Patent Application
20110289610
The present invention relates to a chimeric RNA derived from RNAs of hepatitis C virus (this may be hereinafter referred to as “HCV”) and GB virus B (this may be hereinafter referred to as “GBV-B”), and a chimeric virus having the chimeric RNA.
HCV is the causative factor of chronic hepatitis C and, according to statistics by WHO, it is assumed that 170 million people are infected therewith. HCV is a virus classified to the genus Flavivirus in the family Flaviviridae, and it is considered that its infection is caused via blood or a blood component, followed by its growth in the liver. In an infected patient, a relatively mild symptom is observed at the initial stage of infection, but it frequently becomes chronic and leads to development of chronic hepatitis after a certain length of asymptomatic period. As the period of infection becomes longer, the condition of the disease becomes worse to cause liver cirrhosis, leading to liver cancer at high frequency. In 95% of liver cancer, hepatitis viruses are involved, and 80% of such cases are considered to be due to infection with HCV.
HCV has a plus-strand RNA of about 9600 bases as the genome, and it is assumed, based on analysis of the gene sequence, there are at least 6 types of genotypes. The genome of about 9600 bases works as mRNA in a host cell, and a continuous polyprotein having a length of about 3000 amino acids is synthesized, which is cleaved by signal peptidases and signal peptidyl peptidases of the host and proteases encoded by the HCV genome. As a result, 10 types of proteins, that is, the core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B are produced. In addition to this translational frame (open reading frame), there exist the untranslated regions (UTRs) in the 5′-end and the 3′-end, which are responsible for the functions of translational regulation and regulation of replication of the genome.
Among these, the core, E1 and E2 are structural proteins constituting the virus. The virus genome is considered to be packaged by the core protein to form a capsid, and surrounded by the E1 and E2 envelope proteins anchored to the lipid bilayer membrane, thereby forming a virus particle (virion). The function of p7 is not clear, but it is reported to be indispensable for growth of the virus. NS2 is a metal protease and necessary for cleavage of itself, but other functions are not known. NS3 to NS5B are considered to form a complex which works as an RNA replication apparatus together with host proteins, thereby replicating the genomic RNA.
For therapy of chronic hepatitis C, interferon is widely used. In recent years, by virtue of improvements in the formulation of interferon and improvements in the administration method such as a combination therapy with interferon and ribavirin, the rate of successful elimination of HCV from the body, leading to complete response is gradually increasing. However, the rate of complete response by administration of interferon is still about five tenth, and there are many cases where serious side effects are caused by administration of interferon and where administration of interferon cannot be applied to an elderly patient, so that development of a therapeutic method and an agent effective for HCV is demanded.
Infection with HCV occurs via blood or a blood component in human, and, in terms of non-human organisms, anthropoids (chimpanzee) are infected with HCV and the infection causes hepatitis, leading to chronic hepatitis in some cases. However, none of small experimental animals which can be easily kept is known to be infected with HCV at a high rate.
On the other hand, it was revealed that inoculation of serum, which was collected from a surgeon who developed acute hepatitis, to a small primate tamarin causes hepatitis therein. By analyzing blood of the monkey suffering from post-transfusion hepatitis with unknown etiology by molecular biological techniques, two types of viruses, GBV-A and GBV-B, were identified (Non-patent Document 1). Among these, GBV-B was revealed to be most closely related to HCV in view of the molecular structure and to infect New World monkeys such as tamarin and marmoset, inducing hepatitis (Non-patent Document 2). Since HCV has a narrow range of host species and there is no suitable animal model for HCV, the animal model of GBV-B and tamarin is considered to be useful as an alternative model for infection and growth of HCV. However, although the structural similarity between GBV-B and HCV has been confirmed, GBV-B has an amino acid homology of as small as about 28% with HCV. Therefore, even if the animal model of GBV-B and tamarin is used as it is as a development and evaluation system for drugs which specifically act on HCV, it is impossible to carry out screening of drugs which specifically act on HCV.
In order to construct an animal model for HCV using GBV-B, attempts are being made to prepare an HCV/GBV-B chimeric virus by, using the genes of GBV-B as the basis, replacing a part of the genes of GBV-B with the corresponding genes of HCV, or inserting a part of the genes of HCV into the genes of GBV-B. Rijnbrand et al. showed that GBV-B in which a part of its 5′-UTR was replaced with the corresponding 5′-UTR of HCV can infect tamarin (Non-patent Document 3). Further, Haqshenas et al. prepared a chimeric virus by inserting the hyper variable region 1 (HVR1) of HCV into HVR1 in the envelope protein E2 of GBV-B, and infection of marmoset by the chimeric virus was confirmed (Non-patent Document 4). However, the genes in these chimeric viruses are mostly genes from GBV-B, and the chimeric viruses do not have the replication function as HCV. Therefore, these chimeric viruses cannot be said to be useful for development of therapeutic agents for HCV.
[Patent Document 1] WO 2008/136470 A1
[Non-patent Document 1] “Journal of Virology” (USA) 1995, vol. 69, pp. 5621-5630.
[Non-patent Document 2] “Virology” (USA) 1999, vol. 262, pp. 470-478.
[Non-patent Document 3] “Hepatology” (USA) 2005, vol. 41, pp. 986-994.
[Non-patent Document 4] Journal of General Virology (UK) 2007, vol. 88, pp. 895-902.
Since no animal model other than chimpanzee exists which is infected with HCV and allows growth of HCV, it has been difficult to efficiently develop the drugs. Further, use of chimpanzee in animal experiments is prohibited by application of the animal protection law. From such reasons, the methods of interferon therapy which are widely used at present have been developed and improved directly using patients as subjects, which have been a heavy burden to the patients. Therefore, in development and evaluation of the drugs, use of small animal models in their preclinical tests is important. GBV-B which infects a small primate tamarin is similar to HCV in terms of the gene structure and induces hepatitis symptoms, so that it is expected to be useful as an animal model for HCV. The present invention aims to provide an HCV/GBV-B chimeric virus which can infect tamarin or marmoset and maintain the replication function of HCV, in order to construct an HCV animal model which can be used as a development or evaluation system for HCV drugs.
The present inventors intensively studied on an HCV/GBV-B chimeric virus which can be used for development of drugs which specifically and effectively act on HCV, to prepare an HCV/GBV-B chimeric RNA by linking an RNA of HCV comprising an RNA encoding the NS4B protein having leucine at the 1804th position and lysine at the 1966th position in the amino acid sequence of the polyprotein of HCV to an RNA of GBV-B, which HCV/GBV-B chimeric RNA maintains the replication function as HCV and is capable of persistently infecting tamarin or marmoset and increasing therein.
The replication efficiency of this HCV/GBV-B chimeric RNA in a human liver cancer-derived cell line was higher than that of HCV which is the parent strain. That is, the present inventors discovered that the obtained HCV/GBV-B chimeric RNA sufficiently maintains the replication function of HCV.
Further, when this HCV/GBV-B chimeric RNA was introduced to primary hepatocytes of marmoset, the HCV/GBV-B chimeric RNA increased autonomously in the cells, while persistently releasing the core protein in the cell supernatant. That is, the present inventors discovered that transfection of an HCV/GBV-B chimeric RNA into primary hepatocytes of marmoset enables production of an HCV/GBV-B chimeric virus which is capable of reinfection.
The present invention is based on such discoveries.
Thus, the present invention relates to an HCV/GBV-B chimeric RNA comprising an RNA of HCV and an RNA of GB virus-B, the RNA of HCV comprising an RNA encoding the NS4B protein having leucine at the 1804th position and lysine at the 1966th position in the amino acid sequence of the polyprotein of HCV.
A preferred embodiment of the HCV/GBV-B chimeric RNA of the present invention comprises:
(A) an HCV 5′-side RNA comprising an RNA of the 5′-UTR of HCV;
(B) a GBV-B RNA comprising an RNA encoding the E1 protein and the E2 protein of GBV-B; and
(C) an HCV 3′-side RNA comprising an RNA encoding the NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein, and an RNA of the 3′-UTR, of HCV;
the GBV-B RNA (B) being inserted between the HCV 5′-side RNA (A) and the HCV 3′-side RNA (C).
In another preferred embodiment of the HCV/GBV-B chimeric RNA of the present invention,
the HCV 5′-side RNA (A) comprises an RNA of the 5′-UTR and an RNA encoding a part or all of the core protein;
the GBV-B RNA (B) comprises an RNA encoding a part of the core protein, and the E1 protein, E2 protein and p6 protein; and
the HCV 3′-side RNA (C) comprises an RNA encoding the p7 protein, NS2 protein, NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein, and an RNA of the 3′-UTR;
which HCV/GBV-B chimeric RNA is preferably an RNA having the base sequence shown in SEQ ID NO:55.
