JP2003135078A - Variant of hepatitis c virus ns5a protein and utilization thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a NS5A protein variant in which an ability to inhibit enzymatic activity of double stranded RNA-dependant protein phosphorylase is enhanced and to provide a method for evaluating an anti-HCV agent candidate compound by using the variant and a method for screening the candidate compound. SOLUTION: A specific variant of NS5A protein remarkably suppresses enzymatic activity of the double stranded RNA-dependant protein phosphorylase, compared with a wild type NS5A protein. The method for evaluating a highly sensitive anti-HCV agent candidate compound and the method for screening the candidate compound by using inhibition of enzymatic activity suppression of the RNA dependant protein phosphorylase by the variant as an index are built based on the knowledge. It is expected that these methods are largely useful for identification of the anti-HCV agent candidate compound and the identified compound can become effective anti-HCV agent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a variant of hepatitis C virus NS5A protein having enhanced ability to suppress enzyme activity of double-stranded RNA-dependent protein kinase, and uses thereof.

[0002]

Background Art [0002] Interferon (IFN) is currently known as the only anti-HCV agent showing a significant effect on hepatitis C virus (HCV), but its efficiency is about 30%. Even when used in combination with other adjuvants, it is around 50-60%,
Many patients do not see any effect. In addition, its strong side effects are also a problem. Therefore, the development of anti-HCV agents that replace IFN and the development of adjuvants that enhance the effects of IFN are expected.

In vivo, a defense mechanism against HCV is known. This is a double-stranded RNA-dependent protein kinase (PK) known to be induced by IFN.
It is a mechanism via R). The PKR protein is one of the cAMP-independent serine / threonine phosphatases, and its activation has been shown to require dimerization of the PKR protein itself and binding of dsRNA (double-stranded RNA) ( Kaufma
n, RJ Double-stranded RNA-activated proteinkina
se PKR In "Translational Control of Gene Expressio
n "(N. Sonenberg, JWB Hershey, and MB Mathe
ws, Eds.) pp. 503-528, 2000. Cold Spring Harbor La
boratory Press, Cold Spring Harbor, NY).

Cell death due to inactivation of the translation initiation factor eIF2α protein has been proposed as one of the defense mechanisms against HCV mediated by the PKR protein. According to this, the PKR protein inactivates the eIF2α protein by phosphorylating the eIF2α protein, induces apoptosis of infected cells, and kills the virus along with cell death (JP 2000-135100 A).

Further, as another defense mechanism, the transcription factor NF
Induction of IFN through the κB protein is known. NFκ
The B protein exists in the cytoplasm of the host cell as a complex with the IκBα protein, but the IκBα protein (phosphorylated form) is dissociated from the NFκB protein by the phosphorylation of the IκBα protein by the PKR protein.
The single NFκB protein translocates into the nucleus and induces IFN (Kumar, AJet. Al., Double-stranded RNA-de
pendent protein kinase activates transcription fac
tor NF-κB by phosphorylating IκB. Proc. Natl. Ac
ad. Sci 91: 6288, 1994, Daryoush, MZ et. al., N
F-κB activation by double-stranded RNA activated p
rotein kinase (PKR) is mediated through NF-κB ind
ucingkinase and IκB kinase. Mol. Cell. Biol. 20:
1278, 1999, Yang, Y. et. Al., Deficient signaling
in mice devoid of double-stranded RNA-depindent pr
otein kinase.EMBO J. 14: 6095, 1995, Kumar, A. e
t. al., Deficient cytokine signaling in mouse embr
yo fibroblasts with a targeted deletion in the PKR
gene: role of IRF-1 and NF-κB. EMBO J. 16: 406,
1997, Der, SDet.al., A double-stranded RNA-act
ivated protein kinase-dependent pathway medating s
tress induced apoptosis. Proc. Natl. Acad. USA 94:
3279, 1997).

The NS5A protein is one of the non-structural proteins (Non-Structural Proteins) encoded by the HCV chromosomal RNA, and is known to be a polyphosphorylated form.
Although its function is hard to say,
ISDR (Interferon Sensitivity Det) in NS5A protein
ermining Region; interferon sensitivity determination region)
It has been reported that the relationship between the amino acid region called `` HBV '' and the susceptibility of HCV to IFN (Enomoto, N. et. Al., Mutations in the no
nstructural protein 5A gene and responseto interfe
ron in patients with chronic hepatitis C virus 1b
infection. N. Engl. J. Med. 34: 77-81, 1996). In particular, among the HCVs, the major HCV type 1b is considered to have a low IFN remarkably efficient efficiency, and the above report received attention.

Recently, it has been shown that the NS5A protein binds to the PKR protein to reduce the phosphorylation activity of the PKR protein (Katze GM Gale JM e
t. al., Virology 230: 217-227, 1997). From this, it is considered that the NS5A protein reduces the phosphorylation activity of the PKR protein, thereby interfering with the anti-HCV mechanism mediated by the PKR protein in the living body and further with the anti-HCV action of exogenous IFN.

[0008] To date, using the above findings, a compound that inhibits the binding between the PKR protein and the NS5A protein or inhibits the NS5A protein from suppressing the phosphorylation activity of the PKR protein is screened as a candidate compound for an anti-HCV agent. A patent has been filed for this method (WO 98 /
39487). However, depending on the assay system used, the degree of inhibition of the enzymatic activity of the PKR protein by the NS5A protein is not sufficient to detect the inhibition of the inhibition of the enzymatic activity of the test compound with high sensitivity, and therefore a large number of compounds There is a problem that it is unsuitable for screening.

[0009]

The present invention has been made in view of such circumstances, and an object thereof is to provide a highly sensitive method for evaluating an anti-HCV drug candidate compound and a screening method. . More specifically, the present invention provides
N with enhanced ability to suppress enzyme activity of PKR protein
Mutant of S5A protein and anti-HCV using the mutant
It is an object to provide an evaluation method and a screening method for drug candidate compounds.

[0010]

[Means for Solving the Problems] As a preliminary finding, the present inventors newly identified that the NS5A protein serves as a substrate for the PKR protein. In addition, the NS5A protein
C-terminal deletion contributes to binding to PKR protein (48th
The Annual Meeting of the Japanese Society for Virology). As a result of examining the effect of mutation of the phosphorylation site on the enzymatic activity of PKR protein, the present inventors have surprisingly found that NS5A
It was revealed that the phosphorylation site of the protein, especially the mutant at the C-terminal site, remarkably suppressed the enzymatic activity of the PKR protein as compared with the wild type. Based on this finding, the inventors of the present invention used a mutant of the phosphorylation site of NS5A protein for PK.
Using the inhibition of enzyme activity inhibition of R protein as an index, we have constructed an evaluation method and a screening method for a highly sensitive anti-HCV drug candidate compound that has never existed before. The method is very useful for identifying anti-HCV agent candidate compounds, and it is expected that the identified compounds can be effective anti-HCV agents.

That is, the present invention relates to a mutant of NS5A protein having an enhanced ability to suppress the enzymatic activity of PKR protein and the use thereof, and more specifically provides the following (1) to (34): To do. (1) A DNA encoding a variant of the hepatitis C virus NS5A protein, the variant having an increased ability to suppress the enzyme activity of the double-stranded RNA-dependent protein phosphorylating enzyme due to the mutation. (2) The DNA according to (1), wherein the mutation is a phosphorylation site mutation. (3) The mutation exists on the C-terminal side of the protein, (1)
DNA described in. (4) The DNA according to (1), wherein the mutant consists of the amino acid sequence shown in SEQ ID NO: 2 containing an amino acid sequence containing at least one mutation at the serine residues at positions 2404 and 2406. (5) The DNA according to (4), wherein the mutation in the serine residue is a substitution with an alanine residue. (6) The DNA according to (1), wherein the mutant comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 or 4. (7) A vector having the DNA according to any one of (1) to (6) inserted therein. (8) A transformant carrying the DNA according to any one of (1) to (6) or the vector according to (7). (9) The transformant according to (8), which is capable of inducibly expressing the DNA according to any one of (1) to (6). (10) Double-stranded RNA-dependent protein kinase, NF
expresses κB protein and IκBα protein,
The transformant according to (8) or (9). (11) The transformant according to (10), wherein a DNA functionally linked with a reporter gene is introduced downstream of the NFκB protein binding region. (12) The transformant according to (8) or (9), which expresses IRF-1 protein. (13) The transformant according to (12), wherein a DNA functionally linked to a reporter gene is introduced downstream of the IRF-1 protein binding sequence. (14) The following (i), which is a transformant expressing an IRF-1 protein, in which a DNA functionally linked with a reporter gene is introduced downstream of the IRF-1 protein-binding sequence:
Alternatively, the transformant according to (ii). (I) DNA encoding the hepatitis C virus NS5A protein
Alternatively, a transformant carrying a vector into which the DNA has been inserted. (Ii) D encoding the hepatitis C virus NS5A protein
The transformant according to (i), which can express NA inducibly. (15) The transformant according to (8) or (9), which expresses the ISGF3 protein. (16) The transformant according to (15), wherein a DNA functionally linked to a reporter gene is introduced downstream of ISRE. (17) A transformant expressing the ISGF3 protein, which has a reporter gene functionally linked to the downstream of ISRE
The transformant according to (i) or (ii) below, into which DNA has been introduced. (I) DNA encoding the hepatitis C virus NS5A protein
Alternatively, a transformant carrying a vector into which the DNA has been inserted. (Ii) D encoding the hepatitis C virus NS5A protein
The transformant according to (i), which can express NA inducibly. (18) A variant of hepatitis C virus NS5A protein encoded by the DNA according to any one of (1) to (6). (19) A step of recovering the expressed protein from the transformant or the culture supernatant thereof according to (8),
The method for producing the mutant according to (18). (20) A method for evaluating the anti-hepatitis C virus activity of a test sample, which comprises (a) the mutant according to (18) and the double-stranded RNA-dependent protein phosphorus in the presence of the test sample. And a step of detecting the binding between the NS5A protein variant and the double-stranded RNA-dependent protein phosphorylating enzyme, wherein the test sample contains the NS5A protein variant and the NS5A protein variant. A method in which the test sample is evaluated as having anti-hepatitis C virus activity when the binding to double-stranded RNA-dependent protein kinase is inhibited. (21) A method for evaluating the anti-hepatitis C virus activity of a test sample, which comprises (a) the mutant according to (18) and double-stranded RNA-dependent protein phosphorylation in the presence of the test sample. A test sample, which comprises a step of contacting with an enzyme, and (b) a step of detecting the enzyme activity of the double-stranded RNA-dependent protein kinase If the test sample is
A method evaluated to have hepatitis C virus activity. (22) A method for evaluating the anti-hepatitis C virus activity of a test sample, which comprises (a) a step of providing the transformant according to (11), (b) applying the test sample to the transformant. Comprising contacting, and (c) detecting a reporter activity in the transformant, wherein the test sample has anti-hepatitis C virus activity when the reporter activity is increased. How to be evaluated. (23) A method for evaluating the anti-hepatitis C virus activity of a test sample, which comprises (a) the mutant or double-stranded chain of (18)
The test sample includes a step of adding a test sample to a solution containing RNA-dependent protein kinase, eIF2α protein, and a reporter RNA, and (b) detecting a reporter activity in the solution, wherein the test sample reduces the reporter activity. The method in which the test sample is evaluated as having anti-hepatitis C virus activity when allowed to do so. (24) A method for evaluating the anti-hepatitis C virus activity of a test sample, which comprises (a) a hepatitis C virus NS5A protein in the presence of the test sample or the mutant according to (18).
A step of contacting with IRF-1 protein, and (b) IRF
-1 a method of detecting the activity of a protein, wherein the test sample is evaluated as having anti-hepatitis C virus activity when the test sample increases the activity of the IRF-1 protein. (25) A method for evaluating the anti-hepatitis C virus activity of a test sample, which comprises the step (a) of providing the transformant according to (13) or (14), and (b) the transformant When the test sample increases the reporter activity, the test sample is treated with an anti-antibody when the test sample increases the reporter activity, including the step of contacting the test sample, and (c) detecting the reporter activity in the transformant.
A method evaluated to have hepatitis C virus activity. (26) A method for evaluating the anti-hepatitis C virus activity of a test sample, which comprises (a) a hepatitis C virus NS5A protein in the presence of the test sample or the mutant according to (18):
When a test sample increases the activity of the ISGF3 protein, including the step of contacting with the ISGF3 protein, STAT1 protein, STAT2 protein or IRF-9 protein, and (b) detecting the activity of the ISGF3 protein , A method in which the test sample is evaluated to have anti-hepatitis C virus activity. (27) A method for evaluating the anti-hepatitis C virus activity of a test sample, which comprises the step (a) of providing the transformant according to (16) or (17), and (b) the transformant When the test sample increases the reporter activity, the test sample is treated with an anti-antibody when the test sample increases the reporter activity, including the step of contacting the test sample, and (c) detecting the reporter activity in the transformant.
A method evaluated to have hepatitis C virus activity. (28) A method for screening a compound having anti-hepatitis C virus activity, which comprises: (a) anti-hepatitis C virus for a plurality of test samples, according to any one of (20) to (27) A method comprising the steps of evaluating activity, and (b) selecting a compound evaluated to have anti-hepatitis C virus activity from a plurality of test samples. (29) A compound having anti-hepatitis C virus activity, which is identified by the method according to any one of (20) to (28). (30) A compound having anti-hepatitis C virus activity, which inhibits the binding between the C-terminal side of NS5A protein and double-stranded RNA-dependent protein kinase. (31) A compound having anti-hepatitis C virus activity, which directly interacts with NS5A protein to inhibit the interaction with double-stranded RNA-dependent protein kinase. (32) The compound according to (30) or (31), wherein the NS5A protein is the mutant according to (18). (33) An anti-hepatitis C virus agent comprising the compound according to any one of (29) to (32) as an active ingredient. (34) The anti-hepatitis C virus agent according to (33), which further comprises a combination with another antiviral agent.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention relates to hepatitis C virus NS5A.
A mutant of a protein, which is a double-stranded RN
Provided is a DNA encoding a mutant having an enhanced ability to suppress the enzymatic activity of A-dependent protein kinase.

