WO2010044468A1 - インフルエンザウイルス由来のrnaポリメラーゼpb1-pb2タンパク質の発現系構築と結晶化 - Google Patents
インフルエンザウイルス由来のrnaポリメラーゼpb1-pb2タンパク質の発現系構築と結晶化 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/127—RNA-directed RNA polymerase (2.7.7.48), i.e. RNA replicase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- C—CHEMISTRY; METALLURGY
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- C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/16011—Orthomyxoviridae
- C12N2760/16111—Influenzavirus A, i.e. influenza A virus
- C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/11—Orthomyxoviridae, e.g. influenza virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- G01N2333/91205—Phosphotransferases in general
- G01N2333/91245—Nucleotidyltransferases (2.7.7)
- G01N2333/9125—Nucleotidyltransferases (2.7.7) with a definite EC number (2.7.7.-)
- G01N2333/91275—RNA-directed RNA polymerases, e.g. replicases (2.7.7.48)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
Definitions
- the present invention relates to expression system construction and crystallization of RNA polymerase PB1-PB2 protein derived from influenza virus.
- the present invention also relates to a screening method for substances that can be active ingredients of anti-influenza drugs.
- Influenza is a respiratory infection caused by the influenza virus. Infected patients complain not only of respiratory symptoms such as nasal discharge and cough but also strong systemic symptoms such as high fever, arthralgia and chills, especially for the elderly and infants In the case of death.
- Influenza virus is an RNA virus having minus-strand RNA as its genome. Because influenza virus phenotypes or genomic sequences are frequently mutated, influenza viruses sometimes infect species barriers. In recent years, it has been confirmed that avian and swine influenza viruses infect humans, and there is concern that the infection will spread.
- the influenza virus has hemagglutinin (HA) and neuraminidase (NA) on its surface.
- HA hemagglutinin
- NA neuraminidase
- 16 subtypes of HA and 9 subtypes of NA are known.
- the combination of the subtypes determines the type of influenza virus (for example, H1N1, H3N2, H5N1, H7N7, etc.).
- the anti-influenza virus agent Tamiflu is generally used, but this drug is a drug that suppresses the proliferation by preventing the spread of the virus, and it is not a drug that kills the virus. The problem is not to be.
- the conventional anti-influenza drugs target proteins on the virus surface such as NA or M2.
- Tamiflu oseltamivir
- Relenza zanamivir
- amantadine targets the viral proton channel (M2 protein) and inhibits viral unshelling (Non-patent Document 1).
- influenza viruses have high variability as described above, proteins targeted for drugs are mutated to acquire drug resistance. In fact, resistant influenza viruses against amantadine and oseltamivir have been generated and have become a global problem.
- Influenza virus RNA polymerase plays an important role in virus growth after infection in humans, so it is a target for anti-influenza virus agents, but its mass expression has not been successful.
- the three-dimensional structure information of proteins is indispensable information for the development of anti-influenza virus agents, but such information has not been clarified at present.
- An object of the present invention is to express a large amount of PB1-PB2 chain of RNA polymerase derived from influenza virus.
- Another object of the present invention is to crystallize the PB1-PB2 chain of RNA polymerase derived from influenza virus.
- an object of the present invention is to provide an anti-influenza drug screening method based on information on the crystal structure of the PB1-PB2 chain of RNA polymerase derived from influenza virus.
- Influenza RNA polymerase plays a central role in virus growth and has various functions such as not only replication of viral RNA but also recognition of host RNA and use as a primer.
- the present inventors established an expression system (utilizing E. coli) and a crystallization method using a complex of RNA polymerase PB1-PB2 chains using a gene derived from influenza virus. This is an indispensable method for developing anti-influenza virus drugs targeting RNA polymerase.
- the present inventors succeeded in identifying the structure of the interaction site between the PB1 subunit and the PB2 subunit constituting the RNA polymerase.
- the amino acid sequence involved in this site is highly conserved among virus species, and the above interaction site was found to be useful as a target site for anti-influenza drugs, leading to the completion of the present invention.
- the gist of the present invention is as follows. (1) A complex comprising any one of the following polypeptides (a1), (a2) or (a3) and any one of the following polypeptides (b1), (b2) or (b3).
- space group is P2 1 (9) according crystals.
- the precipitant is potassium phosphate and PEG 4000.
- (b1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4
- (b2) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4 and having the same biological activity as the polypeptide of (b1)
- (b3) a polypeptide encoded by a DNA that hybridizes under stringent conditions with a DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 3, and has the same biological properties as the polypeptide of (b1) Polypeptide having activity (21) The following polypeptide (b4), (b5) or (b6): (b4) a polypeptide comprising the amino acid sequence of SEQ ID NO: 20 (b5) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 20, and having the same biological activity as the polypeptide of (b4) (b6) a polypeptide encoded by
- the PB1 subunit or its partial fragment constituting the RNA polymerase of influenza virus is contacted with the PB2 subunit or its partial fragment, and the PB1 subunit or its partial fragment and the PB2 subunit are contacted
- a method for screening a substance that can be an active ingredient of an anti-influenza drug comprising a step of selecting a substance that inhibits interaction with a unit or a partial fragment thereof.
- a4 a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16
- a5 a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 16 and having the PB1 subunit activity of influenza virus RNA polymerase
- PB1 The method according to (26), wherein the partial fragment of the subunit consists of the polypeptide according to (15).
- the method according to (26), wherein the PB2 subunit is composed of the following polypeptide (b7) or (b8).
- the amino acid residues at the interaction site of the PB1 subunit are the amino acid residues of Leu 695, Lys 698, Phe 699, Val 715, Asp 725, Ile746 and Ile 750 in the amino acid sequence of SEQ ID NO: 16 and SEQ ID NO: 2
- the amino acid residue at the interaction site of the PB2 subunit is Glu 2, Arg 3, Ile 4, Lys 5, Glu 6, Leu 7, Arg 8, Asn 9 in the amino acid sequence of SEQ ID NO: 4, 18 or 20.
- any one of (26) to (30) which is at least one amino acid residue selected from the group consisting of Leu 10. (33)
- the amino acid residue at the interaction site of the PB1 subunit is the amino acid residue of Leu 695, Phe 699, Val 715, Ile746 and Ile 750 in the amino acid sequence of SEQ ID NO: 16 and the amino acid in the amino acid sequence of SEQ ID NO: 2
- the method according to any one of (26) to (30), wherein the method is at least one amino acid residue selected from the group consisting of residues corresponding to the residues.
- PB1-PB2 chain complex of RNA polymerase derived from influenza virus it has become possible to express a large amount of PB1-PB2 chain complex of RNA polymerase derived from influenza virus.
- the present invention it is possible to provide a screening method for substances that can be active ingredients of anti-influenza drugs. Since the amino acid sequence of the interaction site between PB1 and PB2 is a highly conserved region, it can be a target for developing anti-influenza drugs regardless of the phenotype or genomic variation of influenza virus.
- A The whole ribbon figure which shows the structure of a composite_body
- B The same model as (A) is rotated 90 ° around the horizontal axis, showing the separation of the three helices of the N-terminal peptide of PB2.
- C the sequence of the fragments forming the complex, and human (H1N1) influenza virus, avian influenza virus (A / Duck / Hong / Kong / 2000) and H7N7 influenza virus (A / Equine / London / 1416/1973) ) Sequence alignment.
- the red arrow indicates the presence of the PB1 fragment.
- PB2 is indicated by a green ribbon. It was revealed that the PB1-binding surface directly below it was mostly nonpolar. This figure was prepared using CCP 4 mg (29).
- Residues Ile 4, Leu 7 and Leu (10 (shown in green) of PB2 helix 1 form strong hydrophobic contacts with these four PB1 residues. Contact between PB1 and PB2.
- Lys 698 and Asp 725 of PB1 form only salt bridges across the interface.
- the green dashed line indicates a salt bridge connection of length 2.4-3.1mm.
- Nonpolar residues in PB1 are shown in red and single-lined arches indicate 3.4-3.9 cm long hydrophobic contacts. This figure was created using LIGPLOT (30).
- the PB1 residue is shown in yellow and labeled red.
- the PB2 residue is shown in blue and labeled.
- the van der Waals surface of each atom is translucent.
- Electron density map Stereo view of the final electron density map (2mFo-DFc) containing the key residues of the complex.
- PB1 is shown in red and PB2 is shown in blue.
- the map is outlined at 1.3 ⁇ . Interface contact and binding assays.
- FIG. 1 Ribbon diagram showing C ⁇ traces of PB1-C and PB2-N in red and blue, respectively, with residues selected for mutagenesis.
- B Bar graph showing viral genome (vRNA) synthesis levels of various RNA polymerase double mutants in comparison with wild type complex. In the absence of the PB2 subunit, enzyme activity is negligible.
- C shows the level of viral genome replication intermediate (cRNA) produced by the mutant.
- D shows the level of viral mRNA produced by the mutant. RNA synthesis activity of PB1 single mutant or PB2 single mutant.
- A A bar graph showing the results of comparing the level of mRNA synthesis of various RNA polymerase single mutants with wild type polymerase (WT).
- B Production of progeny virus.
- C mRNA production level in MDCK cells infected with wild-type virus or PB1-V715S virus in the presence of cycloheximide.
- D mRNA production level in MDCK cells infected with wild-type virus or PB1-V715S virus in the absence of cycloheximide.
- Each panel shows the amount of mRNA produced (left panel), the amount of cRNA produced (middle panel), and the amount of segment 5 vRNA produced (right panel).
- RNA polymerase PB1-PB2 protein derived from influenza virus The present invention includes the following polypeptide (a1), (a2) or (a3) and the following (b1), (b2) or ( Provided is a complex comprising the polypeptide of b3).
- polypeptide having activity (b1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 (b2) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4 and having the same biological activity as the polypeptide of (b1) (b3) a polypeptide encoded by a DNA that hybridizes under stringent conditions with a DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and has the same biological properties as the polypeptide of (a1) Polypeptide having activity (b1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4 (b2) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4 and having the same biological activity as the polypeptide of (b1) (b3) a polypeptide encoded by a DNA that hybridizes under stringent conditions with a DNA complementary to
- the polypeptide (a1) consists of the amino acid sequence of SEQ ID NO: 2.
- the amino acid sequence of SEQ ID NO: 2 is the amino acid sequence of 678-757 of RNA polymerase PB1 subunit of influenza A / Puerto Rico / 8/34 H1N1 type.
- the polypeptide of (a2) consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2, and has the same biological activity as the polypeptide of (a1) Have
- the total number and position of amino acids to be deleted, substituted or added are not particularly limited.
- the total number of amino acids to be deleted, substituted or added is 1 or more, preferably 1 or several, and the specific range thereof is usually 1 to 10, preferably 1 to 5 for deletion, More preferably, the number is 1 to 2, the substitution is usually 1 to 20, preferably 1 to 10, more preferably 1 to 3, and the addition is usually 1 to 10, preferably 1 to 5.
- the number is more preferably 1-2.
- Examples of the polypeptide (a2) include a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6 or 10.
- the amino acid sequence of SEQ ID NO: 6 is the SQR GILEDEQMYQ KCCNLFEKFF PSSSYRRPVG ISSMVEAMVS RARIDARIDF ESGRIKKEEF AEIMKICSTI E amino acid sequence (678-751) of influenza A virus (A / Duck / Hong Kong / 2986.1 / 2000 (H5N1)) RNA polymerase PB1 subunit It is.
- the amino acid sequence of SEQ ID NO: 10 is the RNA polymerase PB1 subunit SQR GVLEDEQMYQ KCCNLFEKFF PSSSYRRPVG ISSMVEAMVS RARIDARIDF ESGRIKKEEF AEIMKICSTI EE757RQ is there.
- biological activity similar to the polypeptide of (a1) refers to the ability to interact with influenza virus RNA polymerase PB2 subunit or a fragment thereof (eg, a polypeptide comprising the amino acid sequence of SEQ ID NO: 4).
- activity as an antigen, activity as an immunogen, and the like are also included.
- biological activity similar to the polypeptide of (a1) is used in a meaning including “PB1 subunit activity of RNA polymerase” described later.
- the polypeptide of (a3) is a polypeptide encoded by a DNA that hybridizes under stringent conditions with a DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and the polypeptide of (a1) Has similar biological activity.
- “Stringent conditions” can be appropriately selected by those skilled in the art. Examples of hybridization conditions include low stringency conditions. Low stringent conditions include, for example, 42 ° C., 2 ⁇ SSC, 0.1% SDS, and preferably 50 ° C., 2 ⁇ SSC, 0.1% SDS. More preferably, highly stringent conditions are included. Examples of highly stringent conditions include 65 ° C., 2 ⁇ SSC, and 0.1% SDS. Under these conditions, not only DNA having high homology as the temperature is lowered but also DNA having low homology can be obtained comprehensively. On the contrary, it can be expected that only DNA having high homology can be obtained as the temperature is increased.
- the DNA that hybridizes under stringent conditions with the DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 is at least 86% or more, preferably 88% or more, more than the DNA consisting of the nucleotide sequence of SEQ ID NO: 1.
- a DNA having a homology of preferably 90% or more, more preferably 95% or more is mentioned.
- DNA that hybridizes under stringent conditions with DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 include DNA consisting of the nucleotide sequence of SEQ ID NO: 5 or 9.