The present invention also relates to an HCV/GBV-B minus-strand chimeric RNA which is complementary to the HCV/GBV-B chimeric RNA.
The present invention also relates to an HCV/GBV-B chimeric virus comprising the HCV/GBV-B chimeric RNA.
The present invention also relates to an HCV/GBV-B chimeric DNA encoding the HCV/GBV-B chimeric RNA.
The present invention also relates to the HCV/GBV-B chimeric protein translated from the RNA, or from the DNA according to claim 7.
The present invention also relates to an HCV/GBV-B chimeric RNA—replicating cell comprising the HCV/GBV-B chimeric RNA.
The present invention also relates to a non-human animal to which the HCV/GBV-B chimeric RNA, or the HCV/GBV-B chimeric virus according to claim 6 was inoculated.
In the present specification, “HCV/GBV-B chimeric gene” means both an HCV/GBV-B chimeric RNA and an HCV/GBV-B chimeric DNA.
By the HCV/GBV-B chimeric RNA of the present invention, it is possible to provide a replicon RNA which can replicate in a human liver-derived cell in vitro at high efficiency. Further, HCV/GBV-B chimeric virus particles can be produced which can autonomously replicate and is capable of persistent infection and reinfection in primary hepatocytes of marmoset.
By using the HCV/GBV-B chimeric virus of the present invention, an HCV animal model using a small primate such as tamarin or marmoset can be constructed. By using this animal model, it is possible to carry out not only basic research of HCV, but also development and evaluation of drugs to suppress or inhibit aggravation of hepatitis, thereby enabling development and evaluation of more effective prophylactic agents and therapeutic agents for the infection.
The present invention also provides cells in which this HCV/GBV-B chimeric gene can replicate. Using the HCV/GBV-B chimeric replication system of the cells in which this HCV/GBV-B chimeric gene replicates, drugs to suppress growth of HCV can be screened. Further, the animal model is also effective as a method to evaluate the effect of the drugs screened. In this case, it is important to carry out evaluation of the drugs using this method, and, if the method is indispensable for control of the drugs, it can also be used as a method to produce the drugs.
The HCV/GBV-B chimeric RNA of the present invention is constituted by an RNA of HCV and an RNA of GBV-B linked to each other, and comprises an RNA encoding the NS4B protein having leucine at the 1804th position and lysine at the 1966th position in the amino acid sequence of the polyprotein of HCV. The HCV/GBV-B chimeric RNA of the present invention encodes a virus particle that can grow in a hepatocyte of a primate mammal such as tamarin or marmoset.
In the term “leucine at the 1804th position and lysine at the 1966th position”, each of the numbers indicates the amino acid No. in the full length polyprotein of HCV having 3010 amino acids. The leucine at the 1804th position and the lysine at the 1966th position are amino acids contained in the NS4B protein. The present inventors previously reported an RNA of HCV which has a base sequence encoding these amino acids (Patent Document 1), and an NS4B protein containing these amino acids had not been reported until then. Therefore, an HCV polyprotein containing these amino acids, and an RNA replicon containing a polynucleotide encoding these amino acids had not been reported, too.
The polyprotein of HCV comprises, for example, in HCV of genotype 1b having 3010 amino acids, a core protein having the sequence of the 1st to 191st amino acids, E1 protein having the sequence of the 192nd to 383rd amino acids, E2 protein having the sequence of the 384th to 746th amino acids, p7 protein having the sequence of the 747th to 809th amino acids, NS2 protein having the sequence of the 810th to 1026th amino acids, NS3 protein having the sequence of the 1027th to 1657th amino acids, NS4A protein having the sequence of the 1658th to 1711st amino acids, NS4B protein having the sequence of the 1712nd to 1972nd amino acids, NS5A protein having the sequence of the 1973rd to 2419th amino acids, or NS5B protein having the sequence of the 2420th to 3010th amino acids. In the present specification, the polyprotein of HCV means a continuous protein translated from the RNA of HCV, and, for example, in HCV of genotype 1b, it is a protein having 3010 amino acids.
Since the HCV/GBV-B chimeric RNA of the present invention comprises leucine at the 1804th position and lysine at the 1966th position in the full length polyprotein of HCV having 3010 amino acids, it can replicate in a cell derived from human liver, and can autonomously replicate in a primary hepatocyte of marmoset to produce an HCV/GBV-B chimeric virus particle capable of reinfection.
The HCV/GBV-B chimeric RNA may further contain an RNA that does not substantially inhibit replication of, and infection with, the chimeric RNA. For example, it may contain a selection marker gene, a reporter gene or an IRES sequence.
The HCV/GBV-B chimeric RNA of the present invention is not restricted as long as it contains the polynucleotide encoding leucine at the 1804th position and lysine at the 1966th position, and it preferably comprises a 5′-side RNA of HCV, a GBV-B RNA, and a 3′-side RNA of HCV. It is more preferably a chimeric RNA having the nucleotide sequence shown in SEQ ID NO:55.
The 5′-side RNA of HCV comprises at least an RNA of the 5′-UTR of HCV. It may further comprise an RNA encoding a part or all of the core protein of HCV.
In terms of the gene of HCV, for example, the HCV gene of a common genotype 1b is composed of an RNA of the 5′-UTR (1st to 341st positions); followed by an RNA encoding the core protein (342nd to 914th positions), RNA encoding the E1 protein (915th to 1490th positions) and RNA encoding the E2 protein (1941st to 2579th positions), which are structural proteins of the virus, and an RNA encoding the p7 protein (2580th to 2768th positions), RNA encoding the NS2 protein (2769th to 3419th positions), RNA encoding the NS3 protein (3420th to 5312nd positions), RNA encoding the NS4A protein (5313rd to 5474th positions), RNA encoding the NS4B protein (5475th to 6257th positions), RNA encoding the NS5A protein (6258th to 7598th positions) and RNA encoding the NS5B protein (7599th to 9371st positions), which are nonstructural proteins; and further, an RNA of the 3′-UTR (the 9372nd and following positions).
The 5′-UTR is usually composed of 341 nucleotides in the HCV gene of genotype 1b, and the RNA in the 5′-side of HCV preferably comprises the total length of its nucleotide sequence.
Further, the RNA encoding the core protein is composed of the 573 nucleotides at the 342nd to 914th positions, and the RNA in the 5′-side of HCV may comprise all or a part of the nucleotides.
The nucleotide sequence of the RNA in the 5′-side of HCV is not restricted and has a homology of preferably not less than 90%, more preferably not less than 93%, most preferably not less than 95% to the nucleotide sequence of the corresponding region in SEQ ID NO:55. Since the HCV/GBV-B chimeric RNA comprises the 5′-side RNA of HCV, drugs which inhibit the function of the RNA in the 5′-UTR or the RNA encoding the core protein can be screened.
The GBV-B RNA preferably comprises at least an RNA encoding the E1 protein and an RNA encoding the E2 protein. It may further comprise a part or all of the RNA encoding the core protein and/or a part or all of the RNA encoding the p6 protein.
The gene of GBV-B is composed of an RNA of the 5′-UTR (1st to 445th positions); followed by an RNA encoding the core protein (446th to 913rd positions), RNA encoding the E1 protein (914th to 1489th positions) and RNA encoding the E2 protein (1490th to 2284th positions), which are structural proteins of the virus, and an RNA encoding the p6 protein (2285th to 2452nd positions), RNA encoding the p7 protein (2453rd to 2641st positions), an RNA encoding the NS2 protein (2642nd to 3265th positions), RNA encoding the NS3 protein (3266th to 5125th positions), RNA encoding the NS4A protein (5126th to 5290th positions), RNA encoding the NS4B protein (5291st to 6034th positions), RNA encoding the NS5A protein (6035th to 7267th positions) and RNA encoding the NS5B protein (7268th to 9037th positions), which are nonstructural proteins; and further, an RNA of the 3′-UTR (the 9038th and following positions). It is structurally different from the gene of HCV that GBV-B has the region encoding the p6 protein.
The RNA encoding the E1 protein of GBV-B is preferably composed of the 576 nucleotides at the 914th to 1489th positions and comprises the full-length sequence of the nucleotides. The RNA encoding the E2 protein is preferably composed of the 795 nucleotides at the 1490th to 2284th positions and comprises the full-length sequence of the nucleotides.
Since the HCV/GBV-B chimeric RNA comprises an RNA encoding the E1 protein and an RNA encoding the E2 protein, it can infect experimental animals such as tamarin and marmoset, or cells derived from these animals.
