JP2008161080A - Assay method for detecting hepatitis C virus inhibitors - Google Patents

Abstract

The present invention relates to a test capable of detecting and searching for an inhibitor / therapeutic agent of HCV or a drug having an effect of promoting the growth of HCV targeting any process selected from all processes of the HCV growth ring. It is an object to provide a method.
According to a test method, human HCV-derived Huh7 cells, a test specimen and infectious HCV are contacted and then HCV is measured. HCV contains a core protein and two envelope proteins (E1, E2) as well as nonstructural proteins required for replication (NS2, NS3, NS4, NS5), and the HCV RNA genome is upstream of the sequence encoding the core protein. It is based on an infectious HCV replicon genome in which a PolI promoter sequence and a PolI terminator sequence are arranged downstream.
[Selection] Figure 1

Description

  The present invention relates to an assay method for detecting a hepatitis C virus inhibitor that targets any process selected from the entire process of the growth cycle of hepatitis C virus. More precisely, the present invention relates to an assay method capable of searching for various substances that affect any step of HCV growth from HCV infection to replication, translation, HCV protein translation, maturation, modification, assembly, and virus release. .

  Hepatitis C virus (HCV) is a single-stranded RNA virus and belongs to the genus Hepacivirus in the Flaviviridae family. It is classified into 6 genotypes from HCV 1-6. Furthermore, their genotypes are classified into several subtypes (Hepatology, 10, p1321-1324 (1994); Hepatology, 42, p962-973 (2005)). Currently, the full-length genome sequences of a plurality of HCV genotypes have been determined (Science, 244, p359-362 (1989); J. Med. Virol., P334-339 (1992); J. Gen Virol). ., 73, p673-679 (1992); Hepatology, 16, 293-299 (1992)).

  HCV has a single-stranded RNA of about 9.4 kb as its genome. The untranslated region existing at both ends of the genome has a secondary structure, and the 5 ′ end has no cap structure observed in normal mRNA. Instead, rRNA can initiate RNA translation in a cap-independent manner. There is an IRES (Internal Robosomal Entry Site). A huge precursor protein consisting of about 3000 amino acids is translated from viral RNA and incorporated into the core protein and two envelope proteins (E1, E2) that constitute the viral particle by host cell-derived proteolytic enzymes, and into the particle However, nonstructural proteins (NS proteins) necessary for replication are cleaved into proteins by proteolytic enzymes possessed by NS2 and NS3.

  With the introduction of HAART (Highly Active Antiretroviral Therapy), the prognosis of AIDS patients has been greatly improved. As a result, in developed countries including Japan, even in human immunodeficiency virus (HIV) infected people, The number of deaths is increasing rapidly. In particular, the combination of liver damage and liver failure due to co-infected HCV has become an important part of the cause of death. In particular, 97% of HIV and HCV co-infections occur in HIV-infected individuals with non-heated coagulation factor preparations, and treatment of HCV infection is an important medical issue in the post-HAART era. Currently, combination therapy of interferon and ribavirin (Ribavirin (1-β-D-ribofuranosyl-1H-1,2,4-tri-azole-3-carboxamide)) is common for HCV infection. It is effective only for 50% of all patients and is known to cause serious side effects such as cytopenia and hemolytic anemia.

  Until now, the absence of viral culture systems and experimental small animals for HCV has hindered basic research on HCV. Kolyhalov et al. Reported a Kunjin virus replicon system belonging to the same Flaviviridae family as HCV (Non-patent Document 1). This system removes the structural region of Kunjin virus and replaces 3'utr with EMCV's IRES and neomycin resistance gene. The RNA produced from this gene construction is introduced into cultured cells and selectively cultured with neomycin. This is an experimental system in which continuous replication and protein expression of Kunjin virus RNA are observed. In 1999, Lohamann et al. Reported an HCV replicon system (Non-patent Document 2). In this system, a part of the structural region (NS2) of HCV is removed, and an EMCV IRES and a neomycin resistance gene are inserted into that part. By synthesizing RNA in a test tube using this structure as a template, introducing it into Huh7 cells, selectively culturing it in a neomycin selective medium, introducing RNA made from this gene structure into cultured cells, and selectively culturing it with neomycin. The neomycin resistance gene is expressed from the replicated HCV replicon RNA, and only the cells in which the replicon is replicated can survive by the function of the gene to form a proliferated colony. In the replicon-replicating cells, the HCV replicon RNA is replicated at a very high level, and the replicating RNA and the expressed HCV protein can be detected continuously and stably.