- the nucleotide sequence of SEQ ID NO: 5 is the nucleotide sequence of DNA encoding the amino acid sequence of 678-751 of RNA polymerase PB1 subunit of influenza A virus (A / Duck / Hong Kong / 2986.1 / 2000 (H5N1)).
- the nucleotide sequence of SEQ ID NO: 9 is a nucleotide sequence of DNA encoding the amino acid sequence of 678-757 of RNA polymerase PB1 subunit of influenza A virus (A / Equine / London / 1416/1973 (H7N7)).
- Polypeptides encoded by DNA isolated by these hybridization techniques usually have high homology in amino acid sequence with the polypeptide (a1).
- High homology generally refers to a homology of 97% or more, preferably 98% or more, more preferably 99% or more.
- Polypeptide homology was determined using the algorithm described in the literature (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730). I just need it.
- the polypeptide (b1) consists of the amino acid sequence of SEQ ID NO: 4.
- the amino acid sequence of SEQ ID NO: 4 is the amino acid sequence of 1-37 of RNA polymerase PB2 subunit of influenza A / Puerto Rico / 8/34 H1N1 type.
- the polypeptide of (b2) consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4, and has the same biological activity as the polypeptide of (b1) Have
- the total number and position of amino acids to be deleted, substituted or added are not particularly limited.
- the total number of amino acids to be deleted, substituted or added is 1 or more, preferably 1 or several, and the specific range thereof is usually 1 to 10, preferably 1 to 5 for deletion, More preferably, the number is 1 to 2, the substitution is usually 1 to 20, preferably 1 to 10, more preferably 1 to 3, and the addition is usually 1 to 10, preferably 1 to 5.
- the number is more preferably 1-2.
- polypeptide of (b2) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 or 12 can be exemplified.
- the amino acid sequence of SEQ ID NO: 8 is the amino acid sequence of MERIKELRDL MSQSRTREIL TKTTVDHMAI IKKYTSG (1-37) of RNA polymerase PB2 subunit of influenza A virus (A / Duck / Hong Kong / 2986.1 / 2000 (H5N1)).
- the amino acid sequence of SEQ ID NO: 12 is the amino acid sequence of MERIKELRDL MSQSRTREIL TKTTVDHMAI IKKYTSG (1-37) of RNA polymerase PB2 subunit of influenza A virus (A / Equine / London / 1416/1973 (H7N7)).
- Bio activity similar to the polypeptide of (b1) refers to the ability to interact with the influenza virus RNA polymerase PB1 subunit or a fragment thereof (eg, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2). In addition, activity as an antigen, activity as an immunogen, and the like are also included. In addition, the term “biological activity similar to the polypeptide of (b1)” is used in a sense including “PB2 subunit activity of RNA polymerase” described later.
- the polypeptide of (b3) is a polypeptide encoded by a DNA that hybridizes under stringent conditions with a DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 3, and the polypeptide of (b1) Has similar biological activity.
- “Stringent conditions” can be appropriately selected by those skilled in the art. Examples of hybridization conditions include low stringency conditions. Low stringent conditions include, for example, 42 ° C., 2 ⁇ SSC, 0.1% SDS, and preferably 50 ° C., 2 ⁇ SSC, 0.1% SDS. More preferably, highly stringent conditions are included. Examples of highly stringent conditions include 65 ° C., 2 ⁇ SSC, and 0.1% SDS. Under these conditions, not only DNA having high homology as the temperature is lowered but also DNA having low homology can be obtained comprehensively. On the contrary, it can be expected that only DNA having high homology can be obtained as the temperature is increased.
- the DNA that hybridizes under stringent conditions with DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 3 is at least 86% or more, preferably 88% or more, more than the DNA consisting of the nucleotide sequence of SEQ ID NO: 3.
- a DNA having a homology of preferably 90% or more, more preferably 95% or more is mentioned.
- DNA that hybridizes under stringent conditions with DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 3 include DNA consisting of the nucleotide sequence of SEQ ID NO: 7 or 11.
- the nucleotide sequence of SEQ ID NO: 7 is a nucleotide sequence of DNA encoding the 1-37 amino acid sequence of RNA polymerase PB2 subunit of influenza A virus (A / Duck / Hong Kong / 2986.1 / 2000 (H5N1)).
- the nucleotide sequence of SEQ ID NO: 11 is a nucleotide sequence of DNA encoding the amino acid sequence 1-37 of RNA polymerase PB2 subunit of influenza A virus (A / Equine / London / 1416/1973 (H7N7)).
- Polypeptides encoded by DNA isolated by these hybridization techniques usually have high homology in amino acid sequence with the polypeptide (b1).
- High homology generally refers to a homology of 97% or more, preferably 98% or more, more preferably 99% or more.
- Polypeptide homology was determined using the algorithm described in the literature (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730). I just need it.
- polypeptide (a1), (a2) or (a3) can be combined with the polypeptide (b1), (b2) or (b3) to form a complex.
- the present invention provides the following polypeptide (a1), (a2) or (a3) and the following polypeptide (b4), (b5) or (b6): And a complex comprising:
- polypeptides (a1) to (a3) are as described above.
- (B4) polypeptide has the amino acid sequence of SEQ ID NO: 20.
- the amino acid sequence of SEQ ID NO: 20 is the amino acid sequence of 1-86 of RNA polymerase PB2 subunit of influenza A / Puerto Rico / 8/34 H1N1 type.
- the polypeptide of (b5) consists of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 20, and has the same biological activity as the polypeptide of (b4)
- the total number and position of amino acids to be deleted, substituted or added are not particularly limited.
- the total number of amino acids to be deleted, substituted or added is 1 or more, preferably 1 or several, and the specific range thereof is usually 1 to 10, preferably 1 to 5 for deletion, More preferably, the number is 1 to 2, the substitution is usually 1 to 20, preferably 1 to 10, more preferably 1 to 3, and the addition is usually 1 to 10, preferably 1 to 5.
- the number is more preferably 1-2.
- Bio activity similar to the polypeptide of (b4) refers to the ability to interact with influenza virus RNA polymerase PB1 subunit or a fragment thereof (eg, a polypeptide comprising the amino acid sequence of SEQ ID NO: 2). In addition, activity as an antigen, activity as an immunogen, and the like are also included. In addition, the term “biological activity similar to the polypeptide of (b4)” is used in a sense including “PB2 subunit activity of RNA polymerase” described later.
- the polypeptide of (b6) is a polypeptide encoded by a DNA that hybridizes under stringent conditions with a DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 19, and the polypeptide of (b4) Has similar biological activity.
- “Stringent conditions” can be appropriately selected by those skilled in the art. Examples of hybridization conditions include low stringency conditions. Low stringent conditions include, for example, 42 ° C., 2 ⁇ SSC, 0.1% SDS, preferably 50 ° C., 2 ⁇ SSC, 0.1% SDS. More preferably, highly stringent conditions are included. Examples of highly stringent conditions include 65 ° C., 2 ⁇ SSC, and 0.1% SDS. Under these conditions, not only DNA having high homology as the temperature is lowered but also DNA having low homology can be obtained comprehensively. On the contrary, it can be expected that only DNA having high homology can be obtained as the temperature is increased.
- the DNA that hybridizes under stringent conditions with DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 19 is at least 86% or more, preferably 88% or more, more than the DNA consisting of the nucleotide sequence of SEQ ID NO: 19.
- a DNA having a homology of preferably 90% or more, more preferably 95% or more is mentioned.
- the polypeptide encoded by the DNA isolated by these hybridization techniques usually has high homology in amino acid sequence with the polypeptide (b4).
- High homology generally refers to a homology of 97% or more, preferably 98% or more, more preferably 99% or more.
- Polypeptide homology was determined using the algorithm described in the literature (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730). I just need it.
- polypeptide (a1), (a2) or (a3) can be combined with the polypeptide (b4), (b5) or (b6) to form a complex.
- the complex of the present invention comprises a trait into which a DNA encoding a polypeptide of (a1), (a2) or (a3) and a DNA encoding a polypeptide of (b1), (b2) or (b3) are introduced. It can be produced by culturing transformed cells and collecting the desired complex from the culture.
- the complex of the present invention encodes a DNA encoding the polypeptide of (a1), (a2) or (a3) and the polypeptide of (b4), (b5) or (b6) It can be produced by culturing transformed cells into which DNA has been introduced and collecting the desired complex from the culture.
- Transformed cells into which DNA encoding the polypeptide of (a1), (a2) or (a3) and DNA encoding the polypeptide of (b1), (b2) or (b3) are introduced are (a1)
- a recombinant vector containing a DNA encoding the polypeptide of (a2) or (a3) and a DNA encoding the polypeptide of (b1), (b2) or (b3), into an appropriate host cell Can be obtained.
- the present invention also includes a transformed cell into which DNA encoding the polypeptide of (a1), (a2) or (a3) and DNA encoding the polypeptide of (b1), (b2) or (b3) are introduced.
- DNA encoding the polypeptide of (a1), (a2) or (a3) and DNA encoding the polypeptide of (b4), (b5) or (b6) are introduced.
- the transformed cell is a recombinant containing a DNA encoding the polypeptide (a1), (a2) or (a3) and a DNA encoding the polypeptide (b4), (b5) or (b6). It can be obtained by introducing the vector into a suitable host cell.
- the present invention also includes a transformed cell into which a DNA encoding the polypeptide (a1), (a2) or (a3) and a DNA encoding the polypeptide (b4), (b5) or (b6) are introduced. provide.
- a DNA fragment of an appropriate length containing the coding region of the target polypeptide may be prepared.
- Nucleotides may be substituted in the nucleotide sequence of the coding region of the polypeptide of interest so as to be the optimal codon for expression in the host cell.
- the DNA fragment can then be inserted downstream of the promoter of an appropriate expression vector to produce a recombinant vector (eg, MolecularlonCloning 2nd Edition, J. Sambrook et al., Cold Spring HarborLab. Press, 1989). See The DNA fragment may be incorporated into an expression vector so that its function is exhibited.
- the present invention provides a recombinant vector containing a DNA encoding the polypeptide (a1), (a2) or (a3) and a DNA encoding the polypeptide (b1), (b2) or (b3). provide.
- the present invention also provides a recombinant containing a DNA encoding the polypeptide of (a1), (a2) or (a3) and a DNA encoding the polypeptide of (b4), (b5) or (b6).
- Provide vector eg, MolecularlonCloning 2nd Edition, J. Sambrook et al., Cold Spring HarborLab. Press, 1989. See The DNA fragment may be incorporated
- the DNA encoding the polypeptide can be prepared by PCR amplification using influenza virus cDNA.
- the DNA encoding the polypeptide of (a1), (a2) or (a3) includes DNA consisting of the nucleotide sequence of SEQ ID NO: 1, DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 and stringent conditions Examples thereof include DNA that hybridizes underneath.
- the DNA that hybridizes under stringent conditions with the DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 is at least 86% or more, preferably 88% or more, more than the DNA consisting of the nucleotide sequence of SEQ ID NO: 1.
- a DNA having a homology of preferably 90% or more, more preferably 95% or more is mentioned.
- DNA that hybridizes under stringent conditions with DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 include DNA consisting of the nucleotide sequence of SEQ ID NO: 5 or 9.
- the nucleotide sequence of SEQ ID NO: 5 is the nucleotide sequence of DNA encoding the amino acid sequence of 678-751 of RNA polymerase PB1 subunit of influenza A virus (A / Duck / Hong Kong / 2986.1 / 2000 (H5N1)).
- the nucleotide sequence of SEQ ID NO: 9 is a nucleotide sequence of DNA encoding the amino acid sequence of 678-757 of RNA polymerase PB1 subunit of influenza A virus (A / Equine / London / 1416/1973 (H7N7)).
- the DNA encoding the polypeptide of (b1), (b2) or (b3) includes DNA consisting of the nucleotide sequence of SEQ ID NO: 3, DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 3, and stringent conditions Examples thereof include DNA that hybridizes underneath.
- the DNA that hybridizes under stringent conditions with DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 3 is at least 86% or more, preferably 88% or more, more than the DNA consisting of the nucleotide sequence of SEQ ID NO: 3.
- a DNA having a homology of preferably 90% or more, more preferably 95% or more is mentioned.
- DNA that hybridizes under stringent conditions with DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 3 include DNA consisting of the nucleotide sequence of SEQ ID NO: 7 or 11.
- the nucleotide sequence of SEQ ID NO: 7 is a nucleotide sequence of DNA encoding the 1-37 amino acid sequence of RNA polymerase PB2 subunit of influenza A virus (A / Duck / Hong Kong / 2986.1 / 2000 (H5N1)).
- the nucleotide sequence of SEQ ID NO: 11 is a nucleotide sequence of DNA encoding the amino acid sequence 1-37 of RNA polymerase PB2 subunit of influenza A virus (A / Equine / London / 1416/1973 (H7N7)).
- the DNA encoding the polypeptide of (b4), (b5) or (b6) includes DNA consisting of the nucleotide sequence of SEQ ID NO: 19, DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 19, and stringent conditions Examples thereof include DNA that hybridizes underneath.
- plasmids derived from E. coli eg, pBR322, pBR325, pUC12, pUC13
- plasmids derived from Bacillus subtilis eg, pUB110, pTP5, pC194
- yeast-derived plasmids eg, pSH19, pSH15
- ⁇ phage etc.