Further, the RNA encoding the core protein is composed of the 468 nucleotides at the 446th to 913rd positions, and the GBV-B RNA may comprise all or a part of the nucleotides.
Further, the p6 protein is composed of the 168 nucleotides at the 2285th to 2452nd positions, and the GBV-B RNA may comprise all or a part of the nucleotides.
Further, the nucleotide sequence of the GBV-B RNA is not restricted as long as it has the function as the translated protein of GBV-B, that is, as long as the produced chimeric virus has infectivity to experimental animals such as tamarin and marmoset.
The 3′-side RNA of HCV comprises at least an RNA encoding the NS3 protein, RNA encoding the NS4A protein, RNA encoding the NS4B protein, RNA encoding the NS5A protein and RNA encoding the NS5B protein; and an RNA of the 3′-UTR. It may further comprise an RNA encoding the p7 protein of HCV and an RNA encoding the NS2 protein of HCV.
The RNA encoding the p7 protein is composed of the 189 nucleotides at the 2580th to 2768th positions, and may comprise all or a part of the nucleotides. The RNA encoding the NS2 protein is composed of the 651 nucleotides at the 2769th to 3419th positions, and may comprise the total length of the nucleotides.
The RNA encoding the NS3 protein is composed of the 1893 nucleotides at the 3420th to 5312nd positions, and preferably comprises the total length of the nucleotides.
The RNA encoding the NS4A protein is composed of the 162 nucleotides at the 5313rd to 5474th positions, and preferably comprises the total length of the nucleotides.
The RNA encoding the NS4B protein is composed of the 783 nucleotides at the 5475th to 6257th positions, and preferably comprises the total length of the nucleotides.
The RNA encoding the NS5A protein is composed of the 1341 nucleotides at the 6258th to 7598th positions, and preferably comprises the total length of the nucleotides.
The RNA encoding the NS5B protein is composed of the 1773 nucleotides at the 7599th to 9371st positions, and preferably comprises the total length of the nucleotides.
The RNA of the 3′-UTR is the RNA of the 9372nd and following positions, and its length varies depending on the virus strain. It is usually composed of a variable region of 41 nucleotides, a poly-U region whose length varies depending on the strain, and a 3′ X region of 98 nucleotides. The 3′-side RNA of HCV preferably comprises the total length of its 3′-UTR.
The HCV/GBV-B chimeric RNA may comprise, as the RNA encoding the core, an RNA encoding the core of HCV, an RNA encoding the core of GBV-B, or an RNA encoding a chimeric core of HCV and GBV-B. These core proteins translated from the HCV/GBV-B chimeric RNA may be contained in the HCV/GBV-B chimeric virus particle.
The HCV/GBV-B chimeric RNA may comprise all or a part of the RNA encoding the p6 protein of GBV-B, all or a part of the RNA encoding the p7 protein of HCV, and/or all or a part of the RNA encoding the NS2 protein of HCV.
The 3′-side RNA of HCV comprises an RNA encoding leucine at the 1804th position and lysine at the 1966th position in the amino acid sequence of the polyprotein of HCV. These amino acids are contained in the NS4B protein, and it is thought that, by inclusion of an RNA encoding the two amino acid sequences, more preferably, by inclusion of an RNA encoding the NS4B protein having the two amino acid sequences, the HCV/GBV-B chimeric RNA of the present invention can replicate the RNA and produce a chimeric virus in a primary hepatocyte of marmoset.
The nucleotide sequence of the 3′-side RNA of HCV is not restricted, and has a homology of preferably not less than 80%, more preferably not less than 90%, most preferably not less than 95% to the nucleotide sequence of the corresponding region in SEQ ID NO:55.
Further, since the HCV/GBV-B chimeric RNA comprises the 3′-side RNA of HCV, it can be used for screening or evaluation of drugs that inhibit the functions of the nonstructural proteins and of the RNA in the 3′-UTR.
In the HCV/GBV-B chimeric RNA of the present invention, the 5′-side RNA of HCV (A), the GBV-B-RNA (B) and the 3′-side RNA of HCV (C) are preferably linked together in that order. That is, the GBV-B-RNA (B) is preferably inserted between the 5′-side RNA of HCV (A) and the 3′-side RNA of HCV (C).
In the present invention, the genotype of the HCV RNA is not restricted, and preferably genotype 1b of HCV. The HCV gene can be grouped into at least 6 kinds of genotypes based on its nucleotide sequence, and genotype 1b is a subtype belonging to genotype 1. Identification of HCV of genotype 1b is based on the nucleotide sequence of its RNA, and, for example, an HCV with a polynucleotide having a nucleotide sequence showing a homology of not less than 90% to the base sequence of SEQ ID NO:57 is included therein.
The HCV/GBV-B chimeric RNA of the present invention can replicate in a cell, for example, human hepatocyte, tamarin hepatocyte or marmoset hepatocyte. That is, it can also function as a replicon RNA. In cases where it functions as a replicon RNA, the HCV/GBV-B chimeric RNA of the present invention functions as a plus-strand RNA which works as the template for a protein, that is, mRNA. A minus-strand RNA is synthesized from this plus-strand RNA, and, using the minus-strand RNA as a template, a plus-strand RNA can be synthesized. The HCV/GBV-B minus-strand chimeric RNA of the present invention is also useful since it works as a template for an HCV/GBV-B chimeric RNA which is a plus strand.
The HCV/GBV-B chimeric virus of the present invention comprises the HCV/GBV-B chimeric RNA, and may comprise several proteins constituting the virus translated from the HCV/GBV-B chimeric RNA. The proteins constituting the virus are not restricted, and examples thereof include the core protein of HCV; a chimeric core protein composed of a part of the core protein of HCV and a part of the core protein of GBV-B; and the E1 protein and the E2 protein of GBV-B.
The HCV/GBV-B chimeric DNA of the present invention is not restricted as long as it is a DNA corresponding to the HCV/GBV-B chimeric RNA. Examples thereof include a single-stranded cDNA synthesized from the HCV/GBV-B chimeric RNA by reverse transcriptase, a double-stranded DNA composed of the single-stranded cDNA and a complementary strand thereof, and a double-stranded DNA incorporated into a plasmid.
The vector of the present invention is a vector comprising the HCV/GBV-B chimeric DNA. Examples thereof include, but are not limited to, plasmid vectors; linear double-stranded DNA vectors; and virus vectors such as adenovirus vectors, adeno-associated virus vectors, retrovirus vectors and lentivirus vectors; and the vector is preferably a plasmid vector.
The HCV/GBV-B chimeric protein of the present invention is a protein translated from the HCV/GBV-B chimeric RNA, and examples thereof include a single chimeric polyprotein (SEQ ID NO:56) translated from the region in the RNA encoding the protein, or a chimeric core protein composed of a part of the core protein of HCV and a part of the core protein of GBV-B.
The HCV/GBV-B chimeric RNA of the present invention can be prepared using arbitrary genetic engineering techniques. The chimeric RNA can be prepared by, for example, the following method, although the method is not restricted.
A DNA encoding the HCV/GBV-B chimeric RNA is inserted into a cloning vector by a conventional method, to prepare a DNA clone. The obtained DNA is inserted into the downstream of an RNA promoter, to prepare a DNA clone which can produce a replicon RNA. More particularly, for example, a gene is constructed from TPF1 clone (Patent Document 1), which was isolated from a patient suffering from fulminant hepatitis C, by deleting the region from the 156th position in the core protein to the E2 protein. A gene of GBV-B composed of the region from the 124th position in the core protein to the p6 protein is chemically synthesized, and, by inserting and linking the synthesized gene to the portion deleted in HCV, an HCV/GBV-B chimeric gene can be constructed. The RNA promoter is preferably included in the plasmid clone. The RNA promoter is not restricted, and examples thereof include the T7 RNA promoter, SP6 RNA promoter and SP3 RNA promoter, among which the T7 RNA promoter is especially preferred.
Another preferred mode of the present invention is as follows.
That is, in this mode (this may be hereinafter referred to as “E1 fusion type” for convenience), the HCV/GBV-B chimeric RNA comprises:
(A) an HCV 5′-side RNA comprising an RNA of the 5′-UTR of HCV and an RNA encoding the core protein and a part of the E1 protein of HCV;
(B) a GBV-B-RNA comprising an RNA encoding a part of the E1 protein and an RNA encoding the E2 protein of GBV-B; and
(C) an HCV 3′-side RNA comprising an RNA encoding the NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein of HCV, and an RNA of the 3′-UTR of HCV; preferably an RNA encoding the p7 protein, NS2 protein, NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein of HCV, and an RNA of the 3′-UTR of HCV.