  However, until then, all HCV strains that had been reported to grow as replicons were genotype 1 strains. Furthermore, these strains have adaptive mutations that enhance their replication efficiency when grown in cultured cells, and many of these adaptive mutations were not found in HCV strains isolated from patient sera. Moreover, when the applicable range was introduced into an infectious clone and the RNA was inoculated into a chimpanzee liver, it was revealed that the infectivity was lost.

  It has been reported that HCV with different genotypes also has differences in the encoded viral proteins, and only the analysis of the subgenomic replicon derived from genotype 1b HCV can sufficiently elucidate the replication mechanism of HCV. It seems difficult. Furthermore, since the therapeutic effect of interferon varies depending on the genotype of HCV, the development of anti-HCV drugs that exert effects on various types of HCV using only the HCV replication system containing the subgenomic replicon of genotype 1b HCV Is considered particularly difficult. Therefore, it is necessary to prepare HCV replicons of many genotypes and to study HCV replication mechanisms and anti-HCV drugs.

Thereafter, various HCV replicons using genes of HCV strains of other genotypes have been reported. In particular, there are reports on HCV replicons that synthesize RNA of infectious clones using T7 RNA polymerase (Non-patent Documents 3 and 4, Patent Document 1). However, in the conventional replicon system, it was possible to detect any inhibitor that acts in the late stage after HCV infection, but HCV targeting any process selected from all the processes in the growth ring. Therefore, a new assay method has been desired.
J. Virol., 71, 1497-1505 (1997) Science 285, p.110-113 (1999) Nature Medicine 11, Number 7, July p.791-796 (2005) Virus Volume 55 Issue 2 pp.287-296 (2005) International Publication WO2005 / 028652 Pamphlet

  The present invention provides a test method that enables detection and search of an HCV inhibitor / therapeutic agent or a drug having an HCV growth-promoting effect that targets any process selected from the entire process of the HCV growth ring. The task is to do.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have made contact with human hepatoma-derived Huh7 cells, test specimens, and infectious HCV, and then measured HCV. The present inventors have found an assay method for detecting, and completed the present invention. Infectious HCV is due to an infectious HCV replicon obtained from the infectious HCV replicon genome.

That is, this invention consists of the following.
1. An assay method for detecting an HCV inhibitor, which comprises measuring human HCV after contacting human hepatoma-derived Huh7 cells, a test sample and infectious HCV.
2. Assay method for detecting HCV inhibitors comprising the following steps:
1) a step of seeding Huh7 cells derived from human liver cancer;
2) adding the test specimen to the medium containing the seeded cells;
3) further seeding infectious HCV;
4) heating at 37 ± 1 ° C .;
5) A step of collecting the culture supernatant and measuring HCV.
3. 3. The assay method according to item 1 or 2, wherein the human hepatoma-derived Huh7 cell is a Huh7.5.1 cell.
4). Infectious HCV contains a core protein and two envelope proteins (E1, E2) as well as a nonstructural protein (NS protein) required for replication, and the HCV RNA genome has a promoter sequence upstream of the sequence encoding the core protein. 4. The assay method according to any one of items 1 to 3, which is an infectious HCV replicon genome.
5. 5. The assay method according to item 4 above, wherein the promoter arranged in the infectious HCV replicon genome is PolI promoter or T7 promoter.
6). 6. The assay method according to 4 or 5 above, wherein the infectious HCV replicon genome has a terminator arranged downstream of the gene region encoding NS5 protein.
7). 7. The assay method according to any one of 1 to 6 above, wherein the HCV measurement is to measure the amount of HCV core protein.

  According to the assay method for detecting an HCV inhibitor of the present invention, various substances that affect HCV replication or HCV protein translation can be searched and detected. As a result, the assay method of the present invention can be used as a test system for screening a new HCV inhibitor / therapeutic agent from a compound library, and any one selected from the entire process of the HCV growth cycle. Enables screening of new HCV inhibitors / therapeutics targeting the process. For example, HCV inhibitors listed in Table 1 could be screened by the assay method of the present invention.

Hereinafter, the present invention will be described in detail.
1. Infectious HCV
In the present invention, infectious HCV literally refers to HCV having infectivity. As long as the infectious HCV of the present invention has an autonomous replication ability in human hepatoma-derived Huh7 cells, the genotype of HCV may be any of the six types of genotypes 1 to 6, and each of them. The type may be any of several subtypes. Furthermore, HCV isolated from a patient with fulminant hepatitis may be used instead of a general genotype classification.