- Bacteriophages, animal viruses such as retroviruses and vaccinia viruses, insect pathogenic viruses such as baculoviruses, and the like can be used.
- a promoter, enhancer, ribosome binding site, various signal sequences eg, splicing signal, poly A addition signal, etc.
- cloning site eg, translation / transcription terminator, selection marker, SV40 replication origin, etc. may be added to the expression vector. Good.
- the expression vector may be a fusion protein expression vector.
- fusion protein expression vectors are commercially available, and examples thereof include the pGEX series (Amersham Pharmacia Biotech), pET® Expression® Syetem (Novagen), and the like.
- Host cells include bacterial cells (eg, Escherichia, Bacillus, Bacillus, etc.), fungal cells (eg, yeast, Aspergillus, etc.), insect cells (eg, S2 cells, Sf cells, etc.), animal cells ( Examples thereof include CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells), plant cells, and the like.
- bacterial cells eg, Escherichia, Bacillus, Bacillus, etc.
- fungal cells eg, yeast, Aspergillus, etc.
- insect cells eg, S2 cells, Sf cells, etc.
- animal cells examples thereof include CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells
- plant cells and the like.
- a transformed cell introduced with the DNA encoding the polypeptide of (a1), (a2) or (a3) and the DNA encoding the polypeptide of (b1), (b2) or (b3) is cultured in a medium. From the culture, a complex of the polypeptide (a1), (a2) or (a3) and the polypeptide (b1), (b2) or (b3) can be collected.
- a transformed cell into which a DNA encoding the polypeptide of (a1), (a2) or (a3) and a DNA encoding the polypeptide of (b4), (b5) or (b6) are introduced in a medium.
- a complex of the polypeptide (a1), (a2) or (a3) and the polypeptide (b4), (b5) or (b6) can be collected from the culture.
- the complex is secreted into the medium, the medium is recovered, the complex is separated from the medium, and purified.
- the complex is produced in the transformed cell, the cell is lysed, and the complex is separated from the lysate and purified.
- the complex When the complex is expressed in the form of a fusion protein with another protein (functioning as a tag), the fusion protein is separated and purified, and then treated with another enzyme such as FactorXa or enterokinase.
- the target complex can be obtained by cleaving the protein.
- Separation and purification of the complex can be performed by a known method.
- Known separation and purification methods include differences in molecular weight such as methods using solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
- Methods that use, methods that use charge differences such as ion exchange chromatography, methods that use specific affinity such as affinity chromatography, methods that use hydrophobic differences such as reversed-phase high-performance liquid chromatography, etc.
- the complex After the complex is purified to a purity sufficient for crystallization, the complex can be appropriately concentrated to crystallize the complex in the presence of a precipitant.
- the present invention also provides crystals of the composite.
- the precipitant include sodium formate.
- a method for crystallization a batch method, a dialysis method, a vapor diffusion method, or the like can be used. When the batch method is employed, crystallization is preferably performed by the hanging drop method.
- the present invention also provides a polypeptide of (a1), (a2) or (a3), a DNA encoding this polypeptide, a recombinant vector containing this DNA, and a transformed cell into which this DNA has been introduced.
- the present invention also provides a method for culturing transformed cells into which a DNA encoding the polypeptide of (a1), (a2) or (a3) has been introduced, and from the culture to obtain a polypeptide of (a1), (a2) or (a3).
- Also provided is a method for producing a polypeptide comprising collecting the peptide.
- the present invention also provides a polypeptide (b1), (b2) or (b3), a DNA encoding this polypeptide, a recombinant vector containing this DNA, and a transformed cell into which this DNA has been introduced.
- the present invention also provides a method for culturing a transformed cell into which a DNA encoding the polypeptide of (b1), (b2) or (b3) has been introduced, and from the culture to obtain a polypeptide of (b1), (b2) or (b3).
- Also provided is a method for producing a polypeptide comprising collecting the peptide.
- the present invention provides a polypeptide (b4), (b5) or (b6), a DNA encoding this polypeptide, a recombinant vector containing this DNA, and a transformed cell into which this DNA has been introduced. Further, the present invention cultivates a transformed cell into which a DNA encoding the polypeptide of (b4), (b5) or (b6) has been introduced, and from the culture, (b4), (b5) or (b6) Provided is a method for producing a polypeptide, comprising collecting a peptide.
- polypeptide are cell-free. It is also possible to employ a production method based on system protein synthesis. Cell-free protein synthesis can be performed using commercially available kits. Examples of such kits include reagent kits PROTEIOS TM (Toyobo), TNT TM System (Promega), and PG-Mate TM (Toyobo) ) And RTS (Roche Diagnostics).
- PROTEIOS TM Toyobo
- TNT TM System Promega
- PG-Mate TM Toyobo
- RTS Roche Diagnostics
- polypeptide of (b1), (b2) or (b3) and the polypeptide of (b4), (b5) or (b6) may be produced according to known peptide synthesis methods.
- Anti-influenza virus drugs can be screened using the polypeptide of (b5) or (b6) in a binding assay.
- the present invention relates to a screening method for a substance that inhibits the interaction between PB1 subunit and PB2 subunit constituting RNA polymerase of influenza virus.
- the present invention is characterized in that such a substance is selected as one that can be an active ingredient of an anti-influenza drug.
- the influenza virus RNA polymerase subunit is composed of three subunits: PA subunit, PB1 subunit and PB2 subunit, and PB1 binds to PA and PB2 binds to PB1 to form a ternary complex. Thus, it becomes an active substance having the activity of RNA polymerase.
- RNA polymerase plays an essential role in replication of influenza virus genome.
- the amino acid sequence involved in the interaction site between the PB1 subunit and the PB2 subunit is highly conserved among influenza virus species. Therefore, anti-influenza drugs that target this interaction site are different types of viruses (eg, H1N1, H3N2, H5N1, H7N7), hosts (human, birds, swine, etc.) and other proteins. It can be expected to exert its effect regardless of the mutation. Accordingly, the present inventors focused on the subunits PB1 and PB2 of influenza virus RNA polymerase and performed structural analysis of the subunits. As a result, the structure of the interaction site between PB1 and PB2 was successfully analyzed. From this analysis result, it was considered that a substance that inhibits the interaction between PB1 and PB2 inhibits the growth of influenza virus and becomes an active ingredient of anti-influenza drugs. The present invention has been completed based on these findings.
- the present invention is a screening method for substances that can be active ingredients of anti-influenza drugs. Specifically, in the presence of a candidate substance, the PB1 subunit constituting the influenza virus RNA polymerase or a partial fragment thereof is contacted with the PB2 subunit or a partial fragment thereof, and the PB1 subunit or the partial fragment thereof Selecting a substance that inhibits the interaction with the PB2 subunit or a partial fragment thereof.
- RNA polymerase (1) RNA-dependent RNA polymerase complex
- the influenza virus RNA-dependent RNA polymerase complex is a protein complex that binds to each of the eight segments of the influenza virus genome and is essential for viral transcription and replication. It is a complex.
- this complex plays an essential role in order to exert the pathogenicity of the virus. For example, through a cap-snatching reaction, the complex recognizes the cap structure of the host mRNA and cleaves the host mRNA containing the cap structure. Since RNA polymerase complexes play an essential role in viral transcription, replication, and pathogenicity, their amino acid sequences are highly conserved across viral species. On the other hand, since there is no homology with human proteins, drugs targeting this complex are useful in that they can reduce side effects.
- RNA polymerase complex is composed of three subunits, PA, PB1 and PB2. All three of these subunits are required for viral transcription and replication. The structure of these subunits has been reported but limited (Area, E. et al., Proc. Natl. Acad. Sci. USA 101, 308-313 (2004); Torreira, E et al. Nucleic Acids Res. 35, 3774-3783 (2007); Tarendeau, F. et al. Nature Struct. Mol. Biol. 14, 229-233 (2007); Guilligay, D. et al. Nature Struct. Mol. Biol. 15, 500-506 (2008)). This indicates that it was very difficult for those skilled in the art to analyze the X-ray crystal structure of the RNA polymerase complex of influenza virus.
- the PB1 subunit in the present invention includes a polypeptide consisting of the amino acid sequence of SEQ ID NO: 16.
- a polypeptide having an interaction with PB2 may also exist in a variant of the polypeptide.
- a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 16 and having the PB1 subunit activity of influenza virus RNA polymerase is also included in the present invention. It can be used in the method.
- the PB1 subunit in the present invention may be a partial fragment thereof.
- the partial fragment of the PB1 subunit in the present invention include the following polypeptide (a1), (a2) or (a3).
- (a1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 2,
- (a2) a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 2 and having the same biological activity as the polypeptide of (a1)
- a3) a polypeptide encoded by a DNA that hybridizes under stringent conditions with a DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 1, and has the same biological activity as the polypeptide of (a1) Polypeptide having
- each of the polypeptides (a1) to (a3) is as described in “A. Construction and crystallization of RNA polymerase PB1-PB2 protein derived from influenza virus” above.
- biological activity similar to the polypeptide of (a1) is used in the meaning including the following “activity of PB1 subunit of RNA polymerase”.
- PB1 subunit activity of RNA polymerase means binding activity with PB2 subunit. Both of the activity of RNA polymerase obtained by forming a complex by binding PB1 to PB2 and binding to PA, and the activity of PB1 binding to PB2 to form a complex, are described above. Included in “subunit activity”.
- the mutant “RNA polymerase PB1 subunit activity” is at least 30% or more, preferably 50% or more, more preferably 90% or more, compared to the activity of PB1 consisting of the amino acid sequence of SEQ ID NO: 16. It means having activity.
- a substance that inhibits the interaction between subunits can be selected by the screening method of the present invention, so that at least the binding site to PB2 is retained in PB1.
- the amino acid sequence of PB1 may be deleted, substituted, added, or mutated by a combination thereof.
- the PB1 subunit activity does not necessarily mean that it has polymerase activity when PB1 and PB2 are bound.
- the presence or absence of the binding activity between PB1 and PB2 can be detected by using a known method such as an immunoprecipitation method or a pull-down assay.
- PB1 subunit or “PB1” is used to encompass either or both of the full-length polypeptide of RNA polymerase PB1 subunit of influenza virus and partial fragments thereof.
- one or several amino acids in PB1 are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 16 or a partial sequence thereof (for example, the amino acid sequence of SEQ ID NO: 2) as described above.
- a protein having an amino acid sequence mutated by the combination and having the PB1 subunit activity of RNA polymerase is also included.
- amino acid sequence of SEQ ID NO: 16 or a partial sequence thereof as an amino acid sequence in which one or several amino acids are deleted, substituted, or added, or mutated by a combination thereof, for example, (i) 1 to 9 amino acids in the amino acid sequence of SEQ ID NO: 16 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) are deleted.
- Amino acid sequence (ii) 1 to 9 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) amino acids in the amino acid sequence of SEQ ID NO: 16 are other amino acids
- mutant form of PB1 is about 80% or more, preferably 90% or more, more preferably about 95% or more, more preferably about 98% or more, with the amino acid sequence of SEQ ID NO: 16 or the partial sequence thereof.
- examples thereof include an amino acid sequence having an amino acid sequence having homology and having the PB1 subunit activity of RNA polymerase.
- Homology can be performed using a homology search site using the Internet.
- homology searches such as FASTA, BLAST, and PSI-BLAST can be used in Japan DNA Data Bank (DDBJ).
- DDBJ Japan DNA Data Bank
- Leu 695, Lys 698, Phe 699, Val 715, Asp 725, Ile746 and Ile 750 in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 16 preferably Leu 695, Phe 699, Val 715, Ile746 and Ile 750, It is an amino acid necessary to interact with PB2 and maintain binding with PB2. Therefore, it is preferable that at least one amino acid residue selected from the group consisting of the amino acid residues does not have the mutation.
- amino acid residues of the protein are represented by numbers counted from the N-terminus of the full-length amino acid sequence of each subunit alone or in combination with the three-letter code of the number and amino acid.
- amino acid sequence of SEQ ID NO: 16 which is the full-length amino acid sequence of PB1
- the 715th valine residue counted from the N-terminus is expressed as “Val 715” (the same applies to other amino acid residues).
- the amino acid sequence of SEQ ID NO: 2 is identical to the 678th to 757th amino acid residues counted from the N-terminus in the amino acid sequence of SEQ ID NO: 16.
- the amino acid sequence of SEQ ID NO: 2 has residues corresponding to the amino acid residues in the amino acid sequence of SEQ ID NO: 16. Therefore, for the description of amino acid residues in the amino acid sequence of SEQ ID NO: 2, the numbers counted from the N-terminus of the amino acid sequence of SEQ ID NO: 16 are applied. The amino acid sequence represented by this number is called “corresponding residue”. For example, since the 38th valine residue counted from the N-terminal of the amino acid sequence of SEQ ID NO: 2 corresponds to Val 715 in the amino acid sequence of SEQ ID NO: 16, the 38th valine residue corresponds to “Val 715”. “Residue” (the same applies to other amino acid residues in the amino acid sequence of SEQ ID NO: 2).
- a polynucleotide encoding an amino acid sequence having a mutation such as deletion, substitution or addition in one or several amino acids in the amino acid sequence of SEQ ID NO: 2 or 16 is “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989)), “Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997)), Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-92, Kramer and Fritz ( 1987) Method. Enzymol. 154: 350-67, Kunkel (1988) Method. Enzymol. 85: 2763-6, and the like.