The GBV-B RNA (B) is inserted between the HCV 5′-side RNA (A) and the HCV 3′-side RNA (C); the part of the E1 protein encoded by the HCV 5′-side RNA (A) is a part of the N-terminus side of the E1 protein, the part of the E1 protein encoded by the GBV-B RNA (B) is a part of the C-terminus side of the E1 protein; and these both parts are fused together to cover the total length of the E1 protein.
In the E1 fusion type, the RNA of the 5′-UTR and the core protein in the HCV 5′-side RNA (A) are as described above, and these preferably have the nucleotide sequences of the corresponding regions in the nucleotide sequence shown in SEQ ID NO:57 (i.e., the RNA of the 5′-UTR corresponds to the 1st to 341st positions in the nucleotide sequence shown in SEQ ID NO:57, and the coding region of the core protein corresponds to the 342nd to 914th positions in the nucleotide sequence shown in SEQ ID NO:57) or nucleotide sequences having homologies of not less than 90%, more preferably not less than 93%, still more preferably not less than 95%, still more preferably not less than 99% to these nucleotide sequences. The sequences are preferably those with which a virus particle that can grow in a primate hepatocyte can be constructed. The term “homology” of nucleotide sequences means a value calculated by aligning two nucleotide sequences to be compared such that the number of matched bases is the maximum, and dividing the number of the matched bases by the total number of bases, which value is represented in percentage. Upon the above-described alignment, a gap(s) is/are inserted as required into one or both of the two sequences to be compared. Such alignment of sequences can be carried out using a well-known program such as BLAST, FASTA or CLUSTAL W. In cases where a gap(s) is/are inserted, the above-described total number of bases is counted regarding one gap as one base. In cases where the thus counted total number of bases is different between the two sequences to be compared, the homology (%) is calculated by dividing the number of matched bases by the total number of bases in the longer sequence.
In the E1 fusion type, the HCV 5′-side RNA (A) also comprises the region encoding a part of the N-terminus side of the E1 protein. Since the E1 protein of HCV corresponds to the region from the 915th to 1490th positions in the nucleotide sequence shown in SEQ ID NO:57 as described above, the HCV 5′-side RNA (A) preferably has a sequence of a partial region in the 5′-side of this sequence, or a nucleotide sequence having a homology of not less than 90%, more preferably not less than 93%, still more preferably not less than 95%, still more preferably not less than 99% thereto, and can preferably construct a virus particle that can grow in a hepatocyte of primate such as tamarin or marmoset. Here, the region encoding a part of the N-terminus side of the E1 protein is preferably a region of not more than 30 amino acids from the N-terminus of the E1 protein in view of the ability of the virus particle to grow in a primate hepatocyte. In the following Examples, v11-E12 chimeric RNA, which encodes the 11 amino acids from the N-terminus of the E1 protein, and v27-E12 chimeric RNA, which encodes the 27 amino acids from the N-terminus of the E1 protein, were constructed, and both of these were confirmed to have excellent abilities to increase in a primary hepatocyte of marmoset.
The following RNA of GBV-B (GBV-B RNA) (B) encodes a part of the C-terminus side of the E1 protein, and the E2 protein. Here, the term “a part of the C-terminus side” means that the part is located closer to the C-terminus than the partial region of the E1 protein encoded by the HCV 5′-side RNA (A) and, in a preferred mode, the most part of the E1 protein is encoded by the GBV-B RNA (B). The nucleotide sequence of the total length of the GBV-B RNA is shown in SEQ ID NO:99, and the amino acid sequence encoded thereby is shown in SEQ ID NO:100. In the nucleotide sequence shown in SEQ ID NO:99, the E1 protein-coding region corresponds to the 914th to 1489th positions, and the E2 protein-coding region corresponds to the 1490th to 2284th positions. The GBV-B RNA (B) further preferably encodes the p6 protein. In the nucleotide sequence shown in SEQ ID NO:99, the p6 protein-coding region corresponds to the 2285th to 2452nd positions. The GBV-B RNA (B) preferably has the nucleotide sequence of the corresponding region shown in SEQ ID NO:99, or a nucleotide sequence having a homology of not less than 90%, more preferably not less than 93%, still more preferably not less than 95%, still more preferably not less than 99% thereto, and can preferably construct a virus particle that can grow in a primate hepatocyte.
In the E1 fusion type, the E1 protein is encoded by both of the HCV 5′-side RNA (A) and the GBV-B-RNA (B), and the total length of the E1 protein is covered by fusion of the both portions. The region encoded by the both may be partially overlapped with each other. In such a case, the size of the overlapping region is preferably 1 to 24 amino acids. For example, in the v11 chimeric gene prepared in Examples below, the HCV 5′-side RNA (A) encodes the 11 amino acids from the N-terminus of the E1 protein, and, on the other hand, the GBV-B RNA (B) encodes the 3rd and following amino acids from the N-terminus of the E1 protein, so that 8 amino acids are overlapping. Similarly, in v27, the HCV 5′-side RNA (A) encodes the 27 amino acids from the N-terminus of the E1 protein, and, on the other hand, the GBV-B RNA (B) encodes the 3rd and following amino acids from the N-terminus of the E1 protein, so that 24 amino acids are overlapping.
The following HCV 3′-side RNA (C) is the same as described above, and comprises an RNA encoding the NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein, and an RNA of the 3′-UTR; preferably an RNA encoding the p7 protein, NS2 protein, NS3 protein, NS4A protein, NS4B protein, NS5A protein and NS5B protein, and an RNA of the 3′-UTR. As described above, in the nucleotide sequence shown in SEQ ID NO:57, the coding region of the p7 protein is from the 2580th position to the 2768th position; the coding region of the NS2 protein is from the 2769th position to the 3419th position; the coding region of the NS3 protein is from the 3420th position to the 5312nd position; the coding region of the NS4A protein is from the 5313rd position to the 5474th position; the coding region of the NS4B protein is from the 5475th position to the 6257th position; the coding region of the NS5A protein is from the 6258th position to the 7598th position; the coding region of the NS5B protein is from the 7599th position to the 9371st position; and the RNA of the 3′-UTR corresponds to the 9372nd and following positions. The HCV 3′-side RNA (C) preferably has the nucleotide sequences of these regions in the sequence shown in SEQ ID NO:57, or nucleotide sequences having homologies of not less than 90%, more preferably not less than 93%, still more preferably not less than 95%, still more preferably not less than 99% thereto, and can preferably construct a virus particle that can grow in a primate hepatocyte.
Needless to say, the above-described (A), (B) and (C) are linked together such that a continuous reading frame is attained, and the protein encoded thereby encodes a virus particle that can grow in a hepatocyte of primate such as tamarin or marmoset. Preferred examples of the E1 fusion type include the v11-E12 chimeric RNA (SEQ ID NO:93) and the v27-E12 chimeric RNA (SEQ ID NO:95) constructed in the Examples below, and RNAs having homologies of not less than 90%, more preferably not less than 95%, still more preferably not less than 99% thereto, which encode a virus particle that can grow in a primate hepatocyte.
Another preferred mode is an RNA wherein the entire core protein is encoded by the GBV-B RNA (this may be hereinafter referred to as “core GB type” for convenience). That is, the core GB type comprises:
In the core GB type, the RNA of the 5′-UTR in the HCV 5′-side RNA (A) is the same as the RNA in the 5′-UTR of the above-described E1 fusion type, and preferred examples thereof are also the same as those in the E1 fusion type.
In the core GB type, the following core protein is encoded by the GBV-B RNA (B). The core protein-coding region in the GBV-B RNA corresponds to the 446th to 913rd positions in the nucleotide sequence shown in SEQ ID NO:99, and the core protein-coding region of the core GB type has the nucleotide sequence of this region in SEQ ID NO:99, or has a homology of preferably not less than 90%, more preferably not less than 95%, still more preferably not less than 99% to the nucleotide sequence, and preferably encodes a virus particle that can grow in a primate hepatocyte.
The total length of the following E1 protein-coding region is also encoded by the GBV-B RNA (B). As described above, in the nucleotide sequence shown in SEQ ID NO:99, the E1 protein-coding region corresponds to the 914th to 1489th positions; and the E2 protein-coding region corresponds to the 1490th to 2284th positions. The GBV-B RNA (B) further preferably encodes the p6 protein. In the nucleotide sequence shown in SEQ ID NO:99, the p6 protein-coding region corresponds to the 2285th to 2452nd positions. The GBV-B RNA (B) preferably has the nucleotide sequence of the corresponding region in SEQ ID NO:99, or a sequence having a homology of not less than 90%, more preferably not less than 93%, still more preferably not less than 95%, still more preferably not less than 99% thereto, and can preferably construct a virus particle that can grow in a primate hepatocyte.