  In the present specification, HCV isolated from a patient with fulminant hepatitis means HCV isolated from a patient who exhibits a symptom that can be defined as fulminant hepatitis by those skilled in the art. In the present invention, the HCV derived from a patient with fulminant hepatitis may be a virus having not only a naturally derived HCV RNA genome but also an RNA genome obtained by artificially modifying the RNA genome sequence of naturally derived HCV. Specific examples of fulminant strains of HCV include viruses such as JFH-1 strain (Japanese Patent Laid-Open No. 2002-171978), JFH-2.1 strain and JFH-2.2 strain.

  The RNA genome of HCV has a 5 ′ untranslated region (5′NTR or 5′UTR), a translated region composed of a structural region and a nonstructural region, and a 3 ′ untranslated region (3′NTR or 3′UTR). Consists of. The structural region encodes HCV structural proteins such as core protein, E1 protein, and E2 protein, and the nonstructural region encodes nonstructural proteins such as NS2, NS3, NS4, and NS5. Has been.

  The infectious HCV of the present invention is preferably prepared from an HCV gene prepared by modifying the RNA genome of HCV in order to enable assay using cell culture. An HCV produced by such modification is referred to as “infectious HCV replicon” in the present specification, and a genome produced by modification is also referred to as “infectious HCV replicon genome”.

  The infectious HCV replicon of the present invention comprises at least a core protein and two envelope proteins (E1, E2) and nonstructural proteins necessary for replication (NS2, NS3, NS4, NS5), and the genome constituting the HCV, The infectious HCV replicon genome has a promoter sequence placed upstream of the sequence encoding the core protein (see FIG. 1).

  The promoter sequence is not particularly limited as long as it is arranged so that HCV can exhibit autonomous replication ability in human hepatoma-derived Huh7 cells. For example, a PolI promoter sequence or a T7 promoter sequence is particularly preferable. A PolI promoter sequence may be mentioned.

  In one embodiment of the infectious HCV replicon genome according to the present invention, a terminator sequence may be arranged downstream of, for example, a sequence encoding NS protein in addition to the above sequence. Specifically, a PolI terminator sequence or a T7 terminator sequence can be mentioned. Furthermore, one IRES sequence may be included upstream of the sequence encoding the core protein, and one selectable marker gene or reporter gene may be further included therein.

  In the present invention, the “selection marker gene” means a gene that can give a cell selectivity such that only cells in which the gene is expressed are selected. A common example of a selectable marker gene is an antibiotic resistance gene. Examples of selection marker genes suitable in the present invention include zeocin resistance gene, neomycin resistance gene, thymidine kinase gene, kanamycin resistance gene, pyrithyamine resistance gene, adenylyltransferase gene, puromycin resistance gene, etc. A resistance gene, a neomycin resistance gene, and a thymidine kinase gene are preferable, and a zeocin resistance gene is more preferable. However, the selection marker gene in the present invention is not limited to these.

  In the present invention, the “reporter gene” means a marker gene that encodes a gene product that serves as an index of gene expression. General examples of reporter genes include structural genes of enzymes that catalyze luminescence and color reactions. Examples of reporter genes suitable in the present invention include chloramphenicol acetyltransferase gene derived from transposon Tn9, β-glucuronidase or β-galactosidase gene derived from E. coli, luciferase gene, green fluorescent protein gene, jellyfish-derived ichlion gene, secretory type Examples include placental alkaline phosphatase (SEAP) gene. However, the reporter gene in the present invention is not limited to these.

Only one or both of the above selectable marker gene and reporter gene may be contained in the infectious HCV replicon genome of the present invention.
The “IRES sequence” in the present invention means an internal ribosome binding site capable of binding a ribosome inside RNA to initiate translation. Preferable examples of the IRES sequence in the present invention include, but are not limited to, EMCV IRES (internal ribosome binding site of encephalomyocarditis virus), FMDV IRES, HCV IRES, etc., but EMCV IRES, And HCV IRES are more preferred, and EMCV IRES is most preferred.

  The infectious HCV replicon genome according to the present invention may further contain a ribozyme. The ribozyme is inserted so as to link the selectable marker gene, reporter gene, or foreign gene in the 5 ′ replicon RNA with the IRES sequence 3 ′ from that, and the sequence encoding each NS protein. Both can be cut and separated by the self-cleaving activity.