- a mutation introduction kit using site-directed mutagenesis such as Kunkel method or Gapped duplex method, for example, QuikChange TM Site- Using Directed Mutagenesis Kit (Stratagene), GeneTailor TM Site-Directed Mutagenesis System (Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .: Takara Bio) It can be carried out.
- PB1 includes a fusion protein added by another peptide sequence.
- Peptide sequences added to PB1 include identification of proteins such as influenza agglutinin (HA), glutathione S-transferase (GST), multiple histidine tags (6 ⁇ His, 10 ⁇ His, etc.), maltose binding protein (MBP), etc.
- a tag sequence or the like to be facilitated can be selected. Ligation of the tag sequence to PB1 can be easily performed by ordinary genetic engineering techniques.
- PB1 includes a protein encoded by the nucleotide sequence of SEQ ID NO: 15 or a partial sequence thereof (for example, the nucleotide sequence of SEQ ID NO: 1), and a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 15 or a partial sequence thereof.
- a protein encoded by a polynucleotide that hybridizes with a nucleotide under stringent conditions and having the PB1 subunit activity of RNA polymerase is also included.
- the above-mentioned polynucleotide encoding PB1 is used in preparing PB1 or a mutant thereof.
- hybridization conditions can be appropriately selected by those skilled in the art.
- hybridization conditions include low stringency conditions.
- Low stringent conditions include, for example, 42 ° C., 2 ⁇ SSC, 0.1% SDS, and preferably 50 ° C., 2 ⁇ SSC, 0.1% SDS. More preferably, highly stringent conditions are included.
- highly stringent conditions include 65 ° C., 2 ⁇ SSC, and 0.1% SDS. Under these conditions, not only DNA having high homology as the temperature is lowered but also DNA having low homology can be obtained comprehensively. On the contrary, it can be expected that only DNA having high homology can be obtained as the temperature is increased. However, multiple factors other than temperature, such as salt concentration, can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize the same stringency by selecting these factors as appropriate. It is.
- Hybridization can be performed by a known method. Hybridization methods include, for example, “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Laboratory Press (1989)), “Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997)), etc. You can refer to it.
- the polynucleotide that hybridizes under stringent conditions includes, for example, at least 80% or more, preferably 90% or more, more preferably 95%, with the nucleotide sequence of SEQ ID NO: 15 or a partial sequence thereof.
- a polynucleotide containing a nucleotide sequence having 97% or more identity is included.
- a value indicating identity can be calculated by using a known program such as BLAST.
- a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 15 or a partial sequence thereof is, for example, one in the nucleotide sequence of SEQ ID NO: 15 or a partial sequence thereof.
- Examples include polynucleotides containing nucleotide sequences in which mutations such as deletion, substitution or addition have occurred in several nucleic acids.
- nucleotide containing a nucleotide sequence in which mutation such as deletion, substitution or addition has occurred in one or several nucleic acids in the nucleotide sequence of SEQ ID NO: 15 or a partial sequence thereof for example, (i) 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) nucleic acid in the nucleotide sequence of SEQ ID NO: 15 or a partial sequence thereof A nucleotide sequence in which is deleted, (ii) 1 to 10 (eg, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) nucleic acid in the nucleotide sequence of SEQ ID NO: 15 or a partial sequence thereof A nucleotide sequence substituted with another nucleic acid, (iii) 1 to 10 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) nucleic acid is contained in the nucleotide sequence of SEQ ID NO: 15 or
- the polynucleotide encoding PB1 is designed based on, for example, a primer based on the nucleotide sequence of SEQ ID NO: 15 or a partial sequence thereof, and gene amplification technology (PCR) (Current Protocols in Molecular Biology, John Wiley & Sons (1987) Section 6.1-6.4).
- PCR gene amplification technology
- the nucleotide sequence can be confirmed by sequencing by a conventional method. For example, the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) can be used. It is also possible to analyze the sequence using an appropriate DNA sequencer.
- a polynucleotide encoding PB1 is designed with a primer so that a desired sequence can be obtained from the sequence information of the full-length nucleotide sequence or amino acid sequence represented by SEQ ID NO: 15 or 16, or a partial sequence thereof. It can be obtained from a more purified viral genome by reverse transcription and PCR.
- the reverse transcription reaction can be referred to “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989)).
- it can also be obtained by amplifying a desired fragment by a PCR method from a polynucleotide containing a gene encoding PB1 using the above primers. At this time, an appropriate restriction enzyme sequence or the like may be added to the primer.
- PB2 subunit As the PB2 subunit (also referred to as “PB2”) in the present invention, a polypeptide comprising the amino acid sequence of SEQ ID NO: 18 can be mentioned. In addition to the polypeptide consisting of the amino acid sequence of SEQ ID NO: 18, there may be a polypeptide having an interaction with PB1 even if it is a variant of the polypeptide. Accordingly, a polypeptide comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 18 and having the PB2 subunit activity of influenza virus RNA polymerase is also included in the present invention. It can be used in the method.
- the PB2 subunit in the present invention may be a partial fragment thereof.
- Examples of the partial fragment of the PB2 subunit in the present invention include the following polypeptide (b1), (b2) or (b3).
- (b1) a polypeptide comprising the amino acid sequence of SEQ ID NO: 4 (b2) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 4, and A polypeptide having biological activity similar to that of a peptide
- (b3) a polypeptide encoded by DNA that hybridizes under stringent conditions with DNA complementary to DNA consisting of the nucleotide sequence of SEQ ID NO: 3, and A polypeptide having the same biological activity as the polypeptide of (b1)
- the partial fragment of the PB2 subunit in the present invention includes the following polypeptide (b4), (b5) or (b6).
- (b4) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 20 (b5) consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of SEQ ID NO: 20, and a polypeptide of (b4)
- a polypeptide having biological activity similar to that of a peptide (b6) a polypeptide encoded by a DNA that hybridizes under stringent conditions with a DNA complementary to the DNA consisting of the nucleotide sequence of SEQ ID NO: 19, and a polypeptide having the same biological activity as the polypeptide of (b4)
- each of the polypeptides (b1) to (b3) and (b4) to (b6) is as described in “A. Expression system construction and crystallization of RNA polymerase PB1-PB2 protein derived from influenza virus” above. It is. However, the terms "biological activity similar to (b1) polypeptide” and “biological activity similar to (b4) polypeptide” include the following "RNA polymerase PB2 subunit activity”. Used to mean.
- PB2 subunit activity of RNA polymerase means binding activity with PB1 subunit. Both the activity of RNA polymerase acquired by PB2 binding to PB1 to form a complex, and the activity of PB2 binding to PB1 to form a complex are the above-mentioned “RNA polymerase PB2 subunit activity”. included.
- the mutant “RNA polymerase PB2 subunit activity” is at least 30% or more, preferably 50% or more, more preferably 90%, compared to the activity of PB2 consisting of the amino acid sequence shown in SEQ ID NO: 18. It means having activity of more than%.
- the presence or absence of the binding activity between PB2 and PB1 can be confirmed, a substance that inhibits the interaction between subunits can be selected by the screening method of the present invention. Therefore, as long as at least the binding site to PB1 is retained in PB2, the amino acid sequence of PB2 may be mutated by deletion, substitution, addition, or a combination thereof. Further, the PB2 subunit activity does not necessarily mean that it has polymerase activity when PB2 and PB1 are bound. The presence or absence of the binding activity of the PB2 subunit with the PB1 subunit can be determined by using a known method similar to the above.
- one or several amino acids in PB2 are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 18 or a partial sequence thereof (for example, the amino acid sequence of SEQ ID NO: 4 or 20),
- a protein comprising an amino acid sequence mutated by a combination thereof and having the PB2 subunit activity of RNA polymerase is also included.
- amino acid sequence of SEQ ID NO: 18 or a partial sequence thereof as an amino acid sequence in which one or several amino acids are deleted, substituted, or added, or mutated by a combination thereof, for example, (i) 1 to 9 amino acids in the amino acid sequence of SEQ ID NO: 18 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) are deleted.
- Amino acid sequence (ii) 1 to 9 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) amino acids in the amino acid sequence of SEQ ID NO: 18 are other amino acids
- 1 to 9 eg, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1 acids are added to the amino acid sequence of SEQ ID NO: 18.
- PB2 comprises an amino acid sequence in which one or several amino acids in the amino acid sequence of SEQ ID NO: 4 or 20 are deleted, substituted or added, or mutated by a combination thereof, as described above.
- a protein having the PB2 subunit activity of RNA polymerase is also included.
- amino acid sequence of SEQ ID NO: 4 or 20 as an amino acid sequence in which one or several amino acids are deleted, substituted, added, or mutated by a combination thereof, for example, (i) lacking 1 to 9 amino acids in the amino acid sequence of SEQ ID NO: 4 or 20 (eg, 1 to 5, preferably 1 to 3, more preferably 1 to 2 and even more preferably 1) Lost amino acid sequence, (ii) 1 to 9 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2 and even more preferably 1) amino acids in the amino acid sequence of SEQ ID NO: 4 or 20 An amino acid sequence substituted with an amino acid of (iii) 1 to 9 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2, more preferably 1) amino acids are added to the amino acid sequence of SEQ ID NO: 4 or 20. Amino acid sequence, (iv) Amino acid sequences mutated by the combination of (i) to (iii) above.
- mutant form of PB2 has an amino acid sequence of SEQ ID NO: 18 or a partial sequence thereof (for example, the amino acid sequence of SEQ ID NO: 4 or 20) and about 80% or more, preferably 90% or more, more preferably about 95 %, More preferably about 98% or more of the amino acid sequence having the PB2 subunit activity of RNA polymerase.
- Homology can be performed using a homology search site using the Internet.
- homology searches such as FASTA, BLAST, and PSI-BLAST can be used in Japan DNA Data Bank (DDBJ).
- DDBJ Japan DNA Data Bank
- PB2 hybridizes under stringent conditions with a protein encoded by the nucleotide sequence of SEQ ID NO: 17 or a partial sequence thereof, and a polynucleotide comprising a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 17 or a partial sequence thereof. Proteins encoded by soybean polynucleotides and having RNA polymerase PB2 subunit activity are also included. In the present invention, the polynucleotide encoding the PB2 is used when preparing PB2 or a mutant thereof.
- Site-directed mutagenesis method for PB2 tag sequence addition to PB2, definition of stringent conditions, hybridization method, mutation mode, PCR method, etc., nucleotide sequence and amino acid sequence are SEQ ID NO: 17, The same as the above except that it is SEQ ID NO: 18.
- PB2 subunit or “PB2” is used to encompass either or both of the full-length polypeptide of RNA polymerase PB2 subunit of influenza virus and partial fragments thereof.
- interaction means that the constituent factors PB1 and PB2 forming a complex in the RNA polymerase of influenza virus associate and bind.
- interaction type include, but are not limited to, hydrogen bonding, hydrophobic association, and hydrophobic bonding.
- interaction also includes signal transduction between PB1-PB2. Signal transduction between PB1-PB2 is performed, for example, via at least one amino acid residue in the interaction site of PB1 subunit and PB2 subunit.
- the mode in which the candidate substance inhibits the interaction between PB1 and PB2 is not particularly limited.
- the candidate substance binds to the interaction site of either PB1 or PB2, the candidate substance And the like, which bind to either site of PB1 or PB2 and inhibit the interaction of both subunits.
- “In the presence of a candidate substance” means that the test compound can be contacted with PB1 or PB2 or a complex thereof, and the candidate compound is added to a reaction system containing PB1 or PB2 or a complex thereof. It means both adding and culturing with cells containing PB1 or PB2 or a complex thereof (including cells in which these genes are incorporated so that they can be expressed).
- candidate compounds to be screened are not particularly limited, but are preferably compounds having affinity for PB1 or PB2.
- the “interaction site” means an amino acid sequence consisting of at least one amino acid residue among amino acid residues appearing on the contact surface (contact interface) between PB1 and PB2.
- the amino acid residue at the interaction site of the PB1 subunit is not limited as long as it is an amino acid residue included in the amino acid sequence of SEQ ID NO: 2 or 16, but preferably, the above Leu 695, Lys 698, Phe 699 , Val 715, Asp 725, Ile746 and Ile 750, at least one amino acid residue. More preferably, it is at least one amino acid residue selected from the group consisting of Leu 695, Phe 699, Val 715, Ile746 and Ile 750. Even more preferably, Val 715.
- amino acid residues at the interaction site of the PB2 subunit are Glu 2, Arg 3, Ile 4, Lys 5, Glu 6, Leu 7, Arg 8, Asn 9 and Leu 10 in the amino acid sequence of SEQ ID NO: 4, 18 or 20.
- At least one amino acid residue selected from the group consisting of, preferably at least one selected from the group consisting of Glu 2, Ile 4, Leu 7 and Leu 10 in the amino acid sequence of SEQ ID NO: 4, 18 or 20 There are two amino acid residues.
- contact means that a cell into which a gene encoding the subunit is introduced and a candidate substance (test substance) are present in the same reaction system or culture system. Examples include adding a candidate substance to the container, mixing the cells and the candidate substance, and culturing the cells in the presence of the candidate substance.
- Candidate substance is any molecule that can change the RNA polymerase activity of an influenza virus.
- the compounds may be modified by conventional chemical, physical and / or biochemical means such as direct chemical modification such as alkylation, esterification, amidation or random chemical modification. Can be modified to structural analogs.