The structure of the downstream of the E1 protein-coding region (including the HCV 3′-side RNA (C)) is the same as that of the E1 fusion type described above, and the explanations on these regions in the E1 fusion type equally apply as they are.
Needless to say, the above-described regions (A), (B) and (C) are linked together such that a continuous reading frame is attained, and the protein encoded thereby encodes a virus particle that can grow in a hepatocyte of primate such as tamarin or marmoset. Preferred examples of the core GB type include the C6 chimeric RNA (SEQ ID NO:97) constructed in the Examples below, and RNAs having homologies of not less than 90%, more preferably not less than 95%, still more preferably not less than 99% thereto, which encode a virus particle that can grow in a primate hepatocyte.
In the same manner as described above, the E1 fusion type and the core GB type can also be prepared using arbitrary genetic engineering techniques. The chimeric RNA can be prepared by, for example, the following method, although the method is not restricted.
A DNA encoding the HCV/GBV-B chimeric RNA is inserted into a cloning vector by a conventional method, to prepare a DNA clone. The obtained DNA is inserted into the downstream of an RNA promoter, to prepare a DNA clone which can produce a replicon RNA. More particularly, for example, in pTPF1/4B (Patent Document 1, SEQ ID NO:57), which was prepared by mutating the amino acid sequence encoded by TPF1 clone isolated from a patient suffering from fulminant hepatitis C such that the 1804th position is leucine and the 1966th position is lysine, the region encoded by the above-described GBV-B RNA (B) is deleted to construct a DNA. On the other hand, the DNA of the above-described GBV-B RNA (B) is chemically synthesized, and, by inserting and linking the synthesized DNA to the portion deleted in HCV, the HCV/GBV-B chimeric gene can be constructed. The RNA promoter is preferably included in the plasmid clone. The RNA promoter is not restricted, and examples thereof include the T7 RNA promoter, SP6 RNA promoter and SP3 RNA promoter, among which the T7 RNA promoter is especially preferred. Particular methods for construction of preferred examples of the E1 fusion type and the core GB type are described in detail in the Examples below.
The HCV/GBV-B chimeric RNA of the present invention can be prepared from a vector to which the above-described DNA was inserted. Using the DNA clone as a template, the RNA is synthesized by RNA polymerase. The RNA synthesis can be initiated from the 5′-UTR by a conventional method. In cases where the template DNA is a plasmid clone, it is also possible to excise the DNA region linked to the downstream of an RNA promoter from the plasmid clone with a restriction enzyme, followed by synthesizing the RNA using the DNA fragment as a template. The 3′-end of the synthesized RNA is preferably coincident with the 3′-UTR of the virus genomic RNA, with neither addition of another sequence nor deletion. For example, in a preferred mode of the HCV/GBV-B chimeric RNA of the present invention, the template DNA is inserted into a vector which has a T7 RNA promoter at a position corresponding to the upstream of the 5′-UTR of HCV, and an XhoI restriction site at a position corresponding to the end of the 3′-UTR of HCV. After digestion with XhoI, the HCV genomic RNA can be synthesized by T7 RNA polymerase.
The replication cell of the present invention can be prepared by introducing the HCV/GBV-B chimeric RNA into an arbitrary cell. The cell to which the HCV/GBV-B chimeric RNA is introduced is not restricted, and it is preferably a monkey liver-derived cell or a human liver-derived cell. Examples of the monkey liver-derived cell include a marmoset primary hepatocyte and a tamarin primary hepatocyte. Examples of the cell derived from human liver cancer include Huh7 cell, HepG2 cell and Hep3B cell, and IMY-N9 cell, and examples of the other cancer cells include HeLa cell, CHO cell, COS cell, Vero cell and 293 cell.
Transfection of the HCV/GBV-B chimeric RNA into the cell can be carried out by an arbitrary transfection method. Examples of such a method of transfection include electroporation, the particle gun method and the lipofection method. For example, in cases where the transfection is carried out to a Huh7 cell, which is a human liver cancer-derived cell line, electroporation is especially preferred. In cases where the transfection is carried out to a monkey liver-derived cell, the lipofection method is preferred.
By using the replication cell, substances that control infection with HCV can be screened. The term “control infection with hepatitis C virus” means control (e.g., promotion or suppression) of replication of the RNA of HCV or control (e.g., promotion or suppression) of translation of the RNA into proteins.
More particularly, screening of a test substance can be carried out by bringing the test substance into contact with the replication cell and analyzing the level of increase in the HCV/GBV-B chimeric RNA. The term “level of increase in the HCV/GBV-B chimeric RNA” means a change in the rate or the amount of replication of the replicon RNA. More particularly, the amount of the HCV/GBV-B chimeric RNA in the replication cell is detected or measured, followed by comparing it with the amount of the HCV/GBV-B chimeric RNA in a control, that is, a replication cell that was not brought into contact with the test substance, thereby allowing screening of the test substance. Further, screening of the test substance can be carried out also by detecting or measuring the amount of a protein of HCV, a protein of GBV-B, or an HCV/GBV-B chimeric protein in the cell or the supernatant, followed by comparing it with that of a replication cell that was not brought into contact with the test substance. The protein of HCV which can be detected or measured by the screening is not restricted, and it is preferably the core protein, which can be measured also by using a commercially available kit. Further, by automating the screening method, the method can be applied to a high throughput screening method.
The replication cell of the present invention can produce an HCV/GBV-B chimeric RNA, HCV/GBV-B chimeric protein and HCV/GBV-B chimeric virus. Further, the replication of an HCV/GBV-B chimeric RNA in the replication cell of the present invention may be either transient replication or persistent replication. Further, in cases where an HCV/GBV-B chimeric virus is produced, cells can be reinfected with the produced virus.
By inoculating the HCV/GBV-B chimeric RNA or the HCV/GBV-B chimeric virus of the present invention to an experimental animal, a model animal for HCV infection can be prepared.
The non-human experimental animal is not restricted as long as it allows replication of the HCV/GBV-B chimeric virus or it can be infected with the virus, and the non-human experimental animal is preferably a small primate, more preferably marmoset or tamarin.
The method for administration of the HCV/GBV-B chimeric RNA to an experimental animal is not restricted, and examples thereof include intraperitoneal, intramuscular, intraspinal, intracranial, intravenous, intrarespiratory, oral, and intrahepatic administration, and the method for administration is preferably direct intrahepatic administration. The method for administration of the HCV/GBV-B chimeric virus to an experimental animal is also not restricted, and examples thereof include intraperitoneal, intramuscular, intraspinal, intracranial, intravenous, intrarespiratory, oral, and intrahepatic administration, and the method for administration is preferably intravenous administration.
By using an experimental animal wherein replication or infection of the HCV/GBV-B chimeric RNA or the HCV/GBV-B chimeric virus occurred, it is possible to screen or evaluate substances that control infection with HCV.
For example, screening or evaluation of a test substance can be carried out by administering the test substance to an experimental animal and analyzing the level of increase in the HCV/GBV-B chimeric virus, development of hepatitis and/or the like.
The RNA of HCV used for the HCV/GBV-B chimeric RNA of the present invention comprises the RNA encoding the NS4B protein having leucine at the 1804th position and lysine at the 1966th position in the amino acid sequence of the polyprotein of HCV. In a common HCV, the amino acid at the 1804th position is glutamine, and the amino acid at the 1966th position is glutamic acid, but, by their replacement with leucine at the 1804th position and lysine at the 1966th position, the replication efficiency of the RNA surprisingly increases. Therefore, a chimeric virus comprising the above-described RNA is considered to show highly efficient replication and growth in a cell or the living body of tamarin, or in a cell or the living body of marmoset. It is thought that, although the above-described RNA of HCV can replicate in cells of tamarin and marmoset even in cases it is not a chimeric virus, employment of a chimeric virus prepared with an RNA of GBV-B can achieve higher replication efficiency or infection efficiency. Since an RNA of genotype 1b of HCV comprising leucine at the 1804th position and lysine at the 1966th position especially increases replication efficiency, the genotype of the RNA of HCV to be used in the chimeric RNA is preferably 1b. Further, since the HCV/GBV-B chimeric RNA or the HCV/GBV-B chimeric virus of the present invention has the replication function in a Huh-7 cell, they are considered to maintain the replication function as HCV. Therefore, the non-human animal of the present invention is useful for development of prophylactic agents and therapeutic agents for HCV.
The present invention will now be described concretely by way of Examples, but the scope of the present invention is not restricted by these.