Hepatitis C virus having NS5A protein
It is known that there are various types of (HCV).
The type of HCV having a NS5A protein variant in the present invention is not particularly limited, and specifically, 1a type, 1b type,
2a type, 2b type, 3a type, 3b type, 4, type 5, 5 type, etc. (Simmonds,
P. Hepatology, 19: 1321-1324, 1994).

In the present invention, as the NS5A protein mutant having an enhanced ability to suppress the enzymatic activity of the PKR protein, the mutant-introduced protein has a higher PKR protein than the NS5A protein derived from the parent strain. There is no particular limitation as long as it is a mutant containing a mutation that acquires the ability to suppress enzyme activity. NS5A that suppresses the enzymatic activity of PKR protein about 1.5 times or more, more preferably about 1.8 times or more, further preferably about 2.0 times or more higher than the parent strain.
It is a protein variant. The NS5A protein mutant of the present invention may further include a mutation that does not affect the above-mentioned ability of the NS5A protein, as long as it has such a mutation. The term "mutation" used herein means amino acid substitution, deletion,
Insertions, additions, and combinations thereof are included.

The number of amino acid mutations and the mutation site in the NS5A protein mutant of the present invention are not limited as long as the mutant has a higher ability to suppress the enzymatic activity of the PKR protein as compared with the NS5A protein derived from the parent strain. Absent. The number of mutations is typically within 30 amino acids, preferably
It is within 10 amino acids, more preferably within 5 amino acids (for example, within 3 amino acids). The mutation site is preferably a mutation at the phosphorylation site (site undergoing phosphorylation) of the NS5A protein. The phosphorylation site is preferably a phosphorylation site on the C-terminal side of the NS5A protein,
For example, the amino acid sequence of SEQ ID NO: 2 (wild type NS5A
The 1404 type (J4L6S strain) protein has 2404 and 2406 positions of the amino acid sequence from the 1973 position to the C-terminal 2419 position (the amino acid sequence of SEQ ID NO: 2 has an N-terminal amino acid residue of 1).
Serine residues 2404 and 2406 based on the 973rd position, particularly preferably the serine residue at position 2404. The NS5A protein mutant of the present invention having a mutation at the phosphorylation site on the C-terminal side of the NS5A protein is preferably
A variant comprising the amino acid sequence set forth in SEQ ID NO: 3 or 4. Here, "C-terminal side" means within 70 amino acid residues from the C-terminal, preferably within 40 amino acids, and more preferably within 20 amino acid residues. The mutation of the NS5A protein may be a substitution mutation with an amino acid different from the amino acid before substitution, but preferably an amino acid that undergoes phosphorylation (serine residue, threonine residue, tyrosine residue, particularly serine residue). ) To other amino acids, and more preferably a serine residue to alanine.

The NS5A protein mutant of the present invention also includes a peptide derived from hepatitis C virus (HCV) fused with a peptide derived from another source. Other peptides to be fused include, for example, FLAG (Hopp, TP et. Al.,
BioTechnology 6: 1204-1210,1988), 6 × His, 10 × His consisting of 6 His (histidine) residues, human c-myc fragment, VSV-GP fragment, p18HIV fragment, T7-tag, HSV-tag
, E-tag, SV40T antigen fragment, lck tag, α-tubulin
Fragment, B-tag, Protein C fragment, GST (glutathione S-transferase), HA (influenza agglutinin), immunoglobulin constant region, β-galactosidase, MBP (maltose binding protein) and the like.

The DNA encoding the NS5A protein variant of the present invention can be prepared by those skilled in the art, for example, by the site-directed mutagenesis method (Hashimoto-Gotoh, T. et al.
et.al., Gene 152: 271-275, 1995, Zoller, MJ and
Smith, M. Methods Enzymol. 100: 468-500, 1983, Kr
amer, W. et. al., Nucleic Acids Res. 12: 9441-945
6, 1984, Kramer, W. and Fritz, HJ Methods. Enzy
mol. 154: 350-367, 1987, Kunkel, TA et. al., Pr.
oc Natl Acad Sci USA. 82: 488-492, 1985, Kunkel e
t. al., Methods Enzymol. 85: 2763-2766, 1988) and the like. It is also possible to artificially synthesize using a DNA synthesizer or the like. Whether or not the prepared DNA is a DNA encoding an NS5A protein variant with enhanced ability to suppress the enzymatic activity of PKR protein depends on whether the protein encoded by the DNA suppresses the enzymatic activity of PKR protein. Whether or not it can be determined by measuring by the method shown in the examples.

The present invention also provides a vector into which the above DNA is inserted, a transformant carrying the above DNA or the vector, and a method for producing the NS5A protein variant of the present invention by culturing the transformant. .

As the vector of the present invention, those commonly used by those skilled in the art can be used. For example, when Escherichia coli is used as a host in transformation, for example, “ori” for replicating in E. coli, and a gene for selecting transformed E. coli (for example, drugs (ampicillin, tetracycline, kanamycin, crocin, It is desirable to have a resistance gene) such as rhamphenicol on the vector.
2, pBluescript, pCR-Script, pGEM-T, pDIRECT, pT7 and the like.

When the vector is used for the purpose of producing the NS5A protein mutant, the expression vector is particularly useful. Examples of the expression vector include the lacZ promoter (Ward e
t. al., Nature 341: 544-546, 1989), araB promoter (Better et. al., Science 240: 1041-1043, 198).
8), or a vector having a T7 promoter or the like. As such a vector, in addition to the above vector, pGEX-5X-1 (manufactured by Pharmacia), "QIA
express system "(Qiagen), pEGFP, and pET
(In this case, the host is BL21 expressing T7 RNA polymerase.
Are preferred) and the like. The expression vector may also contain a signal sequence for polypeptide secretion. As a signal sequence for protein secretion, for example, when it is produced in the periplasm of E. coli,
pelB signal sequence (Lei, SP et. al., J. Bacterio
l. 169: 4379, 1987) etc. may be used.

Other vectors for producing the NS5A protein variant include, for example, mammalian-derived expression vectors (eg pcDNA3 (Invitrogen), pEGF-BOS).
(Nucleic Acids. Res. 18 (17): 5322, 1990, pEF, pCDM
8), an expression vector derived from an insect cell (for example, "Bac-to-BAC
baculovairus expression system "(manufactured by Gibco BRL), pBacPAK8), plant-derived expression vector (eg pM
H1, pMH2), animal virus-derived expression vectors (eg
pHSV, pMV, pAdexLcw), expression vector derived from retrovirus (eg pZIPneo), expression vector derived from yeast (eg “Pichia Expression Kit” (Invitrogen), pNV11, SP-Q01), Bacillus subtilis derived expression vector (For example, pPL608, pKTH50) and the like.

[0022] For the purpose of expression in animal cells such as CHO cells, COS cells and NIH3T3 cells, a promoter required for intracellular expression, for example, SV40 promoter (Mulligan et. Al., Nature 277: 108, 1979), MMLV-
LTR promoter, EF1α promoter (Mizushima et.
al., Nucleic Acids Res. 18: 5322, 1990), having a CMV promoter and the like is essential, and a gene for selecting transformation into cells (eg, drug (neomycin, G418, etc.) resistance gene). Is more preferable. Vectors having such characteristics include, for example,
Examples include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOP13 and the like.

Furthermore, in order to stably express the gene and to amplify the copy number of the gene in the cell, the CHO cell lacking the nucleic acid synthesis pathway has a DHFR gene which complements it. Examples include a method of introducing a vector (for example, pCHOI) and amplifying it with methotrexate (MTX). In addition, when transient expression of a gene is intended, a gene expressing the SV40 T antigen is placed on the chromosome. Using COS cells that carry SV40 replication origin vector (p
cD) and the like.

The origin of replication includes polyoma virus, adenovirus, bovine papilloma virus (BP
Those derived from V) and the like can also be used. In addition, the expression vector serves as a selectable marker for aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene, dihydrofolate reductase, for amplification of gene copy number in host cell lines. (Dhfr) gene and the like can be included.

The host into which the vector is introduced is not particularly limited, and for example, Escherichia coli or various eukaryotic cells can be used. When using a eukaryotic cell, for example, an animal cell, a plant cell, or a fungal cell can be used as a host. Animal cells include mammalian cells such as CHO, COS,
3T3, myeloma, BHK (baby hamster kidney), HeL
a, Vero, amphibian cells such as Xenopus oocytes (Valle, et. al., Nature 291: 358-340, 198).
1), or insect cells such as Sf9, Sf21, Tn5 are known. As CHO cells, in particular, dhfr-CHO (Proc. Natl. Acad. Sci.
USA 77: 4216-4220, 1980) and CHO K-1 (Proc. Natl. A
cad. Sci. USA 60: 1275, 1968) can be preferably used. Among animal cells, CHO cells are particularly preferable for the purpose of large-scale expression. Examples of plant cells include cells derived from Nicotiana tabacum. Known fungal cells include yeasts such as the genus Saccharomyces, such as Saccharomyces cerevisiae, and filamentous fungi, such as the genus Aspergillus, such as Aspergillus niger. When prokaryotic cells are used, bacterial cells include E. coli, such as JM109, DH5α, HB101,
Examples include XL1Blue and BL21, and other known Bacillus subtilis.

When an animal is used as the host, mammals, plants and insects can be mentioned. As mammals, goat, pig, sheep, mouse and cow can be used (Vicki Glaser, SPECTRUM Biotechnology Appli.
cations, 1993). When plants are used, tobacco can be used, for example. Furthermore, as an insect,
For example, silkworms can be used.

In order to prepare the transformant of the present invention,
The above vector is introduced into the above host. As a method therefor, in the case of introducing the vector into a host cell such as Escherichia coli, for example, calcium chloride method, electroporation method (Chu, G. et. Al., Nucl. Acid Res. 15: 1311-132)
6, 1987) can be used. In the case of introducing the vector into a host cell such as a cultured cell, for example, the calcium phosphate method (Chen, C. and Okayama, H. Mol. Cell. Biol.
7: 2745-2752, 1987), DEAE dextran method (Lopata,
MA et. Al., Nucl. Acids Res. 12: 5707-5717, 198
4, Sussman, DJand Milman, G. Mol. Cell. Biol.
4: 1642-1643, 1985), Cationic ribosomal DOTAP
(Boehringer Mannheim), lipofectin method (Derijard, B. Cell 7: 1025-1037, 1994,
Lamb, BT et. Al., Nature Genetics 5: 22-30, 199
3, Rabindran, SK et.al., Science 259: 230-234,
1993) and the like can be used.

Furthermore, when DNA is introduced into an animal, the DNA
To a suitable vector (eg adenovirus vector (eg pAdexlcw) or retrovirus vector (eg pZIPn
eo) and the like, but not limited thereto. ) And introduced into the living body by, for example, a retrovirus method, a liposome method, a cationic liposome method, an adenovirus method, or the like. When a vector is introduced into insects, for example, it encodes the protein of interest.
It can be carried out by infecting a silkworm with a baculovirus having inserted DNA (Susumu, M. et. Al., Na
ture 315: 592-594, 1985). When introducing DNA into a plant, for example, a DNA encoding a protein of interest is inserted into a plant expression vector, for example, pMON 530, and this vector is transferred to Agrobacterium tumefaciens (Agro
bacterium tumefaciens). The vector can be introduced by infecting this bacterium with tobacco, for example Nicotiana tabacum (Julian K.-C. Ma et.al., Eur. J. Immunol. 24: 1.
31-138, 1994).