- the candidate compound may be a compound identified by a pharmacophore search or a structure comparison program using a computer.
- a compound identified by a pharmacophore search when using a compound identified by a pharmacophore search, a structure comparison program using a computer, etc., based on the result of structural analysis of the binding site of PB1 and PB2, the interaction between these subunits is determined.
- Candidate compounds that inhibit can be screened in Insilico.
- screening can be performed using a multiple target screening (MTS) method, which has a much higher hit rate than ordinary screening methods.
- MTS multiple target screening
- the “salt” refers to a pharmaceutically acceptable salt and is not particularly limited as long as it forms a pharmaceutically acceptable salt with the compound.
- a hydrohalide eg, hydrofluoride, hydrochloride, hydrobromide, hydroiodide, etc.
- an inorganic acid salt eg, sulfate, nitrate, perchlorate
- Acid salt phosphate, carbonate, bicarbonate, etc.
- organic carboxylate eg acetate, oxalate, maleate, tartrate, fumarate, citrate, etc.
- organic sulfonate Eg methanesulfonate, trifluoromethanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate, camphorsulfonate, etc.
- amino acid salts eg aspartate, glutamate
- Screening The screening method of the present invention can be carried out using, for example, biochemical techniques using cells that produce PB1 or PB2, or cell preparations of these cells, and at least one of PB1 and PB2 is It is also possible to use a purified form.
- the “cell preparation” include cell cultures, cultured cell disruptions, cytoplasm fractionated from cultured cells, organelles such as nuclei, and the like.
- Examples of cells that produce PB1 or PB2 include cells used in general genetic engineering techniques. As these cells, those in which at least one of PB1 gene and PB2 gene is introduced and expressed can be used. Gene transfer methods are well known in the art and can be readily performed (see, eg, Sambrook et al., Molecular Cloning, A Laboratory Manual 2nd ed., (Cold Spring Harbor Laboratory Press (1989)).
- the method for producing PB1 and PB2 is as described above.
- the gene encoding PB1 or PB2 (for example, the gene having the nucleotide sequence of SEQ ID NO: 15 or 17 or a partial sequence thereof) is expressed by the protein.
- a vector that is appropriately incorporated into an expression vector in a form suitable for the production is prepared, and a transformant introduced into any of animal cells, plant cells, insect cells, or microorganisms such as yeast and E. coli is prepared, A method for culturing the transformant can be mentioned. It is also possible to employ a production method based on cell-free protein synthesis. Cell-free protein synthesis can be performed using commercially available kits. Examples of such kits include reagent kits PROTEIOS TM (Toyobo), TNT TM System (Promega), and PG-Mate TM (Toyobo) ) And RTS (Roche Diagnostics).
- PB1 or PB2 produced by such a transformant or cell-free protein synthesis can be separated and purified by various separation operations utilizing its physical properties, chemical properties, etc., if desired.
- the purification method include various salt chromatography methods such as ordinary salting-out, centrifugation, ultrasonic crushing, ultrafiltration, gel filtration, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), and dialysis. And combinations thereof.
- PB1 or PB2 a method for producing PB1 or PB2 by transformant or cell-free protein synthesis so that PB1 or PB2 is fused with an affinity tag, and separating and purifying PB1 and PB2. It can be illustrated.
- the screening method of the present invention can be used to select a substance that becomes an active ingredient of an anti-influenza drug by evaluating the replication of the influenza virus or the transcriptional activity of the genome.
- a model virus replicon system (Turan, K. et al., Nucleic Acids Res. 29, 643-652 (2004)) that introduces a model virus genome and viral proteins involved in transcription and replication.
- a model virus replicon system using yeast that can also use genetic techniques can be employed as a method for measuring transcriptional activity (International Publication WO2008 / 139627139A1).
- an in vitro viral genomic RNA synthesis system can also be employed (Kawaguchi, A. and Nagata, K., EMBO J. 26, 4566-4575 (2007)). Those skilled in the art can appropriately select these methods and construct a screening system using transcription activity as an index.
- PB1 and PB2 can also be expressed and used as a fusion protein with a tag or labeled peptide such as FLAG-, HA-, His-, Fc portion of immunoglobulin, GST-, GFP. is there.
- screening can be performed by immunoprecipitation or immunological techniques.
- an antibody used in these techniques an antibody that recognizes the tag can be used.
- immunoprecipitation with antibodies it is also possible to immobilize Ni or glutathione on a solid layer such as beads to supplement the complex of PB1 and PB2.
- the complex can be detected by enzyme activity or fluorescence activity using the characteristics of the fused tag or peptide.
- the constituent factor can be separated and detected by Western blotting.
- PB1 or PB2 When one of PB1 or PB2 is expressed as a fusion protein with a fluorescent protein such as GFP, the fluorescence activity is measured directly after capturing the PB1 / PB2 complex in the solid layer with an antibody that recognizes the other molecule. The interaction (bonding state) between PB1 and PB2 can be evaluated.
- the presence or absence of inhibition of binding between PB1 and PB2 by the candidate substance can be determined by, for example, evaluation based on the absolute amount of the inhibitory effect, evaluation based on comparison with the control, and the like.
- a candidate compound that affects the interaction between PB1 and PB2 is selected based on the measurement result measured in (ii).
- the candidate compound selected in (iii) is identified as a substance that affects the interaction between PB1 and PB2, or an active ingredient of an anti-influenza drug.
- a target substance that inhibits the interaction between PB1 and PB2 can be searched if it is a system that can measure the interaction (binding) between proteins.
- a measurement system a cell system or a cell-free system can be used, and immunological techniques such as ELISA and RIA, Two-Hybrid® System, and the like can be employed.
- a method such as pull-down assay or immunoprecipitation can be used.
- An example of a system for analyzing the bond between PB1 and PB2 kinetically includes a method using a surface plasmon resonance method. In this method, for example, a BIACORE (registered trademark) protein interaction analysis system is used.
- a system that quantitatively analyzes the interaction between PB1 and PB2 it is possible to use cells that produce all of PB1 and PB2 or cell preparations of the cells.
- Screening Kit PB1 and PB2 of the present invention can be provided in the form of a screening kit for a substance that inhibits these interactions, or a substance that can be an active ingredient of an anti-influenza drug.
- the kit of the present invention contains the above PB1 and PB2, but can additionally contain vectors, primers, restriction enzymes, labeling substances, detection reagents and the like necessary for gene expression.
- the labeling substance means an enzyme, a radioisotope, a fluorescent compound, a chemiluminescent compound, and the like.
- the kit of the present invention includes other reagents for carrying out the method of the present invention, such as an enzyme substrate (chromogenic substrate, etc.), an enzyme substrate solution when the label is an enzyme label.
- an enzyme reaction stop solution may be included.
- the kit of the present invention may include a diluent for candidate compounds, various buffers, sterilized water, various cell culture containers, various reaction containers (Eppendorf tubes, etc.), cleaning agents, experimental operation manuals (instructions), and the like. it can.
- Influenza virus RNA-dependent RNA polymerase is a multifunctional heterotrimer that uses a “cap-snatching” mechanism to produce viral mRNA.
- Host cell mRNA is cleaved to produce cap-bearing oligonucleotides.
- This oligonucleotide can be extended using viral genomic RNA as a template. Binding of viral genomic RNA activates cap binding and endonuclease activity. This requires signaling from the RNA-binding PB1 subunit to the cap-binding PB2 subunit and an interface between these two subunits essential for polymerase activity.
- We defined the interaction surface by protein crystallography and examined the effect of contact residue mutation on holoenzyme function. This novel interface plays a critical role in controlling the 250 kDa polymerase, but is surprisingly small and completely conserved between birds and human influenza.
- Influenza kills an average of more than 50,000 people every year in the United States, and in the 1918 pandemic, the number of deaths worldwide is estimated at 50 million.
- Viral RNA polymerase is still not an approved drug target, but recently it has been the focus of new anti-influenza drug development. This is because the viral RNA polymerase is highly conserved among influenza strains that infect both birds and humans. Viral RNA polymerases perform many of the essential processes in the viral life cycle, but many of them and their regulation are still poorly understood (1).
- Influenza A RNA polymerase forms an RNP complex with each of the eight negative-strand RNA genome segments and the nucleoprotein packaged in the mature virion (7).
- the RNP complex moves into the nucleus using a nuclear translocation mechanism (8), where it initiates viral mRNA transcription by a “cap-snatching” process (9).
- This process involves cleaving mRNA cap-containing oligonucleotides from host cell mRNA precursors and extending them into viral mRNA, followed by polyadenylation at the 3 'end (10, 11).
- Polymerases synthesize viral genomic RNA (vRNA) and complementary RNA (cRNA), each with the correct end and no cap, at an appropriate ratio.
- PB1-PB2 interaction domain To further characterize the interaction between PB1 and PB2, we used a coprecipitation assay to bind the C-terminal fragment of PB1 to the N-terminal fragment of PB2. was observed. Only a short region of PB1, residues 678-757, was known to be required for tight binding (21). When this fragment (called PB1-C) was tested with residues 1-37, 1-86, 37-174, 252-490 and 530-759 of PB2, only the 1-37 and 1-86 fragments of PB2 were found. Binding was shown (FIG. 1D).
- Residues 37-177 of PB2 do not bind to the C-terminus of PB1, which is consistent with Perales et al., Who showed that deletion of 27 amino acids at the N-terminus of PB2 dramatically reduced viral RNA polymerase activity (twenty five). Furthermore, they showed that the N-terminal 124 residue of PB2 behaved like a dominant negative inhibitor of viral transcription. Furthermore, PB2-specific monoclonal antibodies against the N-terminus of this protein can suppress the transcription initiation step in vitro, possibly by interfering with binding to PB1 (26, 27). According to our experiments, PB2 residues 530-759, including the proposed second PB1 binding site, were not found to interact with the C-terminus of PB1.
- PB1-C (residues 678-757 on the C-terminal side of PB1) can be purified and crystallized when co-expressed in E. coli with PB2-N (residues 1-37 on the N-terminal side of PB2)
- PB2-N (residues 1-37 on the N-terminal side of PB2)
- a stable complex was formed. Determination of the X-ray crystal structure at 2.1 angstrom resolution reveals that there are two copies of the complex in the asymmetric unit, which forms a single compact domain (FIGS. 1A and B). This domain was very highly conserved among all influenza virus strains (FIG. 1C) and was stable in vitro (FIG. 1D).
- N-terminal fragment of PB2 was easily expressed and could be purified with an N-terminal GST tag, but these fusion proteins did not show binding to PB1 in vitro, indicating that they Suggests that it will not fold. Only the complex was produced by co-expression of the PB1 and PB2 domains. Interface buried throughout the surface area of 1400 ⁇ 2, consistent with tight coupling includes four salt bridge Glu 2 and Lys 698, Arg 3 and Asp 725, Arg 3 and Lys 698, and Glu 6 and Lys 698 (Figure 3a). All eight other hydrogen bonds between the polypeptides contain main chain atoms.
- Helix 1 of PB2-N contributes most of the interaction energy. Helix 1 of PB2-N contributes not only to the four salt bridges of PB1-C, but also to key nonpolar contacts like Ile 4 and Leu 7 ( Figures 3b, 3c and 6A) . These two residues are completely buried at the protein interface.
- mutants in which Leu 7 and Leu 10 were simultaneously substituted with serine also showed a significant decrease in the amount of RNA product produced, similar to the I4S / L7S mutant (FIG. 6B-D).
- two types of double mutants mutants in which Val 715 and Ile 750 of PB1 are substituted with serine (“V715S / I750S”) and mutants in which Ile 746 and Ile 750 of PB1 are substituted with serine (“I746S / I750S ”) was prepared. In both of these PB1 mutants, it was shown that the amount of vRNA produced decreased significantly (FIG. 6B).
- the neighboring Arg 8 on PB2 may form a novel interaction with the Asp 750 carboxylate group in the mutant. Both side chains of Val 715 and Phe 699 are buried close to the side chain of Leu 7. Substitution of PB2 Phe 699 with alanine ("F699A") is expected to introduce substantial cavities within the interface. The significant increase in mRNA production of the F699A mutant in the functional assay may be due to the extra flexibility created by this cavity. As noted above, the very strong enzymatic activity of the V715S mutant cannot be predicted from the structural model, indicating that the polar residues near the protein surface, including Ser 713 and Arg 754, are serine side chains. Suggests that it could be accommodated. The reason why the PB1-PB2 interaction is inhibited or greatly reduced by the mutation from valine to serine cannot be explained from the structural model. Therefore, the present inventors decided to further experiment with the V715S mutation.
- V715S virus (its titer was slightly lower than the wild type titer) (FIG. 7B).
- RNA polymerase is part of the vRNP structure.
- the result that the V715S virus could be isolated indicates that the Val715 mutation does not inhibit the PB1-PB2 interaction.
- CHX cycloheximide
- the primary transcription level from the infected V715S vRNP was significantly reduced compared to the primary transcription level from the wild type (FIG. 7C).
- the RNA synthesis activity of wild type virus or PB1-V715S virus in the absence of cycloheximide was measured. Upon measurement, mRNA production, cRNA production, and segment 5 vRNA production were each evaluated separately. In PB1-V715S virus, the production amount of each RNA was significantly reduced. ⁇ -actin mRNA was used as an internal control in all manipulations. As predicted by the low level of primary transcription, the synthesis of vRNA, cRNA and viral mRNA in V715S virus-infected cells was also reduced in the absence of CHX (FIG. 7D).