  In the infectious HCV replicon genome according to the present invention, a selection marker gene, a reporter gene, a sequence encoding a viral protein on the HCV RNA genome, a foreign gene, and the like are translated from the replicon RNA in a correct reading frame. Are connected to each other. Among these sequences, the protein-encoding sequence is expressed as a fusion protein together with a polyprotein translated from the HCV NS protein-encoding sequence, and then separated into each protein by a protease. They may be connected to each other through a cutting site or the like.

2. Production of infectious HCV replicon genome The infectious HCV replicon genome according to the infectious HCV replicon of the present invention can be produced using any genetic engineering technique known to those skilled in the art.

3. Production of infectious HCV replicon The infectious HCV replicon of the present invention can be produced by introducing the produced infectious HCV replicon genome into cells. The cell into which the infectious HCV replicon genome is introduced is preferably a human hepatoma-derived Huh7 cell. For performing a more sensitive assay, Huh 7.5.1 cells (PNAS, Vol. 102, no. 26 p9294-9299 (2005)) are particularly suitable. The RNA genome can be introduced into cells using any technique known to those skilled in the art. Examples of such introduction methods include electroporation, particle gun method, lipofection method, calcium phosphate method, microinjection method, DEAE sepharose method, and the like, and a method by electroporation is particularly preferable.

  When introducing an infectious HCV replicon genome into cells, the target infectious HCV replicon genome may be introduced alone, or a mixture with other nucleic acids may be introduced. If you want to change the amount of infectious HCV replicon genome while keeping the amount of RNA to be introduced constant, mix the target infectious HCV replicon genome with total cellular RNA extracted from the cells to be introduced and introduce it into the cell. Can be used. The amount of replicon RNA used for intracellular introduction may be determined according to the introduction method to be used, but preferably 1 picogram to 100 micrograms, more preferably 10 picograms to 10 micrograms.

  When introducing an infectious HCV replicon genome into cells to produce HCV, an expression vector capable of expressing the entire HCV genome can be used. Specifically, a sequence containing the full-length cDNA of the infectious HCV RNA genome of the present invention is inserted into a plasmid. For example, clones of JFH-2.1 and JFH-2.2 strains were prepared from HCV strains isolated from patients with fulminant hepatitis by the method described in Non-Patent Document 2, for example, synthesis of PolI promoter sequence and terminator sequence. DNA can be added to the 5 'and 3' ends of each clone and inserted into the plasmid pUC19 plasmid.

  When using an infectious HCV replicon genome containing a selectable marker gene or reporter gene for introduction into the cell, use the expression of the selectable marker gene or reporter gene for the cells into which the genome has been introduced and are continuously replicating Then you can choose. Specifically, for example, the cells subjected to the intracellular introduction treatment of the genome may be cultured in a medium that can be selected by expression of a selection marker gene or a reporter gene.

  As an example, when a zeocin resistance gene or a neomycin resistance gene is included as a selection marker gene in the infectious HCV replicon genome, cells introduced into the cell using the genome are seeded in a culture dish for 16 to 24 hours. After culturing, antibiotics such as zeocin and neomycin are added to the culture dish at a concentration of 0.05 to 3.0 mg / ml, and then the culture is continued about 2 to 4 times a week while the culture medium is changed. From time to time, preferably 10 to 40 days, more preferably 14 to 28 days after culturing, viable cells are stained with crystal violet, so that cells in which the RNA genome has been introduced and are continuously replicated are selected as colonies can do. From the formed colony, cells can be cloned by a conventional method.

  For the established cell clone, detection of the genome replicated in the cell clone from the introduced infectious HCV replicon genome and integration of the selectable marker gene or reporter gene in the introduced genome into the host genomic DNA It is preferable to confirm that the target infectious HCV replicon genome is actually replicated by confirming the presence or absence of the HCV protein and confirming the expression of the HCV protein.

  Detection of the infectious HCV replicon genome replicated in the cell clone from the introduced infectious HCV replicon genome may be performed according to any RNA detection method known to those skilled in the art. For example, total RNA extracted from cell clones can be detected by performing a Northern hybridization method using a DNA fragment specific for the introduced infectious HCV replicon genome as a probe.

  The integration of the introduced infectious HCV replicon genome into the host genomic DNA can be confirmed, for example, by detecting the expression of HCV protein.