The NS5A protein mutant of the present invention can be produced, for example, by culturing the transformant of the present invention. The culture can be performed according to a known method. For example, in the case of culturing animal cells, DMEM, MEM, RPMI1640, IMDM and the like can be generally used as the culture medium. At that time, serum supplement such as fetal calf serum (FCS) may be used in combination, or serum-free culture may be performed. The pH during culture is usually preferably about 6-8.
Culturing is usually performed at about 30 to 40 ° C. for about 15 to 200 hours, and the medium is exchanged, aeration and stirring are added if necessary.

The NS5A protein mutant of the present invention can be isolated from the inside or outside of the host cell (medium etc.) and purified as a substantially pure and homogeneous protein. Separation and purification of proteins may be carried out by using the separation and purification methods used in ordinary protein purification, and are not particularly limited. For example, a chromatography column, filter, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, etc. are appropriately selected,
If combined, proteins can be separated and purified. Alternatively, it is possible to further purify by combining a plurality of these columns.

Examples of the chromatography include, for example, affinity chromatography in which an antibody against the NS5A protein variant of the present invention described below is immobilized on a column, ion exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography. Etc. (Strategies for Protein P
urification and Characterization: A Laboratory Cou
rse Manual. Ed DanielR. Marshak et al., Cold Sprin
g Harbor Laboratory Press, 1996). These chromatography methods include liquid phase chromatography methods such as HPL.
It can be performed using liquid phase chromatography such as C or FPLC. The NS5A protein mutant of the present invention can also be highly purified using these purification methods.

The protein can be optionally modified or partially removed by treating it with an appropriate protein-modifying enzyme before or after purification. Examples of the protein-modifying enzyme include trypsin, chymotrypsin, lysyl endopeptidase, protein kinase, glucosidase and the like.

When the NS5A protein mutant of the present invention is expressed as a fusion protein with a glutathione S-transferase protein or as a recombinant protein to which a plurality of histidines are added, in a host cell (eg, animal cell or E. coli) In particular, the expressed recombinant protein can be purified using a glutathione column or a nickel column. After purification of the fusion protein, the region of the fusion protein other than the protein of interest may be replaced with thrombin or factor if necessary.
It is also possible to remove by cutting with Xa or the like.

Furthermore, it is also possible to recover the protein from a system for producing the protein in an individual. Examples of a system for producing a protein in an individual include a production system using animals and a production system using plants. The desired DNA is introduced into these animals or plants, the protein is produced in the body of the animal or plant, and then the protein is recovered.

For example, the desired DNA is prepared as a fusion gene with a gene encoding a protein which is uniquely produced in milk, such as goat β casein. Then, a DNA fragment containing this fusion gene is injected into a goat embryo, and this embryo is transplanted into a female goat. The protein of interest can be obtained from the milk produced by the transgenic goat born from the goat that received the embryo or its offspring. Hormones may optionally be used in transgenic goats to increase the amount of milk containing proteins produced by the transgenic goats (Ebert, KM et. Al., Bi.
o / Technology 12: 699-702, 1994).

When insects are used, the target protein can be obtained from the body fluid of silkworms infected with baculovirus having a DNA encoding the target protein inserted therein (Susumu, M. et. Al. , Nature 315:
592-594, 1985).

Further, when a plant is used, a method well known to those skilled in the art can be used, for example, from cells derived from Nicotiana tabacum introduced with the vector by the above method (Toki et al., Plant Ph.
The desired protein can be obtained from tobacco (tobacco leaf) regenerated with ysiol 100: 1503-1507, 1995) (Julian K.-C. Ma et. al., Eur. J. Immunol. 24: 13).
1-138, 1994).

The antibody used for purifying or detecting the NS5A protein variant of the present invention can be prepared by a known method. The form of the antibody is not particularly limited. Also included are antisera obtained by immunizing an immunized animal such as a rabbit with an antigen protein, polyclonal and monoclonal antibodies of all classes, human antibodies and humanized antibodies by genetic recombination. Further, the antibody may be an antibody fragment or a modified antibody as long as it binds to the NS5A protein variant.

The antibody can be obtained by using the NS5A protein variant as a sensitizing antigen. The NS5A protein variant used as a sensitizing antigen may be a complete protein or a partial peptide of the protein. In addition, cells expressing the protein, lysates thereof, or chemically synthesized NS5A protein mutants may be used as the sensitizing antigen. The short peptide can be appropriately bound to a carrier protein such as keyhole limpet hemocyanin, bovine serum albumin or ovalbumin to serve as an antigen.

Mammals immunized with the sensitizing antigen include
Although not particularly limited, it is preferable to select in consideration of compatibility with a parent cell used for cell fusion in the production of a monoclonal antibody, generally, rodents such as mouse, rabbit, etc. Primates such as Lagomorpha and Rhesus monkey are used. Methods of immunizing animals with sensitizing antigens are well known to those of skill in the art.

As an example of obtaining a polyclonal antibody, after confirming that the desired antibody level in serum has increased,
The blood of the mammal sensitized with the antigen is removed. Serum is separated from this blood by a known method. As the polyclonal antibody, serum containing the polyclonal antibody may be used, or if necessary, a fraction containing the polyclonal antibody may be further isolated from the serum and used.

As an example of obtaining a monoclonal antibody, after confirming that the desired antibody level is increased in the serum of a mammal sensitized with the above-mentioned antigen, immune cells may be taken out and subjected to cell fusion. In this case, splenocytes are particularly preferred as the immune cells used for cell fusion. The other parent cell to be fused with the immune cell is preferably a mammalian myeloma cell, and more preferably a myeloma cell that has acquired the characteristics for selecting fused cells by a drug. The cell fusion of the immune cells and myeloma cells is basically a known method, for example, the method of Milstein et al. (Galfre, G. and Milstein, C., Methods Enzymol.
73: 3-46, 1981) and the like.

Then, a hybridoma producing a desired antibody is cloned from the hybridoma obtained by cell fusion by a well-known method, and the cloned hybridoma is transplanted into the abdominal cavity of a mouse, and ascites is collected from the mouse.

The obtained antibody can be purified to homogeneity. Separation and purification of the antibody may be carried out by using the separation and purification methods used for ordinary proteins. For example, a chromatography column such as affinity chromatography, a filter, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing, etc. can be appropriately selected and combined to separate and purify the antibody. Yes (Antibodies: A Laboratory Manual. Ed Harlow
and David Lane, Cold Spring Harbor Laboratory, 198
8) is not limited to these. Columns used for affinity chromatography include protein A columns and protein G columns. For example, as a column using a Protein A column, Hyper D,
POROS, Sepharose FF (Pharmacia) and the like can be mentioned.

Examples of methods for measuring the antigen-binding activity of an antibody include measurement of absorbance and enzyme-linked immunosorbent assay (Enz
yme-linked immunosorbent assay (ELISA), EIA (enzyme immunoassay), RIA (radioimmunoassay) or fluorescent antibody method can be used. When using ELISA, NS5A protein variant is added to a plate on which antibodies are immobilized, and then a sample containing the antibody of interest, for example, culture supernatant of antibody-producing cells or purified antibody is added. Add a secondary antibody that recognizes an antibody labeled with an enzyme, such as alkaline phosphatase, incubate the plate, wash it, and then add an enzyme substrate such as p-nitrophenyl phosphate and measure the absorbance to detect antigen binding. The activity can be evaluated. NS5A is the protein added to the plate.
You may use the fragment etc. of a protein variant.

When wild-type mutations confirm the presence of HCV carrying the NS5A protein variant of the present invention, it is suspected that the HCV has high IFN resistance. Therefore, the above mutant (protein or RN that encodes it)
It is considered that the method of confirming the presence thereof using an antibody against A) or the like is effective in detecting HCV, diagnosing the effectiveness of IFN treatment, and the like.

The present invention provides a method for evaluating the anti-HCV activity of a test sample. Evaluation of anti-HCV activity in the method of the present invention, the interaction between the NS5A protein variant and the PKR protein, in particular the binding inhibition is performed as an index, or the inhibition of the enzymatic activity of the PKR protein by the NS5A protein variant is performed as an index. be able to.

In the method using the inhibition of binding between the NS5A protein variant and the PKR protein as an index, first, the NS5A protein variant of the present invention and PKR protein are present in the presence of a test sample.
Contact with protein. Then, the binding between the NS5A protein variant and the PKR protein is detected. When the test sample inhibits the binding between the NS5A protein variant and the PKR protein, it is judged that the test sample has anti-HCV activity.

As the NS5A protein variant in this method, the complete protein can be used, but PKR
It is also possible to use a partial peptide thereof that can bind to a protein. The present inventors have found that the C-terminal side of NS5A protein is a binding region for PKR protein. Therefore, in such a partial peptide, a peptide fragment containing the binding region to the PKR protein existing on the C-terminal side of the NS5A protein variant (the N-terminal amino acid residue of the amino acid sequence of SEQ ID NO: 2 at position 1973) When 2350
Peptide fragment containing at least a variant of the amino acid sequence from position 2419 to position 2419).

An evaluation system using the inhibition of the binding between the NS5A protein variant and the PKR protein as an index can be constructed in vitro or in cells. When done in cells,
Methods known to those skilled in the art can be used. For example, the two-hybrid method using yeast or animal cells (F
ields, S. and Sternglanz, R. Trends. Genet. 10: 28
6-292, 1994, Dalton, S. and Treisman, R. Character
ization of SAP-1, a protein recruited by serum res
ponse factor to the c-fos serum response element.
Cell 68: 597-612, 1992, `` MATCHMARKER Two-Hybrid S
ystem '', `` Mammalian MATCHMAKER Two-Hybrid Assay Ki
t, MATCHMAKER One-Hybrid System (all manufactured by Clontech), HybriZAP Two-Hybrid Vector Syste
m ”(manufactured by Stratagene).

In the yeast two-hybrid system, N
A vector expressing a fusion protein in which either one of the S5A protein variant or the PKR protein or its partial peptide is fused with the SRF DNA binding region or the GAL4 DNA binding region, and the other protein or its partial peptide is expressed as VP16 or A vector fused with a transcriptional activation region such as GAL4 is constructed, introduced into a yeast cell together with a vector encoding a reporter gene, and assayed in the presence of a test sample using the reporter activity as an index. Although the expression of the reporter gene is induced by the binding of the NS5A protein mutant and the PKR protein, when the test sample inhibits the binding of both proteins,
The expression of the reporter gene is suppressed. Examples of reporter genes include HIS3 gene, Ade2 gene, LacZ
Gene, CAT gene, gene encoding luciferase (Luc) (luc gene), PAI-1 (Plasminogen activato
rinhibitor type1) gene and the like, but not limited thereto. A cytotoxic gene can also be expressed as a reporter gene. The evaluation method by the two-hybrid method can be performed using mammalian cells and the like in addition to yeast.

Another example is an assay system using immunoprecipitation. In the presence of the test sample, cells expressing the NS5A protein variant and the PKR protein were cultured, cells were recovered, and then the complex was recovered with an antibody or the like against one of the proteins, and then the other protein was treated with the protein. The binding of both proteins can be evaluated by detection using an antibody or the like.

In such an evaluation system, either or both proteins can be exogenously expressed in cells. When the protein is expressed exogenously in animal cells, for example, the NS5A protein mutant and / or the gene encoding the PKR protein is added to pSV2neo, pcDNA I, pCD8
The gene is expressed in animal cells and the like by inserting it into a vector for expressing foreign genes such as. SV40 early promoter (Rigby In Will
iamson (ed.), Genetic Engineering, AcademicPress,
London, 3: 83-141, 1982), EF-1 α promoter (Kim e
t. al., Gene 91: 217-223, 1990), CAG promoter (Niw
a et. al., Gene 108: 193-200, 1991), RSV LTR promo
ter (Cullen Methods in Enzymology 152: 684-704, 19
87), SRα promoter (Takebe et. Al., Mol. Cell. Bio
l. 8: 466, 1988), CMV immediate early promoter (Se
ed and Aruffo, Proc. Natl. Acad. Sci. USA 84: 3365
-3369, 1987), SV40 late promoter (Gheysen and Fier
s, J. Mol. Appl. Genet. 1: 385-394, 1982), Adenovi
rus late promoter (Kaufman et. al., Mol. Cell. Bio
l. 9: 946, 1989), HSV TK promoter, etc., as long as it is a commonly used promoter. A foreign gene can be expressed by introducing the gene into an animal cell by the method described above.

In addition, the recognition site (epitope) of the monoclonal antibody of which the specificity is known is changed to the NS5A protein mutant and / or the N-terminal or C-terminal of the PKR protein.
By introducing at the end, a fusion protein having a recognition site for a monoclonal antibody can be obtained. As the epitope-antibody system used, commercially available products can be used (Experimental Medicine 13: 85-90, 1995). Vectors capable of expressing a fusion protein with β-galactosidase, maltose binding protein, immunoglobulin constant region, glutathione S-transferase, green fluorescent protein (GFP) and the like via the multiple cloning site are commercially available.