- PB1 and PB2 were used in the polymerase activity assay.
- the V715S mutant shows both significant PB2 binding and greatly reduced enzyme activity, suggesting that slightly altered interaction patterns may have an effect on polymerase efficiency .
- the enzyme activity is not lost because PB1 and PB2 are not bound to each other.
- F699A and I750D mutants show weak PB2 binding, but enhanced enzyme activity.
- T7 RNA polymerase major reorganization occurs during RNA synthesis (S13). Assuming that influenza RNA polymerase, like T7 RNA polymerase, undergoes major reassembly during RNA synthesis, decreased polymerase activity in the V715S mutant indicates structural changes in the polymerase protein, switch inhibition, and structure. It can be explained that this is due to instability.
- the polymerase activity decreases despite binding of PB1 and PB2. This is because the size of valine and serine hardly changes, but the substitution between PB1 and PB2 changes due to hydrogen bonding of the substituted serine with the surrounding water, and the polymerase activity is affected by the difference in the arrangement. It is thought that.
- the influence of the V715S mutation occurs through structural changes and dynamic changes of the complex during the RNA synthesis process.
- the highly conserved sequence at the PB1-PB2 interface is not just because the contact interface folds the two subunits together, but also plays an important role in signal transmission between the subunits. Show that
- helix 1 of PB2-N plays an important role for viral mRNA synthesis by the above functional tests such as transcription activity assay. As shown in FIG. 6, RNA polymerase activity disappeared when this helix (residues 1-12) was deleted. We also performed additional experiments with PB2 mutants. As a result, various interface mutants showed a marked decrease in mRNA. This result is consistent with the results of the pull-down assay using PB1-PB2 complex and Ni-NTA described above.
- PB1 mutants with site-specific mutations showed significantly different results between the enzyme activity test and the pull-down assay.
- the F699A mutant and the I750D mutant are weakly bound to PB2, but the enzyme activity is rather improved.
- the V715S mutant significantly bound to PB2, but the enzyme activity was significantly reduced. This result shows that a slight change in the mode of interaction reveals a significant effect on the efficiency of the polymerase.
- the results shown above demonstrate that the PB1-PB2 interface plays an important role in regulating overall enzyme activity, not just the contact interface where partner proteins assemble.
- the interface of PB1-PB2 occupies a rather low proportion of the entire 250 kDa polymerase complex, but plays an important role in the regulation of the complex.
- the PB1-PB2 interface is completely conserved between avian influenza viruses and human influenza viruses, including species associated with particularly high mortality, but is registered in the Protein Data Bank. Different from any other protein structure. Given the importance of the PB1-PB2 interface for viral replication and high conservation, the PB1-PB2 interface is expected as a target for new anti-influenza drugs to be used against all types of influenza A viruses. The structures presented herein will aid in the search for such compounds.
- Cloning, expression and purification of the PB1-PB2 complex Cloning and purification were performed as previously reported for the PA-PB1 complex (S1).
- a sequence derived from influenza A / Puerto Rico / 8/34 was used (S2).
- a fragment of the PB2 gene encoding residues 1-37, 1-86, 37-174, 252-490 and 530-759 along with a hexahistidine tag and an N-terminal TEV cleavage site was cloned into pET28b.
- the PB1-C coding region was cloned downstream of the PB2 gene with Shine-Dalgarno sequence.
- the obtained co-expression plasmid was transformed into Escherichia coli BL21 (DE3) RILP codon-plus strain, and the cells were induced with 0.5 mM IPTG and cultured at 15 ° C. overnight.
- the PB1-PB2 complex was purified by chromatography using Ni-NTA agarose (Qiagen) followed by SP and Q (GE Healthcare) Sepharose. After Ni-NTA chromatography, the histidine tag was removed by TEV protease digestion, and then the purified complex was concentrated to 5 mg / ml with centricon YM-3 (Millipore) for crystallization.
- Pull-down assay The pull-down assay was performed in the same manner as previously reported (S1). After binding the complex to a nickel affinity column, the complex was eluted with 500 mM imidazole. The eluted protein was analyzed by SDS-acrylamide gel electrophoresis (15%) and Coomassie blue staining.
- Model virus RNP assays were set up as previously reported (S1, S14). HeLa cells were transfected with viral protein expression plasmids encoding PA, PB1 (either wild type or mutant), PB2 (either wild type or mutant), NP, and pHH21-vNS-Luc reporter plasmid .
- This reporter plasmid has a reverse-oriented luciferase gene sandwiched between a 23 nucleotide long 5 ′ terminal promoter sequence and a 26 nucleotide long 3 ′ terminal promoter sequence of influenza virus segment 8. The luciferase gene is placed under the control of the human Pol I promoter.
- luciferase assay Promega
- real-time RT-PCR RNA purified from the cells was reverse transcribed with oligo (dT) 20 and the level of viral mRNA was measured.
- 5′-TATGAACATTTCGCAGCCTACCGTAGTGTT-3 ′ (SEQ ID NO: 13) corresponding to the luciferase coding region at nucleotide positions 351-380 and 5′-CCGGAATGATTTGATTGCCA-3 ′ (SEQ ID NO: 13) complementary to the luciferase coding region at nucleotide positions 681-700
- the synthetic single-stranded cDNA was subjected to real-time quantitative PCR using two specific primers (14).
- NP mRNA transcribed from the expression plasmid was used as an internal control.
- recombinant virus containing a viral genome encoding a segment related to PB1-V715S was produced by the plasmid-based transfection method reported by Neumann et al. (S15). .
- the PB1-V715S genomic segment and the other seven wild type genomic segments were generated by cellular RNA polymerase I. Wild type PB1, PB2, PA, and NP were generated from plasmids encoding these proteins by cellular RNA polymerase II. After incubating the cells for 48 hours after transduction, an aliquot of the cell culture supernatant was used for virus amplification in MDCK cells. At 48 hours after transduction, the culture medium was collected and stored at ⁇ 80 ° C. until use.
- Crystallization and Data Collection Crystals of PB1-PB2 complexes were grown by the hanging drop vapor diffusion method against a crystallization buffer containing 0.1 M potassium phosphate (pH 5.8) and 15% PEG 4,000 at 20 ° C. Diffraction data were collected from crystals cooled to -180 ° C. A crystallization buffer containing 25% glycerol was used to prevent freezing. X-ray diffraction data was collected at the beam line 17A of Photon Factory in Japan. Data sets were collected at three different X-ray energies near the Se-K absorption edge using Selenomethionyl substituted crystals. Data was measured using an ADSC Quantum 270 CCD detector.
- SHELXC and SHELXD were used to locate 12 selenium out of 14 possible Se-Met sites.
- Phase determination was performed using SOLVE (S5). After flattening the solvent, a high quality electron density map with a resolution of 2.1 angstroms was obtained using RESOLVE (S6). The electron density was analyzed, traced using COOT (S7), and the model refined with REFMAC (S8). Spherical electron density peaks are found above 1.3 ⁇ on the
- An anti-influenza virus drug can be developed by using a crystallization method for obtaining expression and three-dimensional structure information of RNA polymerase protein derived from influenza virus.
- SEQ ID NO: 1 shows the nucleotide sequence of DNA encoding 678-757 of RNA polymerase PB1 subunit of influenza A / Puerto Rico / 8/34 H1N1 type.
- SEQ ID NO: 2 shows the amino acid sequence of 678-757 of RNA polymerase PB1 subunit of influenza A / Puerto Rico / 8/34 H1N1 type.
- SEQ ID NO: 3 shows the nucleotide sequence of DNA encoding 1-37 of RNA polymerase PB2 subunit of influenza A / Puerto Rico / 8/34 H1N1 type.
- SEQ ID NO: 7 shows the nucleotide sequence of DNA encoding 1-37 of RNA polymerase PB2 subunit of influenza A virus (A / Duck / Hong Kong / 2986.1 / 2000 (H5N1)).
- SEQ ID NO: 8 shows the amino acid sequence of 1-37 of RNA polymerase PB2 subunit of influenza A virus (A / Duck / Hong Kong / 2986.1 / 2000 (H5N1)).