  The expression of HCV protein can be detected, for example, by reacting an antibody against the HCV protein to be expressed from the introduced replicon genome with a protein extracted from a cell clone. This method can be performed by any protein detection method known to those skilled in the art. Specifically, for example, a protein sample extracted from a cell clone is blotted on a nitrocellulose membrane, and anti-HCV protein is compared thereto. It can be performed by reacting an antibody (for example, an anti-NS3-specific antibody or an antiserum collected from a patient with hepatitis C) and detecting the anti-HCV protein antibody. If HCV protein is detected from the protein extracted from the cell clone, it can be determined that the infectious HCV-derived replicon genome is continuously replicated to express the HCV protein.

  As described above, it is possible to obtain a cell clone (replicon-replicating cell clone) that has been confirmed to continuously replicate the target infectious HCV replicon genome. In the present invention, the replicon RNA can be obtained by any method known to those skilled in the art, for example, extracting RNA from the replicon-replicating cells, and separating the replicon RNA therefrom by electrophoresis. it can. Furthermore, the replicon-replicating cells according to the present invention can be suitably used for producing HCV protein. Those skilled in the art can produce HCV protein from replicon-replicating cells according to a conventional method. Specifically, for example, a replicon-replicating cell is cultured, and a protein can be obtained from the resulting culture (including cultured cells and medium) by a conventional method.

  As a cell for reintroducing the target infectious HCV replicon genome and collecting infectious HCV in the culture supernatant, human hepatoma-derived Huh7 cells are suitable. In order to perform a more sensitive assay, Huh 7.5.1 cells (PNAS, Vol. 102, no. 26 p9294-9299 (2005)) are particularly suitable. Introduction of the infectious HCV replicon genome into cells can be performed using any technique known to those skilled in the art.

4). Assay Method of the Present Invention The assay for detecting the HCV inhibitor according to the present invention can be carried out by contacting human liver cancer-derived Huh7 cells, a test sample and the infectious HCV of the present invention and then measuring HCV. it can.
More specifically, it can be performed as follows.
1) a step of seeding Huh7 cells derived from human liver cancer;
2) adding the test specimen to the medium containing the seeded cells;
3) further seeding infectious HCV;
4) heating at 37 ± 1 ° C .;
5) A step of collecting the culture supernatant and measuring HCV.

The assay method will be described in more detail.
1) As the above-mentioned human hepatoma-derived Huh7 cells, Huh7.5.1 cells (PNAS, Vol.102, no.26 p9294-9299 (2005)) are used in order to perform the assay method of the present invention with higher sensitivity. Particularly preferred. The cells used preferably have passage numbers up to the 43rd. The cells can be suspended in the cell culture medium and seeded in an assay container, such as a 96-well microplate.
2) The test sample may be a high molecular compound or a low molecular compound as long as it is a candidate substance for searching for a substance that promotes or suppresses replication of HCV. Alternatively, it may be a natural product, a natural product derivative, or a culture extract such as radiobacteria. The concentration of the test sample can be appropriately selected.
3) Furthermore, by inoculating the infectious HCV of the present invention, replicate cells containing the test specimen and the infectious HCV can be cultured, and the infectious HCV in the resulting culture can be detected. Infectious HCV can be made as described above.
4) In order to culture infectious HCV in cells, it can be warmed at 37 ± 1 ° C. for 3-10 days, preferably 4-7 days, more preferably 5-6 days.
5) After the above cultivation, the assay of the present invention can be performed by collecting the culture supernatant and measuring HCV. The measurement of HCV is not particularly limited as long as it can confirm the promotion or suppression of HCV replication. For example, the amount of HCV core protein produced can be confirmed by ELISA using a commercially available kit. can do.

  Furthermore, in addition to the assay method described above, in order to more reliably confirm the effect of the test sample on the promotion or suppression of HCV replication, a cytotoxicity test was conducted in parallel to eliminate non-specific effects. Also good. Such an assay method can be used as a screening method for evaluating HCV infection inhibitors, such as HCV therapeutic agents and diagnostic agents. Specifically, the following examples are given.

(1) Search for substances that inhibit HCV growth Examples of substances that inhibit HCV growth include organic compounds that directly or indirectly affect HCV growth, or target sequences of the HCV genome or its complementary strand. And antisense oligonucleotides that directly or indirectly affect the proliferation of HCV or the translation of HCV proteins.