In the case of forming a fusion protein, a method of preparing a fusion protein by introducing only a small epitope portion consisting of several to ten and a dozen amino acids in order to change the properties of the original protein as little as possible It has been reported. For example, polyhistidine (His-tag) (eg 6xHis or 10xHis etc.), influenza agglutinin H
A fragment, human c-myc fragment, FLAG (Hopp, TP et. A
L., BioTechnology 6: 1204-1210, 1988), Vesicular s
fragment of tomatitis virus glycoprotein (VSV-GP), T7
gene10 protein fragment (T7-tag), human herpes simplex virus glycoprotein fragment (HSV-tag), E-tag (epitope on monoclonal phage), SV40T antigen fragment, lck tag, α-tubulin fragment, B -known epitopes such as -tag and Protein C fragments, and monoclonal antibodies that recognize them are used as epitopes for the above screening.
-Available as an antibody system (Experimental Medicine 13: 85-90, 199
Five).

In immunoprecipitation, an immune complex is formed by adding these antibodies to a cell lysate prepared by using an appropriate surfactant. This immune complex
Includes NS5A protein variants, PKR proteins, and antibodies. In addition to using antibodies against the above epitopes, NS
Immunoprecipitation can also be performed using an antibody against the 5A protein variant or the PKR protein. These antibodies can be produced by the method described above.

The immune complex is, for example, Protein A Sepharose or Protein G Sepharo when the antibody is a mouse IgG antibody.
It can be sedimented using se. Also, for example GST
When a fusion protein with such epitopes is prepared, an immune complex can be formed using a substance that specifically binds to these epitopes such as glutathione-Sepharose 4B. For general methods of immunoprecipitation, see, for example, literature (Harlow, E. and Lane, D. Antibodies,
Cold Spring Harbor Laboratory publications, NewYor
k, pp.511-552, 1988) or in accordance therewith.

It is also possible to carry out the assay in vitro using a pull-down assay without using a cell line. For example, in the presence of a test sample, NS5A protein mutant and PKR
After incubating with the protein in a test tube, and recovering the complex with an antibody against one of the proteins or an antibody against the tag fused to these proteins,
The binding of both proteins can be evaluated by detecting the other protein using an antibody against the protein or an antibody against the tag added to the protein. Further, one protein is bound to the support, the other protein is bound, and the test sample is applied thereto. The effect of the test sample can also be evaluated by detecting whether or not the bound protein is dissociated.

The means for detecting or measuring the binding between proteins can be carried out, for example, by using a label attached to the protein. Examples of the type of label include fluorescent labels and radiolabels. Furthermore, a biosensor utilizing the surface plasmon resonance phenomenon can be used to detect the binding between proteins. A biosensor utilizing the surface plasmon resonance phenomenon enables the interaction between proteins to be observed in real time as a surface plasmon resonance signal without labeling with a small amount of protein sample (for example, BIAcor
e, manufactured by Pharmacia). For example, a biosensor such as BIAcore can be used to evaluate the binding between the NS5A protein variant and the PKR protein.

Specific examples of the method for measuring the binding between the PKR protein and the NS5A protein variant include the following methods. First, the GST-K296R protein extract that was forcibly expressed in E. coli (an inactive mutant of PKR protein (K296R) with a GST tag ligated to the N-terminus. Can not be expressed) and a resin (glutathione sepahrose) that specifically adsorbs the GST tag are mixed to form a complex. Then, in the complex
³⁵ S] -Met-labeled NS5A protein (synthesized in vitro) is mixed and allowed to undergo a binding reaction. Further, the above reaction product is electrophoresed and the radioactivity on the gel is measured.

When the test sample inhibits the binding between the NS5A protein variant and the PKR protein by the above method, it is judged that the test sample has anti-HCV activity.

As another method for evaluating the anti-HCV activity of the test sample using the inhibition of the inhibition of the PKR protein enzyme activity by the NS5A protein mutant as an index, the NS5A protein variant may be used to inhibit the PKR protein enzyme activity. Methods of measuring inhibition directly and indirectly are mentioned.

As a method for directly measuring the inhibition of PKR protein enzyme activity inhibition by the NS5A protein mutant,
First, the NS5A protein mutant of the present invention is contacted with the PKR protein in the presence of a test sample. Then, the enzymatic activity of the PKR protein is detected. When the test sample increases the enzymatic activity of PKR protein, the test sample is treated with anti-HC
Judged to have V activity.

An evaluation system using inhibition of PKR protein enzyme activity inhibition by the NS5A protein mutant as an index can be constructed in vitro or in cells. As an in-vitro evaluation system, for example, NS5A by radioactive label ATP
It can be performed by phosphorylating a substrate such as a protein and measuring its radioactivity. Specifically, first, He
PKR protein crudely extracted from La cells and [γ- ³² P] ATP (radioisotope) are mixed and phosphorylated. Then
Antigen (PKR)-
An antibody (PKR antibody) complex is formed. Next, the above complex is mixed with a resin (protein G) that specifically adsorbs an antibody to form an immunoprecipitate (resin-antigen-antibody complex). Further, the above immunoprecipitate is electrophoresed and the radioactivity on the gel is measured.

One embodiment of the method of indirectly measuring the inhibition of the enzymatic activity of PKR protein by the NS5A protein mutant is as follows. The following transformant into which a DNA functionally linked to is introduced is provided. Then, a test sample is brought into contact with the transformant. Then, the reporter activity in the transformant is detected. It is judged that the test sample has anti-HCV activity when the test sample increases the reporter activity.

The reporter gene used in this method is not particularly limited as long as its expression can be detected, and examples thereof include CAT gene, lacZ gene, luc gene, and GFP gene. The expression level of the reporter gene is
Depending on the kind of the reporter gene, it can be measured by a method known to those skilled in the art. For example, when the reporter gene is the CAT gene, the acetylation of chloramphenicol by the gene product is detected, and when the reporter gene is the lacZ gene, a pigment compound of By detecting the color development, and in the case of the luc gene, by detecting the fluorescence of the fluorescent compound by the catalytic action of the gene expression product, further, in the case of the GFP gene, the fluorescence by the GFP protein By detecting, the expression level of the reporter gene can be measured.

In the present invention, “functionally linked” means that the NFκB protein binds to the NFκB protein binding region (region in which the NFκB protein recognition sequence exists, for example, promoter region of the gene encoding IFN), NF so that expression of the reporter gene is induced
It means that the κB protein binding region and the reporter gene are bound. Therefore, even when the reporter gene binds to another gene and forms a fusion protein with another gene product, the expression of the fusion protein is increased by binding the NFκB protein to the NFκB protein binding region. Anything that is induced is included in the meaning of "functionally linked" above. Moreover, in order to enhance the responsiveness of the reporter to the NFκB protein, NFκB
A plurality of protein binding regions may be bound repeatedly.

The transformant used in the present method expresses PKR protein, NFκB protein, and IκBα protein and retains the DNA encoding the NS5A protein mutant of the present invention or the vector into which the DNA is inserted. It is a transformant. More preferably, PKR protein, NF
It is a transformant that expresses κB protein and IκBα protein and that can inducibly express DNA encoding the NS5A protein variant of the present invention. PK
The R protein, NFκB protein, and IκBα protein may be an endogenous protein or an exogenous protein. A cell having the exogenous protein can be prepared by introducing a DNA encoding the protein into the cell by the method described above.

As a system for inducibly expressing the DNA encoding the NS5A protein variant, the initiation or / and termination of gene expression can be artificially manipulated, and thereby the time of gene expression or the amount of gene can be regulated. A system that can be used is desirable, and examples thereof include a Tet-Off gene expression system and a Tet-On gene expression system (both manufactured by CLONTECH).

Transformants capable of inducibly expressing the DNA encoding the NS5A protein variant include:
The inducible NS5A protein mutant-expressing cells and the like described in this example can be exemplified.

A reporter gene was functionally linked to the transformant of the present invention downstream of the NFκB protein binding region.
Examples of the method of introducing DNA include a method of introducing the DNA as a vector and a method of inserting the DNA into a chromosome. The introduction of the vector and the insertion of the DNA into the chromosome can be performed by a method generally used by those skilled in the art, such as an electroporation method or a gene introduction method utilizing homologous recombination.

In another embodiment of the method for indirectly measuring the inhibition of the enzymatic activity of PKR protein by the NS5A protein variant, first, the NS5A protein variant of the present invention, the PKR protein, the eIF2α protein, and the reporter gene are included. The test sample is added to the solution. Then, the reporter activity in the solution is detected. When the test sample decreases the reporter activity, it is judged that the test sample has anti-HCV activity.

In the above method, the NS5A protein mutant, PKR protein, eIF2α protein, prepared in advance,
The reporter RNA is added to the assay system, and the amount of the reporter protein produced by the translation of the reporter RNA is detected by the translation activity of the eIF2α protein.

Those skilled in the art can measure the reporter activity by a generally known method depending on the kind of the reporter protein. By measuring the reporter activity, it is possible to determine whether or not the test sample has anti-HCV activity. For example, reporter RNA
When Luc RNA is used as the enzyme, suppression of the enzymatic activity of the PKR protein can be detected using an increase in the activity of the reporter Luc protein due to an increase in the translation activity of the eIF2α protein as an index.

As another method for evaluating the anti-HCV activity of a test sample, first, the NS5A protein or the NS5A protein variant of the present invention is contacted with the IRF-1 protein in the presence of the test sample. Then, the activity of the IRF-1 protein is detected. It is judged that the test sample has anti-HCV activity when the test sample increases the activity of the IRF-1 protein.

In this method, the NS5A protein or the NS5A protein mutant of the present invention and the IRF-1 protein can be contacted with each other, for example, in a purified state or expressed in a cell extract. Here, the cell extract containing the NS5A protein, the NS5A protein variant of the present invention, and the IRF-1 protein is expressed, for example, in the cell extract contained in the in vitro transcription / translation system, the NS5A protein and the NS5A of the present invention. Examples include protein variants and vectors to which a vector containing a DNA encoding the IRF-1 protein has been added. The in vitro transcription / translation system is not particularly limited, and a commercially available in vitro transcription / translation kit or the like can be used.

The IRF-1 protein activity in the present method preferably includes, but is not limited to, transcription activity. The transcription activity of the IRF-1 protein can be evaluated using, for example, a reporter gene, but is not limited thereto.

As another method for evaluating the anti-HCV activity of a test sample, first, the following transformant in which a DNA functionally linked with a reporter gene is introduced downstream of the IRF-1 protein binding sequence I will provide a. Then, a test sample is brought into contact with the transformant. Then, the reporter activity in the transformant is detected. It is judged that the test sample has anti-HCV activity when the test sample increases the reporter activity.

In the present method, the IRF-1 protein binding sequence is one of the enhancer regions of the gene encoding type I IFN, and the transcription factor of the gene encoding IFN such as IRF-1 protein binds to it. Means an array.

In the present method, "functionally linked" means that the expression of the reporter gene is induced by binding of the IRF-1 protein to the IRF-1 protein binding sequence. It means that the protein binding sequence and the reporter gene are bound. Therefore, even if the reporter gene is linked to another gene and forms a fusion protein with another gene product, IRF-1
The term “functionally linked” is included as long as the expression of the fusion protein is induced by binding of the IRF-1 protein to the protein binding sequence. Further, in order to enhance the responsiveness of the reporter to the IRF-1 protein, a plurality of IRF-1 protein binding sequences may be bound to each other.

The reporter gene used in the present method is not particularly limited as long as its expression can be detected, and examples thereof include the genes described above. The expression level of the reporter gene can be measured by a method known to those skilled in the art depending on the type of the reporter gene.

The transformant used in this method is a trait that expresses the IRF-1 protein and carries the DNA encoding the NS5A protein or the NS5A protein mutant of the present invention, or a vector having the DNA inserted therein. It is a conversion body. More preferably, it is a transformant capable of expressing the IRF-1 protein and inducibly expressing the DNA encoding the NS5A protein or the NS5A protein variant of the present invention. The IRF-1 protein may be an endogenous protein or an exogenous protein. A cell having the exogenous protein can be prepared by introducing a DNA encoding the protein into the cell by the method described above.

As another method for evaluating the anti-HCV activity of a test sample, first, in the presence of the test sample, NS5A protein or the NS5A protein mutant of the present invention and ISGF3 protein, STAT1 protein (p91 or p84) , STAT2 protein (p113) or IRF-9 protein (p48). Then, the activity of the ISGF3 protein is detected. It is judged that the test sample has anti-HCV activity when the test sample increases the activity of the ISGF3 protein.