- SEQ ID NO: 9 shows the nucleotide sequence of DNA encoding 678-757 of RNA polymerase PB1 subunit of influenza A virus (A / Equine / London / 1416/1973 (H7N7)).
- SEQ ID NO: 10 shows the amino acid sequence of 678-757 of RNA polymerase PB1 subunit of influenza A virus (A / Equine / London / 1416/1973 (H7N7)).
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Abstract
Description
(1)下記の(a1)、(a2)又は(a3)のいずれかのポリペプチドと、下記の(b1)、(b2)又は(b3)のいずれかのポリペプチドとを含む複合体。
(a1)配列番号2のアミノ酸配列からなるポリペプチド
(a2)配列番号2のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(a3)配列番号1のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b1)配列番号4のアミノ酸配列からなるポリペプチド
(b2)配列番号4のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b3)配列番号3のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(2)下記の(a1)、(a2)又は(a3)のいずれかのポリペプチドと、下記の(b4)、(b5)又は(b6)のいずれかのポリペプチドとを含む複合体。
(a1)配列番号2のアミノ酸配列からなるポリペプチド
(a2)配列番号2のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(a3)配列番号1のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b4)配列番号20のアミノ酸配列からなるポリペプチド
(b5)配列番号20のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b6)配列番号19のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド
(3) (a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b1)、(b2)又は(b3)のいずれかのポリペプチドをコードするDNAとを含有する組換えベクター。
(4) (a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b4)、(b5)又は(b6)のいずれかのポリペプチドをコードするDNAとを含有する組換えベクター。
(5) (a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b1)、(b2)又は(b3)のいずれかのポリペプチドをコードするDNAとを導入した形質転換細胞。
(6) (a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b4)、(b5)又は(b6)のいずれかのポリペプチドをコードするDNAとを導入した形質転換細胞。
(7)(a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b1)、(b2)又は(b3)のいずれかのポリペプチドをコードするDNAとを導入した形質転換細胞を培養し、培養物から(1)記載の複合体を採取することを含む、(1)記載の複合体の製造方法。
(8)(a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b4)、(b5)又は(b6)のいずれかのポリペプチドをコードするDNAとを導入した形質転換細胞を培養し、培養物から(2)の複合体を採取することを含む、(2)記載の複合体の製造方法。
(9)(1)記載の複合体の結晶。
(10)(2)記載の複合体の結晶。
(11)空間群がP21である(9)記載の結晶。
(12)単位格子が、a=41.12±50Å、b=61.37±50Å、c=45.36±50Åの大きさとβ=103.5±30°の角度を持つ(11)記載の結晶。
(13)(1)又は(2)記載の複合体を沈殿剤の存在下に結晶化させることを含む、(1)又は(2)記載の複合体の結晶の製造方法。
(14)沈殿剤がリン酸カリウム及びPEG 4000である(13)記載の方法。
(15)下記の(a1)、(a2)又は(a3)のいずれかのポリペプチド。
(a1)配列番号2のアミノ酸配列からなるポリペプチド
(a2)配列番号2のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(a3)配列番号1のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(16)(15)記載のポリペプチドをコードするDNA。
(17)(16)記載のDNAを含有する組換えベクター。
(18)(15)記載のポリペプチドをコードするDNAを導入した形質転換細胞。
(19)(15)記載のポリペプチドをコードするDNAを導入した形質転換細胞を培養し、培養物から(15)記載のポリペプチドを採取することを含む、(15)記載のポリペプチドの製造方法。
(20)下記の(b1)、(b2)又は(b3)のいずれかのポリペプチド。
(b1)配列番号4のアミノ酸配列からなるポリペプチド
(b2)配列番号4のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b3)配列番号3のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(21)下記の(b4)、(b5)又は(b6)のいずれかのポリペプチド。
(b4)配列番号20のアミノ酸配列からなるポリペプチド
(b5)配列番号20のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b6)配列番号19のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド
(22)(20)又は(21)記載のポリペプチドをコードするDNA。
(23)(22)記載のDNAを含有する組換えベクター。
(24)(20)又は(21)記載のポリペプチドをコードするDNAを導入した形質転換細胞。
(25)(20)又は(21)記載のポリペプチドをコードするDNAを導入した形質転換細胞を培養し、培養物から(20)又は(21)記載のポリペプチドを採取することを含む、(20)又は(21)記載のポリペプチドの製造方法。
(26)候補物質の存在下で、インフルエンザウイルスのRNAポリメラーゼを構成するPB1サブユニット又はその部分断片とPB2サブユニット又はその部分断片とを接触させ、前記PB1サブユニット又はその部分断片と前記PB2サブユニット又はその部分断片との相互作用を阻害する物質を選択する工程を含む、抗インフルエンザ薬の有効成分となり得る物質のスクリーニング方法。
(27)PB1サブユニットが以下の(a4)又は(a5)のポリペプチドからなるものである(26)に記載の方法。
(a4) 配列番号16のアミノ酸配列からなるポリペプチド
(a5) 配列番号16のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、インフルエンザウイルスのRNAポリメラーゼのPB1サブユニット活性を有するポリペプチド
(28)PB1サブユニットの部分断片が(15)に記載のポリペプチドからなるものである(26)に記載の方法。
(29)PB2サブユニットが以下の(b7)又は(b8)のポリペプチドからなるものである(26)に記載の方法。
(b7) 配列番号18のアミノ酸配列からなるポリペプチド
(b8) 配列番号18のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、インフルエンザウイルスのRNAポリメラーゼのPB2サブユニット活性を有するポリペプチド
(30)PB2サブユニットの部分断片が(20)又は(21)に記載のポリペプチドからなるものである(26)に記載の方法。
(31)PB1サブユニットの相互作用部位のアミノ酸残基が、配列番号16のアミノ酸配列におけるLeu 695、Lys 698、Phe 699、Val 715、Asp 725、Ile746及びIle 750のアミノ酸残基並びに配列番号2のアミノ酸配列における前記アミノ酸残基に対応する残基からなる群から選択される少なくとも1つのアミノ酸残基である(26)~(30)のいずれかに記載の方法。
(32)PB2サブユニットの相互作用部位のアミノ酸残基が、配列番号4、18又は20のアミノ酸配列におけるGlu 2、Arg 3、Ile 4、Lys 5、Glu 6、Leu 7、Arg 8、Asn 9及びLeu 10からなる群から選択される少なくとも1つのアミノ酸残基である、(26)~(30)のいずれかに記載の方法。
(33)PB1サブユニットの相互作用部位のアミノ酸残基が、配列番号16のアミノ酸配列におけるLeu 695、Phe 699、Val 715、Ile746及びIle 750のアミノ酸残基並びに配列番号2のアミノ酸配列における前記アミノ酸残基に対応する残基からなる群から選択される少なくとも1つのアミノ酸残基である(26)~(30)のいずれかに記載の方法。
(34)PB2サブユニットの相互作用部位のアミノ酸残基が、配列番号4、18又は20のアミノ酸配列におけるGlu 2、Ile 4、Leu 7及びLeu 10からなる群から選択される少なくとも1つのアミノ酸残基である、(26)~(30)のいずれかに記載の方法。
(35.)候補物質が、化合物及びその塩、ペプチド、抗体並びに核酸からなる群から選択される少なくとも一種である、(26)~(34)のいずれかに記載の方法。
なお、本明細書において引用した全ての文献、および公開公報、特許公報その他の特許文献は、参照として本明細書に組み込むものとする。また、本明細書は、本願優先権主張の基礎となる2008年10月17日に出願された日本国特許出願(特願2008-268052号)及び2009年5月19日に出願された日本国特許出願(特願2009-121376号)の明細書及び図面に記載の内容を包含する。
本発明は、下記の(a1)、(a2)又は(a3)のポリペプチドと、下記の(b1)、(b2)又は(b3)のポリペプチドとを含む複合体を提供する。
(a2)配列番号2のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(a3)配列番号1のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b1)配列番号4のアミノ酸配列からなるポリペプチド
(b2)配列番号4のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b3)配列番号3のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(a2)配列番号2のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(a3)配列番号1のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b4)配列番号20のアミノ酸配列からなるポリペプチド
(b5)配列番号20のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b6)配列番号19のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド
これらの条件において、温度を下げる程に高い相同性を有するDNAのみならず、低い相同性しか有していないDNAまでも包括的に得ることができる。逆に、温度を上げる程、高い相同性を有するDNAのみを得られることが期待できる。但し、ハイブリダイゼーションのストリンジェンシーに影響する要素としては温度以外にも塩濃度など複数の要素が考えられ、当業者であればこれら要素を適宜選択することで同様のストリンジェンシーを実現することが可能である。配列番号19のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAとしては、配列番号19のヌクレオチド配列からなるDNAと少なくとも86%以上、好ましくは88%以上、より好ましくは90%以上、さらに好ましくは95%以上の相同性を有するDNAが挙げられる。
複合体が培地に分泌される場合には、培地を回収し、その培地から複合体を分離し、精製すればよい。複合体が形質転換された細胞内に産生される場合には、その細胞を溶解し、その溶解物から複合体を分離し、精製すればよい。
1.概要
本発明は、インフルエンザウイルスのRNAポリメラーゼを構成するPB1サブユニットとPB2サブユニットとの相互作用を阻害する物質のスクリーニング方法に関する。本発明は、そのような物質を抗インフルエンザ薬の有効成分になり得るものとして選択することを特徴とするものである。
インフルエンザウイルスのRNAポリメラーゼのサブユニットは、PAサブユニット、PB1サブユニット及びPB2サブユニットの3つのサブユニットから構成され、PAにPB1が、そしてPB1にPB2が結合することにより三者複合体を形成し、RNAポリメラーゼの活性を有する活性体となる。
(1)RNA依存性RNAポリメラーゼ複合体
インフルエンザウイルスのRNA依存性RNAポリメラーゼ複合体は、インフルエンザウイルスゲノムにおける8つのセグメントのそれぞれに結合するタンパク質複合体であり、ウイルスの転写及び複製に必須の複合体である。
RNAポリメラーゼ複合体は、ウイルスの転写、複製、病原性において必須の役割を果たすことから、そのアミノ酸配列はウイルス種間を超えて高度に保存されている。一方、ヒトタンパク質との相同性はないことから、この複合体を標的とする薬剤は副作用を低減できる点で有用である。
これらサブユニットの構造については、いくつか報告があるものの、ごく限られている(Area, E. et al., Proc. Natl. Acad. Sci. USA 101, 308-313 (2004); Torreira, E. et al. Nucleic Acids Res. 35, 3774-3783 (2007);Tarendeau, F. et al. Nature Struct. Mol. Biol. 14, 229-233 (2007); Guilligay, D. et al. Nature Struct. Mol. Biol. 15, 500-506 (2008))。このことは、インフルエンザウイルスのRNAポリメラーゼ複合体のX線結晶構造を解析すること自体が、当業者にとって非常に困難であったことを示す。
本発明におけるPB1サブユニット(「PB1」ともいう)としては、配列番号16のアミノ酸配列からなるポリペプチドが挙げられる。
また、配列番号16のアミノ酸配列からなるポリペプチドのほか、当該ポリペプチドの変異体にも、PB2との相互作用を有するポリペプチドが存在し得る。したがって、配列番号16のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、インフルエンザウイルスのRNAポリメラーゼのPB1サブユニット活性を有するポリペプチドも、本発明の方法に使用することが可能である。
本発明におけるPB1サブユニットの部分断片としては、下記の(a1)、(a2)又は(a3)のポリペプチドが挙げられる。
(a1)配列番号2のアミノ酸配列からなるポリペプチド、
(a2)配列番号2のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(a3)配列番号1のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(i) 配列番号16のアミノ酸配列中の1~9個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)のアミノ酸が欠失したアミノ酸配列、
(ii) 配列番号16のアミノ酸配列中の1~9個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、
(iii) 配列番号16のアミノ酸配列に1~9個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)のアミノ酸が付加したアミノ酸配列、
(iv) 上記(i)~(iii) の組合せにより変異されたアミノ酸配列
などが挙げられる。
但し、配列番号2又は配列番号16のアミノ酸配列におけるLeu 695、Lys 698、Phe 699、Val 715、Asp 725、Ile746及びIle 750、好ましくはLeu 695、Phe 699、Val 715、Ile746及びIle 750は、PB2と相互作用し、PB2との結合を維持するために必要なアミノ酸である。したがって、上記アミノ酸残基からなる群から選ばれる少なくとも1個のアミノ酸残基は、上記変異が生じていないことが好ましい。
また、配列番号2のアミノ酸配列は、配列番号16のアミノ酸配列におけるN末端から数えて678番目から757番目のアミノ酸残基と同一である。従って、配列番号2のアミノ酸配列には、配列番号16のアミノ酸配列におけるアミノ酸残基に対応する残基が存在する。そこで、配列番号2のアミノ酸配列におけるアミノ酸残基の表記については、配列番号16のアミノ酸配列のN末端から数えた番号を適用する。この番号で表記するアミノ酸配列を「対応する残基」という。例えば、配列番号2のアミノ酸配列のN末端から数えて38番目のバリン残基は、配列番号16のアミノ酸配列におけるVal 715に対応するので、前記38番目のバリン残基を「Val 715に対応する残基」という(配列番号2のアミノ酸配列における他のアミノ酸残基についても同様)。
本発明においては、上記PB1をコードするポリヌクレオチドは、PB1又はこれらの変異体を作製する際に使用される。
(i) 配列番号15のヌクレオチド配列又はその部分配列中の1~10個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)の核酸が欠失したヌクレオチド配列、
(ii) 配列番号15のヌクレオチド配列又はその部分配列中の1~10個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)の核酸が他の核酸で置換されたヌクレオチド配列、
(iii) 配列番号15のヌクレオチド配列又はその部分配列に1~10個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)の核酸が付加したヌクレオチド配列、
(iv)上記(i)~(iii)の組合せにより変異されたヌクレオチド配列などが挙げられる。