(2) Evaluation of various substances having antiviral activity in cell culture Examples of the various substances include substances obtained by rational drug design or high-throughput screening (for example, isolated and purified enzymes). It is done.

(3) Evaluation of ability to acquire resistance to drugs such as anti-HCV agents and identification of mutations related to the resistance can be performed.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto.

(Example 1) Assay method 1) Preparation of infectious HCV replicon genome
In accordance with the method described in PNAS, Vol. 102, no. 26 p9294-9299 (2005), a cDNA expression plasmid introduced cell line (Huh7.5.1 / JFH1 zeocin) of the JFH1 strain (genotype 2a) using the RNA PolI promoter / terminator system. Infectious HCV was prepared from the infectious HCV replicon genome shown in FIG. The HCV stock solution was dispensed and stored at -80 ° C.

2) Assay method Huh7 · 5 · 1 cells (passage number up to the 43rd passage) suspended in DMEM medium containing 10% FCS to 1 × 10 ⁴ cells / 100 μl / well Was seeded in a 96-well microplate, and the medium was changed after 1-day culture. To each of these cells, each test compound was added as a specimen so as to be 5 μM. After standing for 15 minutes, the infectious HCV obtained by 1) above (about 0.2 fmol / well as the amount of HCV core protein) was seeded on Huh7 · 5 · 1 cells to infect, and incubated at 37 ° C., 5% CO _2. For 5-6 days. After culturing, the amount of HCV core protein in the supernatant was quantified by an ortho HCV antigen ELISA test, and the HCV proliferation inhibitory effect of each compound was evaluated.

(Experimental example 1) HCV inhibitory effect and cytotoxicity test results
The compounds possessed by Enamine (Namiki Shoji) were assayed according to the method of Example 2. IFN-α (final concentration 100 to 200 Units / ml) was used as a positive control for HCV growth inhibitory factor, and DMSO was used as a solvent as a negative control. Cytotoxicity tests on Huh7 · 5.1 cells were performed by the WST method in which the absorbance of formazan produced by the tetrazolium salt WST-8 was directly measured. In particular, the results of four typical compounds are shown in Table 1.

  According to the assay method for detecting an HCV inhibitor of the present invention, various substances that affect HCV replication or HCV protein translation can be searched and detected. As a result, the assay method of the present invention can be used as a test system for screening a new HCV inhibitor / therapeutic agent from a compound library, and any one selected from the entire process of the HCV growth cycle. It enables screening for new HCV inhibitors / therapeutic agents that target the process.

It is a figure which shows the structure of the infectious HCV replicon genome of this invention, and an expression plasmid. It is a figure which shows the assay method of this invention.

Explanation of symbols

a (Fig. 2) Test specimen to be screened. For example, low molecular weight compound libraries and other test samples (5 μM)
b (Fig. 2) Huh7.5.1 / pHH-JFH1 Zeocin resistant cell culture supernatant

Claims (7)

An assay method for detecting a hepatitis C virus inhibitor comprising measuring human hepatitis C virus after contacting human hepatoma-derived Huh7 cells, a test specimen, and infectious hepatitis C virus. An assay method for detecting a hepatitis C virus inhibitor comprising the following steps:
1) a step of seeding Huh7 cells derived from human liver cancer;
2) adding the test specimen to the medium containing the seeded cells;
3) further disseminating infectious hepatitis C virus;
4) heating at 37 ± 1 ° C .;
5) A step of collecting the culture supernatant and measuring hepatitis C virus. The assay method according to claim 1 or 2, wherein the human hepatoma-derived Huh7 cells are Huh7.5.1 cells. An infectious hepatitis C virus contains a core protein and two envelope proteins (E1, E2) as well as a nonstructural protein (NS protein) required for replication, and the genome of the hepatitis C virus encodes the core protein The assay method according to any one of claims 1 to 3, which is an infectious hepatitis C virus replicon genome in which a promoter sequence is arranged upstream of. The assay method according to claim 4, wherein the promoter sequence arranged in the infectious hepatitis C virus replicon genome is a sequence of PolI promoter or T7 promoter. The assay method according to claim 4 or 5, wherein the infectious hepatitis C virus replicon genome comprises a terminator sequence located downstream of the gene region encoding NS5 protein. The assay method according to any one of claims 1 to 6, wherein the measurement of hepatitis C virus measures the amount of hepatitis C virus core protein.