In this method, the NS5A protein or the NS5A protein mutant of the present invention and the ISGF3 protein, STAT1
The protein, STAT2 protein, or IRF-9 protein can be contacted, for example, in a purified state or expressed in a cell extract. Here, the NS5A protein, the NS5A protein mutant of the present invention, the ISGF3 protein, the STAT1 protein, the STAT2 protein, the cell extract expressing the IRF-9 protein is, for example, a cell extract contained in an in vitro transcription / translation system. , NS5A protein,
NS5A protein mutant of the present invention, ISGF3 protein, STA
Examples thereof include those to which a vector containing DNAs encoding T1 protein, STAT2 protein and IRF-9 protein has been added. The in vitro transcription / translation system is not particularly limited, and a commercially available in vitro transcription / translation kit or the like can be used.

In the present invention, the ISGF3 protein activity preferably includes transcriptional activity, but is not limited thereto. The transcription activity of the ISGF3 protein can be evaluated using, for example, a reporter gene, but is not limited thereto.

As another method for evaluating the anti-HCV activity of a test sample, first, the following transformant in which a DNA functionally linked with a reporter gene is introduced downstream of ISRE is provided. Then, a test sample is brought into contact with the transformant. Then, the reporter activity in the transformant is detected. It is judged that the test sample has anti-HCV activity when the test sample increases the reporter activity.

In the present method, the ISRE is a sequence (Interferon Stimulated Response Elements) common to the enhancer region of the gene induced by IFN, and the transcription factor of the gene induced by IFN such as ISGF3 protein is bound. Means an array.

In the present method, the term “functionally linked” means that the expression of a reporter gene is induced by the binding of ISGF3 protein to ISRE.
And the reporter gene are bound together. Therefore, even when the reporter gene is linked to another gene and forms a fusion protein with another gene product, the ISGF3 protein binds to ISRE to induce the expression of the fusion protein. Anything is included in the meaning of "functionally linked" above. Also,
Multiple ISREs may be bound to increase the responsiveness of the reporter to the ISGF3 protein.

The reporter gene used in this method is not particularly limited as long as its expression can be detected, and examples thereof include the genes described above. The expression level of the reporter gene can be measured by a method known to those skilled in the art depending on the type of the reporter gene.

The transformant used in the present method is a transformant expressing the ISGF3 protein and carrying the DNA encoding the NS5A protein or the NS5A protein mutant of the present invention, or a vector having the DNA inserted therein. Is. More preferred is a transformant capable of expressing ISGF3 protein and inducibly expressing DNA encoding NS5A protein or NS5A protein variant of the present invention. The ISGF3 protein may be an endogenous protein or an exogenous protein. A cell having the exogenous protein can be prepared by introducing a DNA encoding the protein into the cell by the method described above.

The test sample used in the evaluation system of the present invention is not particularly limited and includes, for example, cell culture supernatant, fermentation microbial product, marine organism extract, plant extract, prokaryotic cell extract, eukaryotic single cell extract. Substance or animal cell extract or library thereof, purified or crude protein,
Examples include peptides, non-peptidic compounds, synthetic low molecular weight compounds, and natural compounds.

Further, the PKR protein, NFκB protein, IκBα protein and eIF2 used in the evaluation system of the present invention
α protein, IRF-1 protein, ISGF3 protein, ST
The AT1 protein, STAT2 protein or IRF-9 protein may be a recombinant protein or a naturally occurring protein. The origin of the protein is not limited, and proteins derived from eukaryotes including human can be used. Preferably, it is a protein derived from mammals, and proteins derived from humans and rabbits are used. It may be a mutant, a partial peptide, a fusion protein with another peptide, a soluble protein, or a protein bound to a carrier as long as it has an activity equivalent to that of the purified wild-type protein. As the PKR protein used in the evaluation system of the present invention, an active PKR protein (having increased phosphorylation activity) is usually used. Activation of the PKR protein can be performed by contact with dsRNA as a pseudovirus.

By using the evaluation method of the present invention, anti-HCV activity is evaluated for a plurality of test samples, and compounds having anti-HCV activity are selected to efficiently screen compounds having anti-HCV activity. It becomes possible. The present invention also provides a method for screening a compound having such anti-HCV activity.

The compound having anti-HCV activity identified by the above evaluation method or screening method is a strong candidate for the anti-HCV agent. The present invention also provides an anti-HCV identified by the evaluation method or screening method of the present invention.
A compound having activity is provided.

In addition, the present inventors have shown that the PK of NS5A protein
The binding region for R protein has been previously considered as ISDR
It was found to be on the C-terminal side separately from the region. Therefore, C of NS5A protein or NS5A protein mutant
Compounds that inhibit the binding between the terminal side and PKR protein are anti-H
Can be a CV agent. Such compounds also include, for example, compounds that act directly on the C-terminal side of NS5A protein or NS5A protein mutant. Furthermore, the present invention is an anti-HCV agent containing the compound of the present invention as an active ingredient, and
Anti-HCV consisting of combination of said compound and other antiviral agent
Provide the agent.

When the compound of the present invention is used as a human anti-HCV agent, the compound itself may be directly administered to a patient, or may be formulated into a pharmaceutical preparation by a known pharmaceutical method for administration. For example, tablets, capsules, elixirs, and microcapsules, which are optionally sugar-coated, are orally injected, or a sterile solution or suspension solution of water or other pharmaceutically acceptable liquid is injected. It can be used parenterally in the form of a drug. Further, for example, a pharmacologically acceptable carrier or medium, specifically, sterile water or physiological saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative. It may be considered to be formulated by mixing with a drug, a binder and the like as appropriate and mixing in a unit dose form required for generally accepted pharmaceutical practice.

Additives that can be mixed in tablets and capsules include, for example, gelatin, corn starch, tragacanth gum, binders such as gum arabic, excipients such as crystalline cellulose, corn starch, gelatin,
Puffing agents such as alginic acid, lubricants such as magnesium stearate, sweeteners such as sucrose, lactose or saccharin, flavoring agents such as peppermint, red oil or cherry are used. When the dosage unit form is a capsule, a liquid carrier such as fat may be added to the above materials. Sterile compositions for injection can be formulated using vehicles such as distilled water for injection, according to standard pharmaceutical practice.

Aqueous solutions for injection include, for example, physiological saline, isotonic solution containing glucose and other adjuvants, such as D-
Examples include sorbitol, D-mannose, D-mannitol, sodium chloride, suitable solubilizing agents such as alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate. 80 (TM), HC
You may use together with O-50.

Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. Further, a buffering agent such as a phosphate buffer solution, a sodium acetate buffer solution, a soothing agent such as procaine hydrochloride, a stabilizer such as benzyl alcohol, phenol, and an antioxidant may be added. The prepared injection solution is usually filled in an appropriate ampoule.

Furthermore, when the obtained compound is used as an anti-HCV agent in combination with other antiviral agents, other antiviral agents such as interferon and ribavirin (1-β are used in addition to the above additives. -D-ribofuranosyl-1H-
1,2,4-triazide-3-carboxamide) and the like can also be included for use. Here, the “combination” means simultaneous administration with interferon, or combination with other agents such as administration of interferon after administration of the compound.
It is considered that such an anti-HCV agent can be a highly efficient anti-HCV agent.

Administration of the above-mentioned anti-HCV agents to patients includes, for example, intraarterial injection, intravenous injection, subcutaneous injection, intranasal administration,
It may be performed transbronchially, intramuscularly, transdermally, or orally by methods known to those skilled in the art. The dose varies depending on the body weight and age of the patient, the administration method, etc., but those skilled in the art can appropriately select an appropriate dose. If the compound can be encoded by DNA, it may be considered to incorporate the DNA into a gene therapy vector for gene therapy. The dose and administration method vary depending on the body weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.

The dose of the anti-HCV agent varies depending on the administration subject, symptom, and administration method, but in the case of oral administration, it is generally about 2 g to 0.1 mg per day in an adult (weight 60 kg). It is preferably about 500 mg to 0.1 mg, more preferably about 100 mg to 1 mg.

In the case of parenteral administration, the single dose varies depending on the administration subject, symptoms and administration method. 200 mg to 0.01 mg, preferably about 50 mg
To 0.01 mg, more preferably about 10 mg to 0.1 mg is conveniently administered by intravenous injection. Also in the case of other animals, the amount converted per body weight of 60 kg or the amount converted per body surface area can be administered.

Examples of diseases targeted by the anti-HCV agent of the present invention include hepatitis C and various liver diseases associated therewith (cirrhosis, liver cancer, etc.). There is no particular limitation.

[0105]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. [Example 1] Evaluation of effect of mutation of phosphorylation site of NS5A protein on reduction of enzymatic activity of PKR protein. Based on this finding and the finding that the NS5A protein serves as a substrate for the PKR protein, we paid particular attention to the serine residue of the C-terminal 50 amino acids. In order to understand the role of these phosphorylation sites, each mutant was prepared and the effect on the enzymatic activity of PKR protein was examined.

First, NS5A protein mutants were prepared. From the HCV1b type J4L6S strain, a gene encoding the NS5A protein (Genbank PID: G3098633) was amplified by the PCR method, cloned into the pET22b (Invitorogen) HindIII site, and named pET22b / NS5A DNA. 2401 of NS5A protein was analyzed by PCR on the cloned DNA.
Position, 2404 position, 2406 position, 2409 position, or 2413 position serine residue was replaced with an alanine residue to obtain the DNA
To pET22b / NS5A S2401A DNA and pET22b / NS5A, respectively.
S2404A DNA, pET22b / NS5A S2406A DNA, and pET22b / N
It was named S5A S2409A DNA.

The effect of the NS5A protein mutant on the enzymatic activity of the PKR protein was detected by an in vitro reporter gene assay system utilizing the translation function of the protein translation initiation factor eIF2α protein. In this assay system, suppression of the enzymatic activity of the PKR protein is detected by using as an index the increase in the activity of Luc as a reporter protein due to the increase in the translation activity of the eIF2α protein.

First, the already established method (Hatada
et. al., J. Virol. 73: 2425-2433, 1999).
The KR fraction was prepared. HeLa OY11 cells (3-5 × 10 ⁷ cells) were treated with 1000 IU / ml IFN at 37 ° C for 24 hours, and then PKR
The protein was induced. Next, wash the cells with PBS (-),
After collecting with a scraper, it was centrifuged. For precipitation, 100 mM KCl
Buffer solution (20 mM HEPES (pH 7.5), 100 mM KCl, 4 mM Mg (OA
c) ₂ , 10 mM of 1 mM DTT, 0.5% NP40) was added and suspended. Centrifuge at 10k x g rpm for 10 minutes at 4 ° C, and then supernatant at 100k x gr at 4 ° C.
It was centrifuged at pm for 60 minutes. 0.5M KCl soln B (20mM HE
PES (pH7.5), 500 mM KCl, 4 mM Mg (Oac) ₂ , 1 mM DTT, 0.5%
1 ml of NP40) was added and suspended, and the mixture was ice-bathed for 30 minutes. After sonication, centrifuge at 100k x g rpm for 60 minutes at 4 ° C, and add 1 liter of 50 to the supernatant.
It was dialyzed against mM KCl soln B (excluding NP40) at 4 ° C. overnight. Dialyze the product with DEAE TOYOPEARL (TOHSOH, bed volume 1m
l) It was added to the column and the non-adsorbed fraction was collected and used as the PKR protein fraction. Aliquoted and stored at -80 ° C until use.

Next, the wild-type NS5A RNA and each NS5A mutant type
RNA was prepared. pET22b / NS5A DNA or pET22b / NS5
A S2401A DNA, pET22b / NS5A S2404A DNA, pET22b / NS5A
Restriction enzyme for S2406A DNA and pET22b / NS5A S2409A DNA
It was linearized by digesting with ScaI at 37 ° C for 24 hours. After phenol / chloroform extraction, ethanol precipitation and dissolution in distilled water were performed to prepare 1 μg / μl. Then you got the DN
Using the A sample as a template, RNA was synthesized in vitro using T7 MEGAscript kit (Ambion). Specifically, the substances shown in Table 1 (including T7 MEGAscript kit (Ambion)) were mixed and reacted at 37 ° C. for 3 hours. 115 μl of distilled water (DW) was added to the reaction solution, and 15 μl of NH ₄ OAc was further added to stop the reaction. After extraction with phenol / chloroform, isopropanol was precipitated and dissolved in water to prepare 1 μg / μl.

[0110]

[Table 1] ―――――――――――――――― Linear template DNA 2 (1 μg) × 10 Rxn. Buffer 2 ATP (75mM) 2 CTP 2 GTP 0.4 UTP 2 Cap analog (40mM) 3 Enz. Mix 2 Distilled water (DW) 4.6 ―――――――――――――――― 20 μl total volume ――――――――――――――――

Next, the samples shown in Table 2 (Rabbit Reticuloc
yte Lysate Nuclease Treated kit (including Promega; Catalog No. L4960)) Coaster 96 well black plate
After mixing for 45 minutes at 30 ° C., 15 μl of the sample in Table 3, 75 μl of Steady-Glo (Promega) and PBS (+) 60
Add μl, gently mix the plate for 5 minutes at room temperature, and add micr
Luc activity was measured with obeta (WALLAC).

