lys Ala Thr Ala Ala Cys Arg     290 295 300 Ala Ala Lys Leu Gln Asp Cys Thr Met Leu Val Asn Gly Asp Asp Leu 305 310 315 320 Val Val Ile Cys Glu Ser Ala Gly Thr Gln Glu Asp Ala Ala Ala Leu                 325 330 335 Arg Ala Phe Thr Glu Ala Met Thr Arg Tyr Ser Ala Pro Pro Gly Asp             340 345 350 Pro Pro Gln Pro Glu Tyr Asp Leu Glu Leu Ile Thr Ser Cys Ser Ser         355 360 365 Asn Val Ser Val Ala His Asp Ala Ser Gly Lys Arg Val Tyr Tyr Leu     370 375 380 Thr Arg Asp Pro Thr Thr Pro Leu Ala Arg Ala Ala Trp Glu Thr Ala 385 390 395 400 Arg His Thr Pro Ile Asn Ser Trp Leu Gly Asn Ile Ile Met Tyr Ala                 405 410 415 Pro Thr Leu Trp Ala Arg Met Ile Leu Met Thr His Phe Phe Ser Ile             420 425 430 Leu Leu Ala Gln Glu Gln Leu Glu Lys Ala Leu Asp Cys Gln Ile Tyr         435 440 445 Gly Ala Cys Tyr Ser Ile Glu Pro Leu Asp Leu Pro Gln Ile Ile Glu     450 455 460 Arg Leu His Gly Leu Ser Ala Phe Thr Leu His Ser Tyr Ser Pro Gly 465 470 475 480 Glu Ile Asn Arg Val Ala Ser Cys Leu Arg Lys Leu Gly Val Pro Pro                 485 490 495 Leu Arg Thr Trp Arg His Arg Ala Arg Ser Val Arg Ala Lys Leu Leu             500 505 510 Ser Gln Gly Gly Arg Ala Ala Thr Cys Gly Arg Tyr Leu Phe Asn Trp         515 520 525 Ala Val Arg Thr Lys Leu Lys Leu Thr Pro Ile Pro Ala Ala Ser Gln     530 535 540 Leu Asp Leu Ser Gly Trp Phe Val Ala Gly Tyr Ser Gly Gly Asp Ile 545 550 555 560 Tyr His Ser Leu Ser Arg Ala Arg Pro Arg Trp Phe Pro Leu Cys Leu                 565 570 575 Leu Leu Leu Ser Val Gly Val Gly Ile Tyr Leu Leu Pro Asn Arg             580 585 590 <210> 14 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Primer for siRNA <400> 14 gatcccccat cagttgcact gcctggttca agagaccagg cagtgcaact gatgtttttg 60 gaaa 64 <210> 15 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Primer for siRNA <400> 15 agcttttcca aaaacatcag ttgcactgcc tggtctcttg aaccaggcag tgcaactgat 60 gggg 64 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Sense Primer 5'-UTR-F for HCV 5'-UTR <400> 16 gcgtctagcc atggccttag tatgagtgtc 30 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Antisense Primer 5'-UTR-R for HCV 5'-UTR <400> 17 accacaaggc ctttcgcgac ccaacactac 30  

Claims (12)

Peptides P1 (SEQ ID NO: 1), P2 (SEQ ID NO: 2), P3 (SEQ ID NO: 3), P4 (SEQ ID NO: 4), P5 (SEQ ID NO: 5), P6 (SEQ ID NO: 6), P7 Any one peptide selected from the group consisting of (SEQ ID NO: 7) and P8 (SEQ ID NO: 8), wherein the peptide specifically binds to the HCV NS5B protein, a fragment thereof, or a derivative thereof. The peptide, fragment thereof, or derivative thereof according to claim 1, which is a peptide P1 having the amino acid sequence of SEQ ID NO: 1. The nucleotide sequence of SEQ ID NO: 9 encoding the peptide P1 of claim 2, a fragment or derivative thereof. A protein comprising the amino acid sequence of SEQ ID NO: 1 (SEQ ID NO: 1), a fragment thereof or a derivative thereof. The protein of claim 4, wherein the protein is a PRK2 protein of SEQ ID NO: 10. 6. HCV NS5B protein finger domain comprising 1 to 187 portions of amino acids of SEQ ID NO: 13 which specifically binds to the PRK2 protein of claim 5. PRK2 protein that binds to the finger domain of HCV NS5B protein and regulates phosphorylation of HCV NS5B protein. A method of modulating HCV replication by inhibiting expression of PRK2 protein. SiRNA to inhibit expression of PRK2 protein. The siRNA according to claim 9, which is pSUPER-PRK2. A fusion protein of siRNA and PTD (protein transduction domain) of claim 9 or 10 for inhibiting HCV replication. Peptide having at least 80% homology with amino acid sequence 518 to 525 region of SEQ ID NO: PRK2 protein of claim 5.

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