(A) Extraction of RNA from Serum
From 250 μL of serum collected from a fulminant hepatitis patient in the acute stage, RNA was purified using High Pure Viral Nucleic Acid Kit (Roche diagnostics corporation) according to the method recommended by the manufacturer.
(B) Synthesis of cDNA and Amplification of cDNA by PCR
A primer XR58R was added to the purified RNA, and reverse transcription was carried out with SuperSucript II reverse transcriptase (Invitrogen) according to the method recommended by the manufacturer at 42° C. for 1 hour, to obtain cDNA. RNase H (Invitrogen) was added to the obtained reaction solution, and the reaction was allowed to proceed at 37° C. for 30 minutes, to degrade the RNA. The resulting reaction solution was subjected to polymerase chain reaction (PCR) using the HC-LongA1 primer and the 1b9405R primer, and Takara LA Taq DNA polymerase (Takara Shuzo Co., Ltd.), by the thermal cycle reaction of 30 cycles of 94° C. for 20 seconds and 68° C. for 9 minutes, to amplify cDNA. Further, a part of the obtained reaction solution was subjected to PCR using the HC85F and HC9302R primers to amplify the HCV cDNA.
(C) Cloning of cDNA
The amplified DNA fragment was separated by electrophoresis using 0.7% agarose gel, and the DNA fragment was recovered using QIAquick gel purification kit (QIAGEN) according to the method recommended by the manufacturer. The recovered DNA fragment was subjected to a ligation reaction with pGEM-T easy vector (Promega), and the DH5α strain was transformed with the resulting plasmid. An ampicillin-resistant transformant was selected and cultured using 2YT medium. From the cultured bacterial cells, the plasmid was purified using Wizard Plus SV Miniprep DNA Purification System.
The nucleotide sequence of the HCV cDNA was determined using a primer designed based on the nucleotide sequence of genotype 1b of HCV. The reaction was carried out using CEQ DTCS Quick Start Kit (Beckman Coulter) according to the method recommended by the manufacturer, followed by analysis using CEQ2000 XL DNA analysis system (Software version 4.0.0, Beckman Coulter). The obtained data were analyzed using Sequencher (Version 4.1.2, Gene Codes Corporation). The obtained HCV clone was designated pTPF1-0193.
(E) Obtaining cDNA of 5′-UTR and Determining Its Base Sequence
Further, from the RNA obtained in the above-described step (A), a cDNA of the end of the 5′-UTR was obtained by the 5′-RACE method. The reaction was carried out using 5′ RACE System for Rapid Amplification of cDNA Ends, Version2.0 (Invitrogen), according to the instructions attached to the kit.
As the antisense primer for the synthesis of cDNA, Chiba-as was used. cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen) and purified with an S.N.A.P column, followed by TdT-tailing reaction to add dCTP to the cDNA. Using the 5′ RACE Abridged Anchor primer and the KY78 primer attached to the kit, and Takara LA Taq DNA polymerase (Takara Shuzo Co., Ltd.), the 1st PCR was carried out. Using a part of the PCR product, as a template, and the UTP primer and the KM2 primer attached to the kit, the 2nd PCR was carried out using Takara LA Taq DNA polymerase (Takara Shuzo Co., Ltd.), to obtain a PCR product. This PCR product was cloned into pGEM-T easy vector, and the nucleotide sequence of the product was determined according to the above-described step (D). The HCV cDNA clone containing the 1st to 709th positions in the obtained sequence was designated pTPF1-0007.
(F) Obtaining cDNA of 3′-UTR and Determining Its Nucleotide Sequence
From the RNA obtained in the above-described step (A), a cDNA of the end of the 3′-UTR was obtained by the 3′-RACE method. First, Poly(A) was added to RNA of a patient using Poly(A) Tailing Kit (Ambion) according to the instructions attached to the kit. The above-described steps (B) to (D) were carried out in the same manner except that the dT-Adp primer was used instead of the XR58R primer; the 3UTR-1F primer and the Adp primer were used as the primers for the 1st PCR; and the XR58F primer and the Adp primer were used as the primers for the 2nd PCR. The obtained HCV cDNA clone was designated pTPF1-8994.
The obtained HCV strain was designated the TPF1 strain. The TPF1 strain was an HCV having a total length of 9594 bases. The polynucleotide of the TPF1 strain obtained had a coding region encoding continuous 3010 amino acids between the 342nd position and the 9374th position thereof.
The total length of the polynucleotide of the HCV TPF 1 strain was inserted into the downstream of the T7 promoter sequence of pBluescript II SK(+) (the resulting plasmid is hereinafter referred to as pTPF1).
Subsequently, a part of the region encoding structural proteins and nonstructural proteins of pTPF1 was replaced with a neomycin resistance gene (neomycin phosphotransferase, NPT-II) and EMCV-IRES (the internal ribosomal entry site of encephalomyocarditis virus), to construct a plasmid DNA pRepTPF1. This construction was carried out according to a reported process (Lohmann et al., Science, (1999) 285, p. 110-113).
More particularly, pTPF1 was cleaved with restriction enzymes AgeI and BsrGI, and, to the cleaved site, a fragment prepared by PCR amplification of the sequence of the region from the 5′-UTR to the core region derived from pTPF1 and the neomycin resistance gene derived from pcDNA3.1(+), followed by cleavage thereof with restriction enzymes AgeI and PmeI; and a fragment prepared by PCR amplification of the sequence of the region from EMCV-IRES to the NS3 region, followed by cleavage thereof with restriction enzymes PmeI and BsrGI; were inserted by ligation. This plasmid DNA pRepTPF1 was digested with XbaI and used as a template for synthesis of RNA using Megascript T7 kit (Ambion). The RNA was purified by the method recommended by the manufacture.
Human liver cancer cells (Huh7, JCRB0403) were cultured under 5% carbon dioxide at 37° C. in culture medium prepared by adding fetal bovine serum (FBS) to 10%, and penicillin and streptomycin to 50 U/mL and 50 μg/mL, respectively, to Dulbecco's modified Eagle medium (D-MEM, IWAKI). The cells before confluence were peeled off from the culture dish by trypsin/EDTA treatment, and trypsin was then inactivated by resuspending the cells in serum-containing medium. After washing the cells twice with PBS, they were resuspended in Cytomix (120 mM potassium chloride, 10 mM potassium phosphate, 5 mM magnesium chloride, 25 mM HEPES, 0.15 mM calcium chloride, 2 mM EGTA, pH7.6) supplemented with 1.25% DMSO and transferred to an electroporation cuvette with a gap of 0.4 cm.
After adding an appropriate amount of the RNA to the cells, the cells were sufficiently cooled on ice for 5 minutes. Using an electroporator (Bio-Rad), the cells were pulsed at 960 uF, 250V. The cells were immediately resuspended in 8 mL of medium, and an aliquot thereof was plated. After a given period of culture, G418 (neomycin) was added to the culture plate to a concentration of 1 mg/mL. Thereafter, culture was continued while replacing the culture medium at 4-day intervals. About 20 days after the plating, colonies of live cells were cloned from the culture plate, and culture was continued. By such cloning of colonies, cells wherein the pRepTPF1 replicon RNA is autonomously replicating could be established. Whether or not replication of the replicon RNA occurred was determined by analyzing the copy number of replicated replicon RNA contained in cellular RNA by quantitative RT-PCR.
Whether or not autonomous replication of the replicon RNA was occurring was assayed whether or not the minus strand of the 5′-UTR region of the HCV RNA could be detected in the cells. Specific quantification of the minus strand was carried out in the same manner as in the method of specific detection of minus strand RNA described in Japanese Patent Application No. 8-187097.
In the cells to which the RNA synthesized in vitro using pRepTPF-1 as a template was introduced by electroporation, a significant amount of minus strands could be detected, and therefore autonomous replication of the replicon RNA in the cells was confirmed.
From the replicon RNA-replicating cell line established according to Reference Example 2 by synthesizing RNA in vitro using pRepTPF1 as a template and transfecting it to Huh7 cells, intracellular RNA was extracted using ISOGEN (Nippon Gene Co., Ltd.) under conditions recommended by the manufacturer.
From this intracellular RNA, DNA corresponding to almost the entire region of the replicon RNA was amplified in the same manner as in the case of obtaining the gene from TPF1 in Reference Example 1. More particularly, using the extracted intracellular RNA as a template and SuperSucript II reverse transcriptase (Invitrogen) and the XR58R primer, cDNA corresponding to the replicon RNA was synthesized.