As a result of the examination, the Luc activity was significantly increased in the NS5A2404 protein mutant and the NS5A2406 protein mutant as compared to the wild-type NS5A protein (eIF2
α protein activity is increased).
That is, it was revealed that these protein mutants had a significantly higher ability to suppress the enzymatic activity of the PKR protein than the wild-type protein (Fig. 1).

[0113]

[Table 2] ―――――――――――――――――――― Rabbit reticulocyte lysate 10.5 μl Amino acid mixture except Leu (1 mM) 0.1 Amino acid mixture excluding Met (1 mM) 0.1 NS5A RNA 1 (final concentration 0, 0.1, 0.3, 1 μg) Distilled water (DW) 0.3 ―――――――――――――――――――― Total volume 12 μl ――――――――――――――――――――

[0114]

[Table 3] ―――――――――――――――――――――――――― PKR protein fraction (1 μg / μl) 1 (final concentration 1 μg) Luc RNA (0.1 μg / μl) 0.2 (final concentration 20 ng) poly (I). (C) (dsRNA) (100 μg / ml) 0.15 (final concentration 1 μg / ml) Distilled water (DW) 1.65 ―――――――――――――――――――――――――― Total volume 15 μl ――――――――――――――――――――――――――

Example 2 Using different assay systems,
Evaluation of the effect on the enzymatic activity reduction of PKR protein by mutation of the phosphorylation site of NS5A protein, the present inventors, in the NS5A2404 protein mutant strong inhibitory activity of the enzymatic activity of PKR protein, the effect on the enzymatic activity of PKR protein, Evaluation was performed using another assay system that was newly constructed. In this assay system, suppression of the enzymatic activity of the PKR protein by the NS5A protein is evaluated by using the decrease in the activity of the reporter Luc protein due to the decrease in the transcriptional activity of the transcription factor NFκB protein as an index.

[0116] The present inventors firstly expressed inducible NS5A protein mutant expressing cells (Saos-2 Tet-Off cells: expressing NS5A protein mutants, PKR protein, and IκBα / NF).
(a source of κB complex protein) was prepared. p
ET22b / NS5A S2401A, S2404A, S2406A, S2409A, S2413A
The NS5A region was amplified by PCR using DNA as the template and pTRE
Each NS5A amplified fragment was inserted into the XbaI site of the expression vector (manufactured by Clontech). Then, the obtained recombinant plasmid was introduced into Saos-2 Tet Off cells using FuGENE6 (transfection reagent manufactured by Roche).

Specifically, Saos-2 Tet O is applied to a 60 mm petri dish.
ff cells were seeded. (Medium: 10% FCS-DMEM supplemented w
ith 10ng / ml Doxicyclin, 100 μg / ml hygromycin
B, 10000u / ml Penicillin, 10mg / ml Streptomycin) FuG
ENE6 3 μl and OPTI-MEM (GibcoBRL, transfection reagent) 5
0 μl was mixed and left at room temperature for 5 minutes. In the obtained solution, pLXSN
(neomycin (G418) resistant gene expression plasmid) 0.2 μg pT
RE / NS5A (E) (NS5A C-terminal mutant expression plasmid, NS5A C
1.8 μg of a mixture of Etag (Amersham Pharmacia) -expressing sequence ligated at the end) was mixed and left at room temperature for 15 minutes. This solution was added to a petri dish and incubated at 37 ° C for 24 hours, then 1 mg / ml G41
8 (Gibco BRL) was added. After culturing at 37 ° C. for 4 weeks, G418 resistant colonies were selected, and induction of NS5A expression by removal of Doxicyclin was detected by western blot using an Etag antibody (Santa Cruz). The obtained cells were cultured under the conditions of 10% FCS DMEM (low glucose), 1 mg / ml GENETICIN (G418), 100 μg / ml hygromycin B (Hyg) and 50 ng / ml Doxicyclin (Dox). In this example, cells after 16 passages were finally used.

Next, a DNA containing a reporter gene was prepared. Specifically, a promoter region of a gene encoding IFN was ligated upstream of a gene encoding Luc to prepare a DNA, which was named NFκB / Luc.

Next, NFκB / Luc was introduced into inducible NS5A-expressing cells, and Luc activity was measured. Specifically, cells were seeded in 5 225 cm ² flasks containing Dox-containing medium (5 × 10 ⁶ cells per flask, p16 (cell passage number 16)). After culturing for 1 week, the cells were subcultured to 20 225 cm ² flasks containing Dox-containing medium (5 × 10 ⁶ cells per flask, p19).
Next, NFκB / Luc was introduced under the following conditions. OPTI-MEM 15
00 μl / flask FuGENE 660 μl was added and left at room temperature for 5 minutes. Further, 20 μg of NFκB / Luc was introduced and left at room temperature for 15 minutes. 1500 μl / flask was added to the above mixture, and the mixture was cultured at 37 ° C. for 12 hours. 96 well plate (half ar
ea, black, catalog number costar9722) (5 × 10
³ cells / well / 45 μl), Dox was removed (blank: NS5A (-) 4w
ell, control: NS5A (+) 4 well). Next, it was cultured at 37 ° C for 24 hours to induce NS5A. Furthermore, 5 μl per well
1 mg / ml pIC (polyinosinic acid-polycytidylic aci
d; dsRNA (double-stranded RNA) aqueous solution and 1.25 μl per well
10 μM sample was added (final 100 μg / ml pIC, 0.5% DMS
O) and cultured at 37 ° C. for 24 hours. Steady Glo (Promega) 1
50 μl was added per well and Luc activity was measured.

[0120] As a result of the examination, in the NS5A2404 protein mutant, the Luc activity was decreased (N
It was revealed that the FκB protein activity was significantly decreased (FIG. 2). That is, it was demonstrated in different assay systems that the NS5A2404 protein mutant had a significantly higher ability to suppress the enzymatic activity of the PKR protein than the wild-type protein.

Example 3 Evaluation of Efficacy as Evaluation Method and Screening Method of Anti-HCV Agent Candidate Compound In the same assay system as in Example 2, NS5A2404 protein mutant was used to suppress the enzymatic activity of PKR protein The effects of activity and expression level of NS5A protein mutant were examined. The activity of PKR protein and the expression level of NS5A protein mutant were controlled by the pIC treatment time and the induction time of NS5A protein mutant, respectively. As a result of the investigation, depending on the pIC treatment time and the induction time of the NS5A protein mutant, the NS5A2404 protein mutant
It was found that the degree of decrease in Luc activity was increased (Figs. 3 to 5). That is, by the NS5A2404 protein variant
It was found that the inhibition of the enzymatic activity of the PKR protein depends on the activity of the PKR protein and the expression level of the NS5A protein mutant. When evaluating and screening an anti-HCV agent candidate compound that inhibits the NS5A protein variant from inhibiting the PKR protein enzyme activity, it is desirable that the NS5A protein variant exhibits a high degree of inhibition of the PKR protein enzyme activity. This example shows that the assay system using the NS5A protein mutant is effective as a highly sensitive evaluation method and screening method for anti-HCV agent candidate compounds.

In an assay system similar to that of the Examples, when the anti-HCV agent acts directly on the NS5A protein (including the mutant), for example, when the agent binds to NS5A, when the NS5A protein is not expressed, It does not change the Luc amount. On the other hand, when the drug acts on other elements including PKR protein, for example, when it binds to PKR protein, even when NS5A protein is not expressed
Change the Luc amount.

A drug that directly acts on a virus-derived protein is expected to have fewer side effects than one that acts on a human endogenous substance, which is one of desirable modes. In the present assay system, the NS5A protein ( It is possible to evaluate or screen an anti-HCV agent that directly acts on (including mutants).

Example 4 NS5A Protein, ISDR Deletion NS
Comparison of binding strength between 5A protein mutant and C-terminal deleted NS5A protein mutant and PKR protein In order to reconfirm the binding between NSDRA of IS5A protein and PKR protein, an ISDR deletion mutant (Fig. 6B) was prepared. I confirmed. Specifically, GST-K296 was forcibly expressed in E. coli.
R protein extract (wild-type PKR protein cannot be forcibly expressed in cells because it suppresses translation, so an inactive mutant of PKR protein (K296R) with a GST tag linked to the N-terminus was used). Resin that specifically adsorbs GST tag (gl
utathionesepahrose) was mixed to form a complex.

Next, NS ³⁵ labeled with [ ³⁵ S] -Met was added to the complex.
A protein was mixed, and NS5A protein and PKR protein were allowed to react with each other. Furthermore, the binding complex of NS5A protein and PKR protein was washed with PBS (-) containing 1% NP40, the resulting immunoprecipitate was electrophoresed, and the radioactivity on the gel was measured. As a result of the examination, even in the ISDR deletion mutant, almost the same binding as the wild type was observed (FIG. 7). So IS
In order to identify the NS5A-PKR binding region other than the DR region, an NS5A protein having about 69 amino acids deleted from the C-terminus was prepared, and the binding between the protein and the PKR protein was measured. It turned out (Figure 7). Therefore, it was revealed that the PKR protein binds to the C-terminal region of NS5A protein. This result shows that the NS5A protein
It is a finding clearly showing that the C-terminal region has a greater contribution to the binding to the PKR protein than the conventionally known ISDR region.

Considering the statistical findings that HCV1b type has low efficiency of IFN (IFN resistance) and high HCV2b type (IFN sensitivity), and the findings obtained in this example, NS
It is speculated that agents that act on the C-terminus of the 5A protein, such as agents that inhibit the binding between the C-terminus of the NS5A protein and the PKR protein, reduce IFN resistance.

Example 5 Evaluation of Interaction between NS5A Protein and IRF-1 Protein Induction of IFN through the transcription factor IRF-1 protein is known (Nature, 337 (6204), 270-2, 1989. ). Also, IFN
The IRF-1 protein binding sequence is present in the promoter region of the gene. Furthermore, it was suggested that NS5A protein may exert an effect on HCV drug resistance by suppressing IRF-1 protein activity (PNAS, 99 (7), 4650-5, 200
2).

The present inventors have reported that the interaction of the NS5A protein mutant with the IRF-1 protein was reported by the Lucif reporter.
The erase (Luc) protein enzyme activity was evaluated as an index. Specifically, first, in the same manner as in Example 2, preparation of inducible NS5A protein mutant-expressing cells (Saos-2 Tet-Off cells: expressing NS5A protein mutant and serving as a source of IRF-1). I went.

Next, a DNA containing a reporter gene was prepared. Specifically, IRF-1 protein binding sequence, which is the promoter of the gene encoding IFN, was ligated upstream of the gene encoding Luc protein to prepare DNA, which was named IRF1 / Luc. Next, IRF1 / Luc was introduced into cells expressing the inducible NS5A protein mutant, and Luc activity was measured. Specifically, cells were seeded in a 225 cm ² flask containing Dox-containing medium (5 × 10 ⁶ cells per flask, p16
(Cell passage number 16)). After culturing for 1 week, the cells were subcultured in a 225 cm ² flask containing a medium containing Dox (5 × 10 ⁶ cells per flask, p19). Next, IRF1 / Luc was introduced under the following conditions. OPTI-MEM 1500 μl / flask FuGENE6 60 μl
Was added and left at room temperature for 5 minutes. Furthermore, 20 IRF1 / Luc
μg was introduced and left at room temperature for 15 minutes. 1500 above mixture
μl / flask was added, and the mixture was cultured at 37 ° C. for 12 hours. 96 well plate (full area, black, catalog number cos
tar3916) (1 × 10 ⁴ cells / well / 80 μl) and Dox was removed (blank: NS5A (-) 4well, control: NS5A (+) 4wel
l). Next, it was cultured at 37 ° C for 24 hours to induce the NS5A protein mutant. In addition, 1000 IU / ml IFN per well
(Sumiferon 300 (registered trademark)) was cultured at 37 ° C. for 24 hours. Steady Glo (Promega) was added at 50 μl per well, and Luc activity was measured. Here, a clone having a low expression level and a clone having a high expression level when the NS5A protein mutant was induced were selected from a plurality of clones (FIG. 8), and the same test was performed for each clone. After inducing the expression of each clone for 24 hours, a cell lysate was prepared and subjected to electrophoresis, and the expression of NS5A protein was detected by Western blotting.

As a result of the examination, a decrease in Luc activity was confirmed in NS5A (+) as compared with NS5A (-) (Fig. 9). Also, NS5A
Clones with low expression of protein variants (Fig. 9A)
Of the NS5A protein mutants, the stronger Lu was compared with the clones with higher expression levels of NS5A protein mutants.
A decrease in c activity was seen. That is, the presence of the NS5A protein variant mediated the interaction of IFN with the IRF-1 protein.
It was proved to suppress the transcription of. Therefore, by canceling the interaction of the NS5A protein mutant with the IRF-1 protein, it becomes possible to provide an anti-HCV agent by a novel mechanism.

From the above results, the NS5A protein is
It was suggested that it suppresses both two transcription factors, IRF-1 protein and NFκB protein, required for gene expression.