A part of this cDNA was amplified by PCR and cloned into the pGEM-T easy vector, and the sequence of the clone was determined. As a result, substitution of A to T at the 5752nd nucleotide position and G to A at the 6237th nucleotide position were found. These resulted in mutation of Q (glutamine) to L (leucine) at the 1804th amino acid position and E (glutamic acid) to K (lysine) at the 1966th amino acid position, respectively.
Subsequently, the influence of the amino acid substitutions on replication of the replicon RNA was studied. First, to the HCV RNA replicon pRepTPF1 prepared in Reference Example 2, the adaptive mutations at the 1804th amino acid position (Q to L) and the 1906th amino acid position (E to K) were introduced using Quick Mutagenesis Kit (Stratagene) according to the method recommended by the manufacturer. This replicon RNA in which the amino acid substitutions were introduced was designated pRep4B.
Plasmid DNAs of pRepTPF1, which does not have the nucleotide sequence that cause the mutations, and pRep4B, which has the amino acid mutations, were digested with XbaI, and RNAs were synthesized using these DNAs as templates and Megascript T7 kit (Ambion). The RNAs were purified by the method recommended by the manufacturer. Each of the purified RNAs was transfected to Huh7 cells, and the cells were cultured in the presence of G418 for about 20 days, and then the live cells were stained with crystal violet. The numbers of colonies stained were counted, and the number of colonies per 1 μg of the transfected replicon RNA was calculated.
When 1 μg of RepTPF1 RNA was transfected, a single G418-resistant colony was selected, and when 1 μg of Rep4B RNA was transfected, 104 colonies were selected. That is, the nucleotide mutations that cause the amino acid mutations in the replicon RNA were considered to be adaptive mutations that increase the replication efficiency of the replicon RNA in Huh7 cells.
The full-length HCV DNA pTPF1 prepared in Reference Example 2 was digested with a restriction enzyme SfiI, and, to the cleaved site, a fragment prepared by digesting pRep4B with the restriction enzyme SfiI was inserted by ligation, thereby preparing a full-length HCV DNA pTPF1/4B to which the adaptive mutations were inserted. Its nucleotide sequence (described as RNA) is shown in SEQ ID NO:57, and the amino acid sequence encoded thereby is shown in SEQ ID NO:58.
The above-described pTPF1/4B comprising the gene of HCV whose growth in a human liver cancer cell line had been confirmed was subjected to polymerase chain reaction (PCR) in the presence of the Agel primer 5′-GGAACCGGTGAGTACACCGGAATTGCCAGG-3′ (SEQ ID NO:101) and the SplI primer 5′-ACCCGTACGCCATGCGCCAGGGCCCTGGCAG-3′ (SEQ ID NO:102) using Takara EX Taq DNA polymerase (Takara Shuzo Co., Ltd.), by the thermal cycle reaction of 20 cycles of 94° C. for 20 seconds and 68° C. for 30 seconds, to amplify the region from the 5′-UTR to the 156th position in the core protein in the TPF1 genome.
The amplified fragment was separated by 0.7% agarose gel electrophoresis, and the DNA fragment was recovered using QIAquick gel purification kit (QIAGEN) according to the method recommended by the manufacturer. The recovered TPF1 fragment was subjected to a ligation reaction with pGEM-T easy vector (Promega) according to the method recommended by the manufacturer, and the DHSa strain was transformed with the resulting plasmid. A transformant which was resistant to ampicillin and formed a white colony by plate culture on agar medium was selected, and cultured using 2YT medium to which ampicillin was added to 100 μg/mL From the cultured bacterial cells, the plasmid was purified using Wizard Plus SV
Miniprep DNA Purification System.
The sequence reaction of the TPF1 fragment incorporated in the purified plasmid was carried out using primers suitable for the vector and the HCV sequence and CEQ DTCS Quick Start Kit (Beckman Coulter) according to the method recommended by the manufacturer, followed by analysis using CEQ2000 XL DNA analysis system (Software version 4.0.0, Beckman Coulter). Based on the obtained data, sequence data were combined and analyzed using Sequencher (Version 4.1.2, Gene Codes Corporation), and the nucleotide sequence of pTPF1-AgeSpI was confirmed.
On the other hand, in terms of the genes including those of the envelope proteins of GBV-B, the genes in the region from the 124th position in the core protein to the p6 protein were constructed using the following synthetic genes.
For phosphorylation of the 5′-end of each synthetic gene, the phosphorylation reaction was carried out using T4 Polynucleotide Kinase (Takara Shuzo Co., Ltd.). These phosphorylation products were mixed together and slowly cooled from 95° C. to room temperature, thereby allowing annealing of the respective synthetic genes, followed by carrying out ligation reaction using Takara Ligation Kit (Takara Shuzo Co., Ltd.). This ligation product was subjected to blunting of the ends of the double-stranded DNA using Klenow Fragment (Takara Shuzo Co., Ltd.). This double-stranded DNA was cloned into the pGEM-T easy vector, and its nucleotide sequence was determined, thereby confirming that the DNA is the desired GBV-B gene.
In order to convert the XbaI site in pTPF1/4B to an XhoI site, a gene mutation was introduced to pTPF1/4B such that T at the 9594th nucleotide position was mutated to C using Quick Mutagenesis Kit (Stratagene) according to the method recommended by the manufacturer. The plasmid to which this mutation was introduced was designated pTPF1/4B-Xho.
By ligating the AgeI-SplI fragment prepared by cloning from pTPF1/4B by PCR and digestion with the restriction enzymes; the BbvCI-RsrII fragment prepared from pTPF1/4B by digestion with the restriction enzymes; and the SplI-BbvCI fragment prepared from the GBV-B gene by digestion with the restriction enzymes; into the vector prepared by digestion of pTPF1/4B-Xho with AgeI-RsrII, an HCV/GBV-B chimeric plasmid pTPF/GBB-C156E12, which has the GBV-B envelope proteins, was constructed.
In the presence of the above-mentioned AgeI primer and the EcoRV (v11) primer 5′-GATATCGTACAGCCCGGATACGTTGCGCAC-3′ (SEQ ID NO:103), pTPF1/4B was subjected to PCR using Takara EX Taq DNA polymerase (Takara Shuzo Co., Ltd.), by the thermal cycle reaction of 20 cycles of 94° C. for 20 seconds and 68° C. for 30 seconds, to amplify the region from the 5′-UTR to the 11th position in the E1 protein in the TPF1 genome. The amplified fragment was ligated into the pGEM-T easy vector in the same manner as in the case where the HCV gene fragment (pTPF1-AgeSpl) was obtained in Example 1, followed by determination of the sequence of the fragment according to a conventional method. As a result, the base sequence of pTPF1-AgeEcoR(v11) was confirmed.
On the other hand, in terms of the genes including those of the the envelope proteins of GBV-B, in the presence of the SnaBI primer 5′-TACGTAACTGACCCAGACACAAATACCACA-3′ (SEQ ID NO:104) and the BbvCI primer 5′-CCTCAGCCATGGGCACAAACCCTAAAAGGG-3′ (SEQ ID NO:105), pTPF/GBB-C156E12 was subjected to PCR using Takara EX Taq DNA polymerase (Takara Shuzo Co., Ltd.), by the thermal cycle reaction of 20 cycles of 94° C. for 20 seconds and 68° C. for 90 seconds, to amplify the region from the 3rd position in the E1 protein to the p6 protein in the GBV-B genome. The amplified fragment was ligated into the pGEM-T easy vector, followed by determination of the sequence thereof. As a result, the base sequence of pGBV-B SnaBbv was confirmed.
The AgeI-EcoRV (v11) fragment prepared by cloning from pTF1/4B by PCR and digestion with the restriction enzymes and the SnaBI-BbvCI fragment prepared by digestion of the GBV-B gene with the restriction enzymes were ligated into the vector prepared by digestion of pTPF1/4B-Xho with AgeI-BbvCI, thereby constructing an HCV/GBV-B chimeric plasmid having the GBV-B envelope proteins, pTPF/GBB -v11E12.
In the presence of the above-mentioned Agel primer and the EcoRV (v27) primer 5′-GATATCCGCTGCCTCATACACAATGCTTGA-3′ (SEQ ID NO:106), the above-described pTPF1/4B was subjected to PCR using Takara EX Taq DNA polymerase (Takara Shuzo Co., Ltd.), by the thermal cycle reaction of 20 cycles of 94° C. for 20 seconds and 68° C. for 90 seconds, to amplify the region from the 5′-UTR to the 27th position in the E1 protein in the TPF1 genome. The amplified product was ligated into the pGEM-T easy vector in the same manner as in the case where the HCV gene fragment (pTPF1-AgeSpl) was obtained in Example 1, followed by determination of the sequence of the fragment according to a conventional method. As a result, the base sequence of pTPF1-AgeEcoR(v27) was confirmed.