[Example 6] Evaluation of interaction between NS5A protein and ISGF3 complex Induction of PKR protein via the transcription factor ISGF3 protein is known. In addition, in the promoter region of the PKR gene, ISRE (Interfero
n Stimulated Response Elements) exists. Furthermore, the ISGF3 protein is STAT1 (p91 or p84), STAT2 (p91
113), a heterotrimer of IRF9 (p48) (Virology 22
7 (1), 119-130, 1997, PNS,, 91 (11), 1994).

The present inventors evaluated the interaction of the NS5A protein variant with the ISGF3 protein, using the reporter Luc protein as an indicator. First, DNA containing a reporter gene was prepared. Specifically, ISRE, which is the promoter of the gene encoding the PKR protein, was ligated upstream of the gene encoding the Luc protein to prepare a DNA, which was named ISRE / Luc. Next, as in Example 5,
ISRE / Luc was introduced into cells expressing the inducible NS5A2404 protein mutant, and Luc activity was measured.

As a result of the examination, a decrease in Luc activity was confirmed in NS5A (+) as compared with NS5A (-) (Fig. 10). Also, NS5
A clone with low expression level of A protein mutant (Fig. 10).
Comparing A) with many clones (Fig. 10B), NS5A
A stronger decrease in Luc activity was observed in the clones in which the amount of protein mutants expressed was higher. That is, it was demonstrated that the presence of the NS5A protein mutant represses the transcription of the PKR protein via the ISGF3 protein. Therefore, NS5A
By canceling the interaction of the protein variant with the ISGF3 protein or its constituent proteins (STAT1, STAT2, IRF-9), it becomes possible to provide an anti-HCV agent by a novel mechanism.

The results of Examples 5 and 6 demonstrate that the NS5A protein is anti-HCV of IFN via a PKR protein independent pathway.
It strongly suggests the possibility of inhibiting the activity.

[0136]

INDUSTRIAL APPLICABILITY The present invention provides a mutant of NS5A protein having an enhanced ability to suppress the enzymatic activity of PKR protein. Furthermore, an unprecedented highly sensitive anti-HCV agent candidate compound evaluation method and screening method, which use as an index the inhibition of the enzymatic activity of PKR protein using the mutant, were provided. The method of the present invention is very useful for identifying an anti-HCV agent candidate compound, and it is expected that the identified compound can be an effective anti-HCV agent.

[0137]

[Sequence list]                                SEQUENCE LISTING <110> Japan Tobacco, Inc. <120> Mutants of hepatitis C virus NS5A protein, and use thereof <130> J1-A0102 <140> <141> <150> JP2001-225613 <151> 2001-07-26 <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 1341 <212> DNA <213> Hepatitis C virus <220> <221> CDS <222> (1) .. (1341) <400> 1 tcc ggc tcg tgg cta agg gat gtt tgg gat tgg ata tgc acg gtg ttg 48 Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Ile Cys Thr Val Leu   1 5 10 15 act gac ttc aag acc tgg ctc cag tcc aaa ctc ctg ccg cgg tta ccg 96 Thr Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu Leu Pro Arg Leu Pro              20 25 30 gga gtc cct ttc ctg tca tgc caa cgc ggg tac aag gga gtc tgg cgg 144 Gly Val Pro Phe Leu Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg          35 40 45 ggg gac ggc atc atg caa acc acc tgc cca tgc gga gca cag atc gcc 192 Gly Asp Gly Ile Met Gln Thr Thr Cys Pro Cys Gly Ala Gln Ile Ala      50 55 60 gga cat gtc aaa aac ggt tcc atg agg atc gta ggg cct aga acc tgc 240 Gly His Val Lys Asn Gly Ser Met Arg Ile Val Gly Pro Arg Thr Cys  65 70 75 80 agc aac acg tgg cac gga acg ttc ccc atc aac gca tac acc acg gga 288 Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly                  85 90 95 cct tgc aca ccc tcc ccg gcg ccc aac tat tcc agg gcg cta tgg cgg 336 Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg             100 105 110 gtg gct gct gag gag tac gtg gag gtt acg cgt gtg ggg gat ttc cac 384 Val Ala Ala Glu Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His         115 120 125 tac gtg acg ggc atg acc act gac aac gta aag tgc cca tgc cag gtt 432 Tyr Val Thr Gly Met Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val     130 135 140 ccg gcc ccc gaa ttc ttc acg gag gtg gat gga gtg cgg ttg cac agg 480 Pro Ala Pro Glu Phe Phe Thr Glu Val Asp Gly Val Arg Leu His Arg 145 150 155 160 tac gct ccg gcg tgc aaa cct ctt cta cgg gag gac gtc acg ttc cag 528 Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Glu Asp Val Thr Phe Gln                 165 170 175 gtc ggg ctc aac caa tac ttg gtc ggg tcg cag ctc cca tgc gag ccc 576 Val Gly Leu Asn Gln Tyr Leu Val Gly Ser Gln Leu Pro Cys Glu Pro             180 185 190 gaa ccg gac gta aca gtg ctt act tcc atg ctc acc gat ccc tcc cac 624 Glu Pro Asp Val Thr Val Leu Thr Ser Met Leu Thr Asp Pro Ser His         195 200 205 att aca gca gag acg gct aag cgt agg ctg gct aga ggg tct ccc ccc 672 Ile Thr Ala Glu Thr Ala Lys Arg Arg Leu Ala Arg Gly Ser Pro Pro     210 215 220 tct tta gcc agc tca tca gct agc cag ttg tct gcg cct tct ttg aag 720 Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys 225 230 235 240 gcg aca tgc act acc cac cat gac tcc ccg gac gct gac ctc atc gag 768 Ala Thr Cys Thr Thr His His Asp Ser Pro Asp Ala Asp Leu Ile Glu                 245 250 255 gcc aac ctc ttg tgg cgg cag gag atg ggc gga aac atc act cgc gtg 816 Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val             260 265 270 gag tca gag aat aag gta gta att ctg gac tct ttc gaa ccg ctt cac 864 Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Glu Pro Leu His         275 280 285 gcg gag ggg gat gag agg gag ata tcc gtc gcg gcg gag atc ctg cga 912 Ala Glu Gly Asp Glu Arg Glu Ile Ser Val Ala Ala Glu Ile Leu Arg     290 295 300 aaa tcc agg aag ttc ccc tca gcg ttg ccc ata tgg gca cgc ccg gac 960 Lys Ser Arg Lys Phe Pro Ser Ala Leu Pro Ile Trp Ala Arg Pro Asp 305 310 315 320 tac aat cct cca ctg cta gag tcc tgg aag gac ccg gac tac gtc cct 1008 Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro                 325 330 335 ccg gtg gta cac gga tgc cca ttg cca cct acc aag gct cct cca ata 1056 Pro Val Val His Gly Cys Pro Leu Pro Pro Thr Lys Ala Pro Pro Ile             340 345 350 cca cct cca cgg aga aag agg acg gtt gtc ctg aca gaa tcc aat gtg 1104 Pro Pro Pro Arg Arg Lys Arg Thr Val Val Leu Thr Glu Ser Asn Val         355 360 365 tct tct gcc ttg gcg gag ctc gcc act aag acc ttc ggt agc tcc gga 1152 Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Gly     370 375 380 tcg tcg gcc gtt gat agc ggc acg gcg acc gcc ctt cct gac ctg gcc 1200 Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Ala Leu Pro Asp Leu Ala 385 390 395 400 tcc gac gac ggt gac aaa gga tcc gac gtt gag tcg tac tcc tcc atg 1248 Ser Asp Asp Gly Asp Lys Gly Ser Asp Val Glu Ser Tyr Ser Ser Met                 405 410 415 ccc ccc ctt gaa ggg gag ccg ggg gac ccc gat ctc agc gac ggg tct 1296 Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser             420 425 430 tgg tct acc gtg agt gag gag gct agt gag gat gtc gtc tgc tgc 1341 Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val Cys Cys         435 440 445 <210> 2 <211> 447 <212> PRT <213> Hepatitis C virus <400> 2 Ser Gly Ser Trp Leu Arg Asp Val Trp Asp Trp Ile Cys Thr Val Leu   1 5 10 15 Thr Asp Phe Lys Thr Trp Leu Gln Ser Lys Leu Leu Pro Arg Leu Pro              20 25 30 Gly Val Pro Phe Leu Ser Cys Gln Arg Gly Tyr Lys Gly Val Trp Arg          35 40 45 Gly Asp Gly Ile Met Gln Thr Thr Cys Pro Cys Gly Ala Gln Ile Ala      50 55 60 Gly His Val Lys Asn Gly Ser Met Arg Ile Val Gly Pro Arg Thr Cys  65 70 75 80 Ser Asn Thr Trp His Gly Thr Phe Pro Ile Asn Ala Tyr Thr Thr Gly                  85 90 95 Pro Cys Thr Pro Ser Pro Ala Pro Asn Tyr Ser Arg Ala Leu Trp Arg             100 105 110 Val Ala Ala Glu Glu Tyr Val Glu Val Thr Arg Val Gly Asp Phe His         115 120 125 Tyr Val Thr Gly Met Thr Thr Asp Asn Val Lys Cys Pro Cys Gln Val     130 135 140 Pro Ala Pro Glu Phe Phe Thr Glu Val Asp Gly Val Arg Leu His Arg 145 150 155 160 Tyr Ala Pro Ala Cys Lys Pro Leu Leu Arg Glu Asp Val Thr Phe Gln                 165 170 175 Val Gly Leu Asn Gln Tyr Leu Val Gly Ser Gln Leu Pro Cys Glu Pro             180 185 190 Glu Pro Asp Val Thr Val Leu Thr Ser Met Leu Thr Asp Pro Ser His         195 200 205 Ile Thr Ala Glu Thr Ala Lys Arg Arg Leu Ala Arg Gly Ser Pro Pro     210 215 220 Ser Leu Ala Ser Ser Ser Ala Ser Gln Leu Ser Ala Pro Ser Leu Lys 225 230 235 240 Ala Thr Cys Thr Thr His His Asp Ser Pro Asp Ala Asp Leu Ile Glu                 245 250 255 Ala Asn Leu Leu Trp Arg Gln Glu Met Gly Gly Asn Ile Thr Arg Val             260 265 270 Glu Ser Glu Asn Lys Val Val Ile Leu Asp Ser Phe Glu Pro Leu His         275 280 285 Ala Glu Gly Asp Glu Arg Glu Ile Ser Val Ala Ala Glu Ile Leu Arg     290 295 300 Lys Ser Arg Lys Phe Pro Ser Ala Leu Pro Ile Trp Ala Arg Pro Asp 305 310 315 320 Tyr Asn Pro Pro Leu Leu Glu Ser Trp Lys Asp Pro Asp Tyr Val Pro                 325 330 335 Pro Val Val His Gly Cys Pro Leu Pro Pro Thr Lys Ala Pro Pro Ile             340 345 350 Pro Pro Pro Arg Arg Lys Arg Thr Val Val Leu Thr Glu Ser Asn Val         355 360 365 Ser Ser Ala Leu Ala Glu Leu Ala Thr Lys Thr Phe Gly Ser Ser Gly     370 375 380 Ser Ser Ala Val Asp Ser Gly Thr Ala Thr Ala Leu Pro Asp Leu Ala 385 390 395 400 Ser Asp Asp Gly Asp Lys Gly Ser Asp Val Glu Ser Tyr Ser Ser Met                 405 410 415 Pro Pro Leu Glu Gly Glu Pro Gly Asp Pro Asp Leu Ser Asp Gly Ser             420 425 430 Trp Ser Thr Val Ser Glu Glu Ala Ser Glu Asp Val Val Cys Cys         435 440 445 <210> 3 <211> 11 <212> PRT <213> Hepatitis C virus <400> 3 Asp Leu Ser Asp Gly Ala Trp Ser Thr Val Ser   1 5 10 <210> 4 <211> 11 <212> PRT <213> Hepatitis C virus <400> 4 Ser Asp Gly Ser Trp Ala Thr Val Ser Glu Glu   1 5 10

[Brief description of drawings]

FIG. 1 is a diagram showing a comparison between a wild-type NS5A protein and each NS5A protein mutant in a translation system reporter gene assay. The vertical axis shows the value of Luck activity in a sample containing neither PKR nor NS5A protein, which is 100%.
Represents the value (%) of the relative Luc amount. The abscissa control shows samples that do not contain NS5A protein (including PKR protein). Wild type indicates a sample containing a wild-type NS5A protein of HCV1b type (1973aa-2419aa). S2401A, S2404A, S2406A, S2409A, and S2413A are wild type NS5A proteins 2401, 2404, 240.
The sample containing the protein variant which substituted the serine residue of 6,2409, and 2413 positions by the alanine residue is shown.