The AgeI-EcoRV (v27) fragment prepared by cloning from pTF1/4B by PCR and digestion with the restriction enzymes and the fragment prepared by digestion of the above-mentioned pGBV-B SnaBbv with the restriction enzymes SnaBI-BbvCI were ligated into the vector prepared by digestion of pTPF1/4B-Xho with AgeI-BbvCI, thereby constructing an HCV/GBV-B chimeric plasmid having the GBV-B envelope proteins, pTPF/GBB-v27E12.
The gene fragment having the 5′-UTR of TPF1 and the region from the core protein to the 125th position in E1 protein of GBV-B were constructed using the following synthetic genes.
For phosphorylation of the 5′-end of each synthetic gene, phosphorylation reaction was carried out using T4 Polynucleotide Kinase (Takara Shuzo Co., Ltd.). These phosphorylation products were mixed together and slowly cooled from 95° C. to room temperature, thereby allowing annealing of the respective synthetic genes, followed by carrying out ligation reaction using Takara Ligation Kit (Takara Shuzo Co., Ltd.). This ligation product was subjected to blunting of the ends of the double-stranded DNA using Klenow Fragment (Takara Shuzo Co., Ltd.). This double-stranded DNA was cloned into the pGEM-T easy vector, and its nucleotide sequence was determined, thereby confirming that the DNA is composed of the 5′-UTR of TPF1 and the genes in the region from the core protein to the 125th position in E1 protein of GBV-B.
The gene fragment prepared by digestion of the above synthesized TPF1 5′-UTR and the genes in the region from the core protein to the 125th position in El protein of GBV-B was ligated into the vector prepared by digestion of the HCV/GBV-B chimeric plasmid pTPF/GBB-C156E12 with restriction enzymes AgeI-AvrII, thereby constructing an HCV/GBV-B chimeric plasmid pTPF/GBB-C6, which has the region from the core protein to the p6 protein of GBV-B.
pTPF/GBB-C156E12 constructed in Example 1 was digested with XhoI, and, using the resulting digestion product as a template, RNA was synthesized using Megascript T7 kit (Ambion) or AmpliScribe T7-Flash transcription kit (Epicentre). The RNA was purified according to the method recommended by the manufacturer.
Human liver cancer cells (Huh7, JCRB0403) were cultured under 5% carbon dioxide at 37° C. in culture medium prepared by adding fetal bovine serum (FBS) to 10%, and penicillin and streptomycin to 50 U/mL and 50 μg/mL, respectively, to Dulbecco's modified Eagle medium (D-MEM, IWAKI). The cells before confluence were peeled off from the culture dish by trypsin/EDTA treatment, and trypsin was then inactivated by resuspending the cells in serum-containing medium. After washing the cells twice with PBS, they were resuspended in Cytomix (120 mM potassium chloride, 10 mM potassium phosphate, 5 mM magnesium chloride, 25 mM HEPES, 0.15 mM calcium chloride, 2 mM EGTA, pH7.6) supplemented with 1.25% DMSO and transferred to an electroporation cuvette with a gap of 0.4 cm.
After adding 10 μg of the RNA to the cells, the cells were sufficiently cooled on ice for 5 minutes. Using an electroporator (Bio-Rad), the cells were pulsed at 960 μF, 250V. The cells after transfection were immediately resuspended in 10 mL of medium, and 1 mL each thereof was plated on a 12-well plate (22.1 mm diameter), and then the culture was begun. The culture supernatant was collected 4 hours, 24 hours, 48 hours and 72 hours after the beginning of the culture. The collected culture supernatant was centrifuged at 2 k rpm for 10 minutes, and the resulting supernatant was collected. The measurement was carried out with 100 μl of the supernatant using a kit for the HCV core antigen (FUJIREBIO INC., Lumipulse).
As shown in
An attempt was made in order to evaluate whether or not the HCV/GBV-B chimeric genotype, which could replicate in Huh7 cells in Example 2, can replicate in primary hepatocytes of marmoset. RNA of pTPF/GBB-C156E12 was synthesized in the same manner as in Example 2.
Primary hepatocytes of marmoset (BIOPREDIC INTERNATIONAL) were cultured according to the method recommended by the manufacturer. More particularly, frozen primary hepatocytes of marmoset were melt in a water bath at 37° C., and suspended in 30 mL of Leibovitz's L-15 medium (Invitrogen) supplemented with 1% GlutaMAX-I Supplement (Invitrogen), which had been prewarmed to 37° C. The cell suspension was centrifuged at 1 k rpm (160×g) for 1 minute and the supernatant was removed, and the resulting cell pellet was resuspended in William's medium E (Invitrogen) supplemented with 1% GlutaMAX-I Supplement (Invitrogen), 4 μg/mL Bovine insulin and 10% fetal bovine serum (FBS) such that a density of about 6×105 cells/mL was attained. The resuspended cells were plated on a collagen type I-coated 24-well plate (15.6 mm diameter) in an amount of 0 5 mL each, followed by culture under 5% carbon dioxide at 37° C.
To the primary hepatocytes of marmoset cultured for 1 day, 2 μg/well of purified TPF/GBB-C156E12 and 4 μL/well of a gene transfection reagent HilyMax (DOJINDO) were added, followed by culture under 5% carbon dioxide at 37° C. for 4 hours. Gene transfection was carried out according to the method recommended by the manufacturer. Thereafter, the cells were washed 3 times with PBS, and cultured in William's medium E (Invitrogen) supplemented with 1% Glutamax-I supplement (Invitrogen), 4 μg/mL bovine insulin and 50 μM hydrocortisone hemisuccinate (growth medium) under 5% carbon dioxide at 37° C. The culture supernatant was collected at 4 hours, 24 hours, 48 hours, 72 hours, 96 hours, 144 hours, 192 hours, 240 hours, 288 hours and 336 hours during the culture. The collected culture supernatant was subjected to centrifugation at 2 k rpm for 10 minutes, and the resulting supernatant was collected. The measurement was carried out with 100 μL of the supernatant using a kit for the HCV core antigen (FUJIREBIO INC., Lumipulse).
As shown in
pTPF/GBB-C156E12, pTPF1/GBB-v11E12, pTPF/GBB-v27E12 and pTPF/GBB-C6 constructed in Example 1 were digested with XhoI, and RNAs were synthesized in the same manner as in Example 2.
The primary hepatocytes of marmoset were cultured by the method described in Example 3. The resuspended cells were plated on a collagen type I-coated 6-well plate (34.6 mm diameter) in an amount of 2 mL each, followed by culture under 5% carbon dioxide at 37° C.
To the primary hepatocytes of marmoset cultured for 1 day, 5 μg/well of the 4 types of purified TPF/GBB chimeric RNAs and 15 μL/well of a gene transfection reagent HilyMax were added, followed by culture under 5% carbon dioxide at 37° C. for 4 hours. Gene transfection was carried out according to the method recommended by the manufacturer. Thereafter, the cells were washed 3 times with PBS, and the growth medium was then added thereto, followed by beginning culture under 5% carbon dioxide at 37° C. The culture supernatant was collected 48 hours later, and the collected culture supernatant was centrifuged at 2 k rpm for 10 minutes, followed by collecting the resulting supernatant. The collected culture supernatant was stored at −80° C. until use for the infection test.
Further, as the cells to be infected, primary hepatocytes of marmoset were newly melt, and cultured in a collagen type I-coated 6-well plate. After 1 day of culture, 500 μL of the culture supernatant (5-fold diluted) collected 48 hours after the gene transfection was added to the cells, and culture was carried out under 5% carbon dioxide at 37° C. for 6 hours. Thereafter, the cells were washed 3 times with PBS, and the growth medium was added thereto, followed by beginning culture under 5% carbon dioxide at 37° C. The culture supernatant was collected at 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours, 168 hours, 192 hours and 216 hours. The collected culture supernatant was centrifuged at 2 k rpm for 10 minutes, and the resulting supernatant was collected. Whether or not reinfection with the TPF1/GBB chimera occurred was determined by measuring the number of genome of the chimeric virus contained in the culture supernatant by quantitative RT-PCR.
As shown in
By infecting a small primate such as tamarin or marmoset with the HCV/GBV-B chimeric virus of the present invention, an animal model for HCV can be constructed. By using this animal mode, development and evaluation of drugs to suppress or inhibit aggravation of hepatitis can be carried out, and therefore development and evaluation of more effective prophylactic agents and therapeutic agents for the infection are possible.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2008-184179 | Jul 2008 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2009/062786 | 7/15/2009 | WO | 00 | 3/11/2011 |