[Fig. 2] NS in transcription-based reporter gene assay
It is a figure which shows the comparison of 5A2404 protein mutant (A) and wild type NS5A protein (B). The vertical axis shows the relative value when the Luc activity of the sample to which pIC is not added is 1. The horizontal axis is from the left, control, NS5A (-), NS5A
(+). The control is a sample without pIC added. NS5A (−) represents a sample containing no NS5A protein, and NS5A (+) represents a sample containing an NS5A protein.

FIG. 3 is a diagram showing reduction in phosphorylation activity of PKR protein by NS5A2404 protein mutant. The vertical axis is Luc
The actual measured value (RLV) of the activity is shown. From the left, the horizontal axis is the sample without pIC and without NS5A protein variant, the sample without pIC and with NS5A protein variant, the sample with pIC and with NS5A protein variant, the pIC and without NS5A protein variant. The sample of addition is shown. A
Is 30 hours after the induction of the NS5A protein mutant and 13 hours after the addition of pIC. B is the NS5A protein mutant induction 30
After 24 hours and 24 hours after the addition of pIC.

FIG. 4 is a diagram showing reduction in phosphorylation activity of PKR protein by NS5A2404 protein mutant. The vertical axis is Luc
The actual measured value of the activity is shown. The horizontal axis is from the left with no pIC added and NS5
Sample without A protein mutant, NS without pIC and NS
5A protein mutant added sample, pIC added and NS5A
A sample with addition of a protein variant and a sample with addition of pIC and no addition of NS5A protein variant are shown. A is 43 hours after induction of NS5A protein mutant and 9 hours after addition of pIC. B is 43 hours after induction of NS5A protein mutant and 24 hours after addition of pIC.

FIG. 5 is a diagram showing reduction in phosphorylation activity of PKR protein by NS5A2404 protein mutant. The vertical axis is Luc
The actual measured value of the activity is shown. The horizontal axis is from the left with no pIC added and NS5
Sample without A protein mutant, NS without pIC and NS
5A protein mutant added sample, pIC added and NS5A
A sample with addition of a protein variant and a sample with addition of pIC and no addition of NS5A protein variant are shown. A is 54 hours after the NS5A protein variant induction and 13 hours after the addition of pIC. B is 54 hours after induction of NS5A protein mutant and 24 hours after addition of pIC.

FIG. 6A is a diagram showing a comparison of amino acids of NSVA proteins of HCV1b type and HCV2a type. * Indicates HCV1b type and HCV
It shows that the amino acid residues of the 2a type are the same. B)
FIG. 4 is a diagram showing primary structures of a wild-type NS5A protein (NS5A1b type), an ISDR deletion mutant, and a C-terminal deletion mutant. V3 in Fig. A is a motif name of the primary amino acid sequence, and it is reported that this region regulates IFN resistance (Nousbaum, J. et. Al., Prospective characteriza
tion of full-length hepatitis Cvirus NS5A quasispe
cies during induction and combination antiviral th
erapy. J Virol. 74: 9028-9038, 2000, Duverlie, G.
et.al., Sequence analysis of the NS5A protein of
European hepatitis C virus 1b isolates and relatio
n to interferon sensitivity. J Gen Virol. 79: 1373
-1381, 1998) is known.

FIG. 7 is a diagram comparing the binding strengths of wild-type NS5A protein (NS5A1b type), ISDR deletion mutant, and C-terminal deletion mutant with PKR protein. The vertical axis shows the precipitation rate of the complex of wild-type NS5A protein and PKR protein (wild-type NS5A
The relative value when the binding rate of A protein and PKR protein) is set to 100% is shown. From the left, the horizontal axis is the wild-type NS5A protein, ISDR deletion mutant, C-terminal deletion mutant and HCV2a.
A sample containing type NS5A protein is shown.

FIG. 8 is a photograph comparing the expression levels of NS5A protein among clones. A clone with a low expression level (A) and a clone with a high expression level (B) are shown.

FIG. 9 shows the effect of NS5A protein on IRF-1 activity. The vertical axis shows the measured value of Luc activity as an index of IRF-1 activity. The horizontal axis shows the time after IFN treatment. NS5
A (+) indicates a sample containing NS5A protein,
NS5A (-) indicates a sample containing no NS5A protein. FIG. 9A and FIG. 9B show the results for clones with low and high expression levels of NS5A protein, respectively.

FIG. 10 shows the effect of NS5A protein on ISGF3 activity. The vertical axis shows the measured value of Luc activity as an index of ISGF3 activity. The horizontal axis shows the time after IFN treatment. N
S5A (+) indicates a sample containing NS5A protein, and NS5A (-) indicates a sample not containing NS5A protein. FIG. 10A and FIG. 10B show the results for clones with low and high expression levels of NS5A protein, respectively.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI Theme Coat (Reference) C07K 14/18 C12N 1/15 4C084 C12N 1/15 1/19 4H045 1/19 1/21 1/21 C12P 21/02 C 5/10 C12Q 1/48 Z C12P 21/02 G01N 33/15 Z C12Q 1/48 33/50 Z G01N 33/15 33/53 M 33/50 33/566 33/53 33/576 Z 33/566 C12N 15/00 ZNAA 33/576 5/00 A (72) Inventor Toru Noguchi 1-1 No. 1 Murasakicho, Takatsuki City, Osaka Prefecture Japan Tobacco Inc. Pharmaceutical Research Institute F Term (reference) 2G045 AA34 AA35 AA40 BA11 BB50 DA12 DA13 DA14 DA36 FB02 4B024 AA11 BA80 CA01 CA06 DA03 EA04 FA06 FA10 HA01 4B063 QA05 QQ27 QQ95 QR07 QR58 QR77 QR80 QS05 QS38 QX02 4B064 AG01 CA10 CA19 CC24 DA13 4B065 A46 AC25 CAA CABA CAA CAB 20 MA02 NA05 NA15 ZA752 ZB332 ZC202 4H045 AA10 AA20 AA30 BA10 BA50 CA02 EA50 FA72 FA74

Claims (34)

[Claims] 1. A DNA encoding a variant of the hepatitis C virus NS5A protein, the variant having an increased ability to suppress the enzyme activity of the double-stranded RNA-dependent protein phosphorylating enzyme by the mutation. 2. The DNA according to claim 1, wherein the mutation is a phosphorylation site mutation. 3. The DNA according to claim 1, wherein the mutation is present on the C-terminal side of the protein. 4. The DNA according to claim 1, wherein the variant consists of an amino acid sequence containing at least one mutation in the serine residues at positions 2404 and 2406 in the amino acid sequence of SEQ ID NO: 2. 5. The DNA according to claim 4, wherein the mutation in the serine residue is a substitution with an alanine residue. 6. The DNA according to claim 1, wherein the variant comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 3 or 4. 7. A vector having the DNA according to any one of claims 1 to 6 inserted therein. 8. A transformant carrying the DNA according to any one of claims 1 to 6 or the vector according to claim 7. 9. The transformant according to claim 8, which is capable of inducibly expressing the DNA according to any one of claims 1 to 6. 10. The transformant according to claim 8, which expresses a double-stranded RNA-dependent protein kinase, NFκB protein, and IκBα protein. 11. The transformant according to claim 10, wherein a DNA functionally linked to a reporter gene is introduced downstream of the NFκB protein binding region. 12. The transformant according to claim 8 or 9, which expresses IRF-1 protein. 13. The transformant according to claim 12, wherein a DNA functionally linked to a reporter gene is introduced downstream of the IRF-1 protein binding sequence. 14. A transformant expressing an IRF-1 protein, wherein the DNA functionally linked to a reporter gene is introduced downstream of the IRF-1 protein binding sequence, and the following (i) or (ii): ). (I) DNA encoding the hepatitis C virus NS5A protein
Alternatively, a transformant carrying a vector into which the DNA has been inserted. (Ii) D encoding the hepatitis C virus NS5A protein
The transformant according to (i), which can express NA inducibly. 15. The transformant according to claim 8 or 9, which expresses the ISGF3 protein. 16. The transformant according to claim 15, wherein a DNA functionally linked to a reporter gene is introduced downstream of ISRE. 17. A transformant expressing an ISGF3 protein, wherein the DNA functionally linked to a reporter gene is introduced downstream of ISRE, the following (i) or (i):
The transformant according to i). (I) DNA encoding the hepatitis C virus NS5A protein
Alternatively, a transformant carrying a vector into which the DNA has been inserted. (Ii) D encoding the hepatitis C virus NS5A protein
The transformant according to (i), which can express NA inducibly. 18. The DNA according to any one of claims 1 to 6.
A variant of hepatitis C virus NS5A protein encoded by. 19. The method for producing the mutant according to claim 18, which comprises a step of recovering the expressed protein from the transformant according to claim 8 or the culture supernatant thereof. 20. A method for evaluating the anti-hepatitis C virus activity of a test sample, which comprises (a) the mutant according to claim 18 and double-stranded RNA dependence in the presence of the test sample. Contacting with a protein phosphatase, and (b) the NS
A step of detecting the binding between the 5A protein variant and the double-stranded RNA-dependent protein kinase, wherein the test sample binds the NS5A protein variant and the double-stranded RNA-dependent protein kinase When the test sample is inhibited, the test sample is evaluated as having anti-hepatitis C virus activity. 21. A method for evaluating the anti-hepatitis C virus activity of a test sample, comprising: (a) the mutant and double-stranded RNA-dependent protein of claim 18 in the presence of the test sample. A test sample comprising contacting with a phosphorylase, and (b) detecting the enzymatic activity of the double-stranded RNA-dependent protein phosphorylase, A method in which the test sample is evaluated as having anti-hepatitis C virus activity when the enzyme activity is increased. 22. A method for evaluating the anti-hepatitis C virus activity of a test sample, comprising: (a) providing the transformant according to claim 11, and (b) testing the transformant. When the test sample increases the reporter activity, the test sample contains anti-hepatitis C virus activity, which comprises a step of contacting the sample, and (c) detecting a reporter activity in the transformant. Methods that are evaluated as having. 23. A method for evaluating the anti-hepatitis C virus activity of a test sample, which comprises (a) the mutant of claim 18, the double-stranded RNA-dependent protein kinase, eIF2α.
When the test sample reduces the reporter activity, the test sample includes a step of adding the test sample to a solution containing a protein and a reporter RNA, and (b) a step of detecting the reporter activity in the solution. Is evaluated to have anti-hepatitis C virus activity. 24. A method for evaluating the anti-hepatitis C virus activity of a test sample, comprising the step of: (a) in the presence of the test sample;
The step of contacting the hepatitis B virus NS5A protein or the mutant of claim 18 with an IRF-1 protein, and (b) the step of detecting the activity of the IRF-1 protein, wherein the test sample is the IRF-1. A method in which the test sample is evaluated as having anti-hepatitis C virus activity when the activity of the protein is increased. 25. A method for evaluating the anti-hepatitis C virus activity of a test sample, the method comprising: (a) providing the transformant according to claim 13 or 14, and (b) providing the transformant. The method comprises contacting a test sample, and (c) detecting a reporter activity in the transformant, wherein when the test sample increases the reporter activity, the test sample contains anti-hepatitis C virus. A method that is evaluated to have activity. 26. A method for evaluating the anti-hepatitis C virus activity of a test sample, comprising the step of: (a) in the presence of the test sample, C
The hepatitis B virus NS5A protein or the mutant according to claim 18 and ISGF3 protein, STAT1 protein, STAT2
A step of contacting a protein or an IRF-9 protein, and (b) detecting the activity of the ISGF3 protein, wherein the test sample increases the activity of the ISGF3 protein, A method evaluated to have hepatitis C virus activity. 27. A method for evaluating the anti-hepatitis C virus activity of a test sample, comprising: (a) a step of providing the transformant according to claim 16 or 17, and (b) the transformant. The method comprises contacting a test sample, and (c) detecting a reporter activity in the transformant, wherein when the test sample increases the reporter activity, the test sample contains anti-hepatitis C virus. A method that is evaluated to have activity. 28. A method for screening a compound having anti-hepatitis C virus activity, which comprises (a) 20.
27. A method of evaluating anti-hepatitis C virus activity of a plurality of test samples by the method according to any one of 27, and (b) evaluation of having anti-hepatitis C virus activity from the plurality of test samples. Selecting the compound described above. 29. A compound having anti-hepatitis C virus activity, which is identified by the method according to any one of claims 20 to 28. 30. C-terminal side of NS5A protein and double-stranded RNA
A compound having anti-hepatitis C virus activity, which is characterized by inhibiting the binding to a dependent protein kinase. 31. A compound having anti-hepatitis C virus activity, which is characterized by inhibiting the interaction with double-stranded RNA-dependent protein kinase by acting directly on NS5A protein. 32. The compound according to claim 30 or 31, wherein the NS5A protein is the mutant according to claim 18. 33. An anti-hepatitis C virus agent containing the compound according to any one of claims 29 to 32 as an active ingredient. 34. The anti-hepatitis C virus agent according to claim 33, which comprises a combination with another antiviral agent.