本発明において、ヌクレオチド配列の確認は、慣用の方法により配列決定することにより行うことができる。例えば、ジデオキシヌクレオチドチェーンターミネーション法(Sanger et al.(1977)Proc. Natl. Acad. Sci. USA 74: 5463)等により行うことができる。また、適当なDNAシークエンサーを利用して配列を解析することも可能である。
本発明におけるPB2サブユニット(「PB2」ともいう)としては、配列番号18のアミノ酸配列からなるポリペプチドが挙げられる。
また、配列番号18のアミノ酸配列からなるポリペプチドのほか、当該ポリペプチドの変異体であっても、PB1との相互作用を有するポリペプチドが存在し得る。したがって、配列番号18のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、インフルエンザウイルスのRNAポリメラーゼのPB2サブユニット活性を有するポリペプチドも、本発明の方法に使用することが可能である。
本発明におけるPB2サブユニットの部分断片としては、下記の(b1)、(b2)又は(b3)のポリペプチドが挙げられる。
(b1)配列番号4のアミノ酸配列からなるポリペプチド
(b2)配列番号4のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b3)配列番号3のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b4)配列番号20のアミノ酸配列からなるポリペプチド
(b5)配列番号20のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b6)配列番号19のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド
PB2サブユニットのPB1サブユニットとの結合活性の有無については、前記と同様の公知の方法を用いることにより、判断することができる。
(i) 配列番号18のアミノ酸配列中の1~9個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)のアミノ酸が欠失したアミノ酸配列、
(ii) 配列番号18のアミノ酸配列中の1~9個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、
(iii) 配列番号18のアミノ酸配列に1~9個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)のアミノ酸が付加したアミノ酸配列、
(iv) 上記(i)~(iii) の組合せにより変異されたアミノ酸配列
などが挙げられる。
(i) 配列番号4又は20のアミノ酸配列中の1~9個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)のアミノ酸が欠失したアミノ酸配列、
(ii) 配列番号4又は20のアミノ酸配列中の1~9個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、
(iii) 配列番号4又は20のアミノ酸配列に1~9個(例えば、1~5個、好ましくは1~3個、より好ましくは1~2個、さらに好ましくは1個)のアミノ酸が付加したアミノ酸配列、
(iv) 上記(i)~(iii) の組合せにより変異されたアミノ酸配列
などが挙げられる。
PB2についての部位特異的変異誘発法、PB2へのタグ配列の付加、ストリンジェントな条件の定義、ハイブリダイゼーションの方法、変異の態様、PCR法などは、ヌクレオチド配列及びアミノ酸配列がそれぞれ配列番号17、配列番号18である点を除き、前記と同様である。
本発明において、「相互作用」とは、インフルエンザウイルスのRNAポリメラーゼにおいて複合体を形成する構成因子PB1とPB2とが会合して結合することを意味する。相互作用の種類として、例えば、水素結合、疎水的会合、疎水結合などが挙げられるがこれらに限定されるものではない。
また、「相互作用」には、PB1-PB2間のシグナル伝達も含まれる。PB1-PB2間のシグナル伝達は、例えば、PB1サブユニット及びPB2サブユニットの相互作用部位における少なくとも一つのアミノ酸残基を介して行われる。
また、本発明において、「相互作用部位」とは、PB1とPB2との接触面(接触界面)に現れるアミノ酸残基のうち、少なくとも1つのアミノ酸残基からなるアミノ酸配列をいう。
よりさらに好ましくは、Val 715である。
本明細書において「候補物質」とは、インフルエンザウイルスのRNAポリメラーゼ活性を変化させることのできる任意の分子である。例えば、天然又は合成の低分子化合物ライブラリー由来の化合物、遺伝子ライブラリーの発現産物(ペプチド、タンパク質等)、天然又は合成のオリゴ核酸、天然又は合成のペプチドライブラリー由来のペプチド、抗体、細菌由来の物質(細菌から代謝により放出される物質等)、微生物、植物細胞抽出液、動物細胞抽出液、培養液(微生物、植物細胞、動物細胞等の培養物)由来の化合物、土壌中の化合物、ファージディスプレイライブラリー等に含まれる化合物などが挙げられる。化合物は、従来の化学的手段、物理的手段及び/又は生化学的手段により改変したものであってもよく、たとえばアルキル化、エステル化、アミド化などの直接的化学修飾又はランダムな化学修飾に付して構造的類似体に改変させることができる。
本発明のスクリーニング方法は、PB1若しくはPB2を産生する細胞、又はこれらの細胞の細胞調製物を用いて、例えば生化学的手法により行うことができ、また、PB1及びPB2のうち少なくとも一つは精製された形態のものを使用することも可能である。「細胞調製物」としては、細胞の培養物、培養細胞の破砕物、培養細胞から分画された細胞質、核などのオルガネラなどが挙げられる。また、PB1又はPB2を産生する細胞としては、一般の遺伝子工学的手法で使用される細胞が挙げられる。これらの細胞は、PB1遺伝子及びPB2遺伝子のうち少なくとも一つが導入されて発現しているものを使用することができる。遺伝子の導入法は当分野で周知であり、容易に実施することができる(例えば、Sambrookら、Molecular Cloning, A Laboratory Manual 2nd ed., (Cold Spring Harbor Laboratory Press (1989)を参照)。
また、PB1又はPB2を調製するその他の方法として、PB1又はPB2がアフィニティータグを融合した形になるように形質転換体又は無細胞系タンパク質合成で産生させ、PB1及びPB2を分離、精製する方法を例示することができる。
(i)候補化合物の存在下及び非存在下で、PB1とPB2とを接触させ、
(ii)候補化合物の存在下及び非存在下におけるPB1とPB2との相互作用をそれぞれ測定し、
(iii)前記(ii)で測定された測定結果に基づき、PB1とPB2との相互作用に影響を与える候補化合物を選択する。
前記(iii)で選択された候補化合物を、PB1とPB2との相互作用に影響を与える物質、あるいは抗インフルエンザ薬の有効成分であると同定する。
PB1とPB2との間の結合を速度論的に解析する系としては、例えば表面プラズモン共鳴法などを使う方法も挙げられる。この方法では、例えばBIACORE(登録商標)タンパク質相互作用解析システムなどが使用される。
PB1とPB2との相互作用を量的に解析する系においては、PB1及びPB2の全てを産生する細胞又は当該細胞の細胞調製物を用いて行なうことが可能である。
本発明のPB1及びPB2は、これらの相互作用を阻害する物質、又は抗インフルエンザ薬の有効成分となり得る物質のスクリーニング用キットの形態で提供することができる。本発明のキットは上記PB1及びPB2を含むが、その他に、遺伝子発現に必要なベクター、プライマー、制限酵素、標識物質、検出用試薬などを含めることができる。標識物質とは、酵素、放射性同位体、蛍光化合物及び化学発光化合物等を意味する。本発明のキットは、上記の構成要素のほか、本発明の方法を実施するための他の試薬、例えば標識物が酵素標識物の場合は、酵素基質(発色性基質等)、酵素基質溶解液、酵素反応停止液などを含めることができる。さらに、本発明のキットには、候補化合物用希釈液、各種バッファー、滅菌水、各種細胞培養容器、各種反応容器(エッペンドルフチューブ等)、洗浄剤、実験操作マニュアル(説明書)等を含めることもできる。
インフルエンザウイルスRNA依存性RNAポリメラーゼは多機能性ヘテロ三量体であり、”cap-snatching”機構を用いてウイルスmRNAを生産する。宿主細胞mRNAが切断されて、cap-bearingオリゴヌクレオチドが生産される。このオリゴヌクレオチドはウイルスゲノムRNAを鋳型として用いて伸長することができる。ウイルスゲノムRNAが結合すると、cap結合性及びエンドヌクレアーゼ活性が活性化する。それには、RNA結合性PB1サブユニットからcap結合性PB2サブユニットへのシグナル伝達と、ポリメラーゼ活性に必須のこれら2つのサブユニット間の界面が必要である。本発明者らは、タンパク質結晶学により相互作用表面を規定し、接触残基の突然変異がホロ酵素の機能に及ぼす効果を試験した。この新規な界面は、250 kDaポリメラーゼを制御するのに重大な役割を果たすが、驚くほど小さく、トリとヒトインフルエンザの間で完全に保存されている。
PB1とPB2との相互作用をより詳細にキャラクタライズするために、本発明者らは、共沈アッセイを用いて、PB1のC末端断片とPB2のN末端断片との結合を観察した。PB1の短い領域、残基678-757だけが緊密な結合に必要であることがわかっていた(21)。この断片(PB1-Cと呼ぶ)をPB2の残基1-37、1-86、37-174、252-490及び530-759とともに試験したところ、PB2の1-37と1-86断片のみが結合を示した(図1D)。PB2の残基37-177はPB1のC末端と結合せず、これは、PB2のN末端の27アミノ酸の欠失によりウイルスRNAポリメラーゼ活性が劇的に消失したことを示したPeralesらと一致する(25)。さらに、彼らは、PB2のN末端124残基がウイルス転写のドミナントネガティブインヒビターのような挙動をすることを示した。さらに、このタンパク質のN末端に対するPB2特異的モノクローナル抗体は、おそらく、PB1への結合を妨害することにより、in vitroで転写開始工程を抑制できる(26, 27)。
本発明者らの実験によれば、提案されている第二のPB1結合部位を含むPB2残基530-759は、PB1のC末端と相互作用することが見出されなかった。これらの結果は、明らかに、PB1のC末端とPB2のN末端が緊密で必須のサブユニット界面を形成することを示している。相互作用する断片は、PB1の80残基とPB2の37残基という、各サブユニットからのとりわけ短い配列である。これらの断片は、サブユニット間における重要な伝達に関与するが、これらの断片の分子量は、合わせても複合体の全分子量のわずか6%程度を含むに過ぎない。
このモデルの機能について試験するために、種々のPB2変異体を作製し、これら変異体のウイルスRNA合成レベルとin vitroでの複合体の安定性に対する効果を調べた(図6B-D)。機能アッセイにおいて、PB2の非存在下ではRNA産物が検出できなかった。また、PB2のヘリックス1を欠失させると、RNAポリメラーゼ活性は消失した。
また、Ile 4及びLeu 7をセリン残基に置換したPB2-N変異体(「I4S/L7S」)を用いて実験を行ったところ、そのRNA産物の産生量は大きく減少することが示された(図6B-D)。また、Leu 7及びLeu 10を同時にセリンに置換した変異体(「L7S/L10S」)もI4S/L7S変異体と同様に、RNA産物の産生量は大きく減少した(図6B-D)。
さらに、2種類の二重変異体(PB1のVal 715及びIle 750をセリンに置換した変異体(「V715S/I750S」)並びにPB1のIle 746及びIle 750をセリンに置換した変異体(「I746S/I750S」))を調製した。これらPB1変異体の双方において、vRNAの産生量は顕著に減少することが示された(図6B)。これらの変異体においては、cRNA及びmRNAの産生量も有意に減少したが、その減少の程度はvRNAに比較すると低いことが示された(図6C及びD)。これらの結果は、Leu 7が疎水性コアに埋もれている構造モデルからも理解できる。
非極性残基であるVal 715の側鎖は、極性残基であるLeu 7の側鎖の近くに埋もれている。しかしながら、Val 715の側鎖は、タンパク質表面の極性残基(Ser 713及びArg 754などを含む)の近くに存在するので、セリン側鎖に置換しても大きな影響を与えることがないと考えられる。また、Ile 750は、この構造モデルのタンパク質表面近くに位置することから、極性残基であるセリン残基に置換しても、PB1-PB2結合を阻害することなく、この位置に存在することができると考えられる。
一つの残基を置換した変異体PB2-Nを用いてさらに実験を行った。ウイルスmRNA産生量はHeLa細胞において評価した。RNA合成活性は、I4D変異体(4番目のアミノ酸残基をイソロイシンからアスパラギン酸に置換した変異体。以下同様)において有意に低下した。しかしながら、mRNA産生量は、Leu 7をアスパラギン酸に置換した変異体(L7D)においてさらに顕著に減少した(図7A)。同様の実験は、PB1のLeu 695をアスパラギン酸に置換した変異体(L695D)、Ile 750をアスパラギン酸に置換した変異体(I750D)、Phe 699をアラニンに置換した変異体(F699A)、及びVal715をセリンに置換した変異体(V715S)に対しても行った。これらの変異体は、80%の減少を示したV715Sを除き、いずれの変異体もmRNAの産生量の有意な減少を示さなかった(図7A)。
Leu 695及びIle 750はどちらも溶媒である水に近接する位置にある。おそらく、この水は、PB1-PB2結合を妨害することなく、アスパラギン酸残基がLeu 695又はIle 750のいずれかと置き換わることを許容するのであろう。PB2上の近隣のArg 8は、変異体においてAsp 750のカルボキシレート基との新規な相互作用を形成するのかもしれない。Val 715及びPhe 699の両方の側鎖はLeu 7の側鎖に近接して埋もれている。PB2のPhe 699をアラニンに置換すると(「F699A」)、界面内に実質的な空洞が導入されることが予想される。機能アッセイにおいてF699A変異体のmRNA産生量が有意に増加したのは、この空洞により生じた余分なフレキシビリティが原因かもしれない。上述の通り、V715S変異体の酵素活性が非常に強く低下したことは構造モデルからは予想できず、このことは、Ser 713及びArg 754を含むタンパク質表面での近くの極性残基がセリン側鎖を収容できるのであろうことを示唆している。バリンからセリンへの変異によりPB1-PB2相互作用が阻害又は大きく減少する理由は、構造モデルからは説明することができない。そこで、本発明者らは、V715S変異についてさらに実験を行うこととした。
逆遺伝学(reverse genetics)に基づく手法により、PB1ゲノムセグメントにV715S変異を有する組換えウイルス(「V715Sウイルス」という)を作製した。V715Sウイルスは、V715S変異を有するセグメント以外の7つのセグメントは全て野生型である。V715Sウイルスを実験に用いることにより、感染したvRNPからの一次転写レベルに対する単一部位突然変異(single-site mutation)の効果を分析することが可能となった。
野生型ウイルス又はPB1-V715SウイルスをそれぞれMOI=1でMDCK細胞に感染させた。感染から24時間後、細胞上清を採取し、MDCK細胞を用いてプラーク力価を決定した。
本発明者らは、V715Sウイルスを回収することに成功した(その力価は、野生型の力価よりわずかに低かった)(図7B)。RNAポリメラーゼはvRNP構造の一部である。従って、V715Sウイルスを単離することができたという結果は、Val715変異によりPB1-PB2相互作用が阻害されていないことを示す。
CHXを用いる前記試験方法により、本発明者らは、ウイルスゲノムの複製や三量体ポリメラーゼ複合体形成の効率とは独立して、ウイルスの転写活性を評価することができた。
次に、NP mRNAに特異的なプライマーセットを用いてリアルタイム定量PCRアッセイを実施した。
その結果、感染したV715S vRNPからの一次転写レベルは、野生型からの一次転写レベルと比較して顕著に減少することが示された(図7C)。
また、シクロヘキシミド非存在下における野生型ウイルス又はPB1-V715SウイルスのRNA合成活性を測定した。測定に際し、mRNAの産生量、cRNAの産生量、セグメント5 vRNAの産生量をそれぞれ別々に評価した。PB1-V715Sウイルスにおいて、各RNAの産生量は有意に減少した。βアクチンmRNAは、全ての操作において内部コントロールとして用いた。
一次転写レベルが低いことから予測できるとおり、V715Sウイルス感染細胞におけるvRNA、cRNA及びウイルスmRNAの合成は、CHXの非存在下でも減少した(図7D)。
in vitro及びin vivo機能アッセイの結果により、PB1のVal 715残基がRNA合成反応における二以上のステップに関与していることが強く示された。V715S変異が単にPB1-PB2結合をブロックしているにすぎないという可能性を排除するため、プルダウンアッセイを行った。プルダウンアッセイは、ヒスチジンタグが融合したPB2-NとPB1との複合体を共発現させ、この複合体をNi-NTAカラムに結合させることにより行った。
このプルダウンアッセイの結果は、上述した機能アッセイの結果とは対照的なものであった。本実施例では、イミダゾールで溶出する前に複合体を洗浄し、PB1の損失又は保持をゲル電気泳動で測定した。フリーのPB2-Nは不安定で、このアッセイでは検出されなかった。L695D、F699A及びI750D変異体は全てPB2-Nへの結合を示さなかったが、V715S変異体はPB2-Nへの結合を示した。これは、構造モデルから予想された通りである(図5C)。ポリメラーゼアッセイの結果とプルダウンアッセイの結果との間に相関がないのは、おそらく一部には、後者が平衡結合の試験ではなく、パートナータンパク質の解離速度に依存するという事実によるのであろう。プルダウンアッセイの結果は、V715S変異がPB1-PB2結合をブロックしないことを明確に示す。PB1とPB2との弱められた相互作用は、用いたアッセイ条件下では、酵素活性と矛盾しないように見える。ポリメラーゼ活性アッセイでは全長PB1とPB2を用いた。V715S変異体は、かなりのPB2結合と非常に減少した酵素活性の両方を示すが、このことは、わずかに変化した相互作用様式がポリメラーゼの効率に効果を及ぼすかもしれないことを示唆している。この場合、PB1とPB2が互いに結合していないために、酵素活性は喪失していない。F699A及びI750Dの変異体は弱いPB2結合を示すが、酵素活性は増強している。これらの対照をなす結果は、PB1-PB2界面が、パートナータンパク質が一体となる受動的な結合表面であるばかりでなく、それが酵素活性全体を制御する重要な役割を果たすことを示している。
一つのポリメラーゼサブユニットにおける変異が他のサブユニットの機能に影響すること、及び当該変異が別のサブユニットにおける補償変異(compensating mutation)により抑制されることは、既に報告されている(S11、S12)。
これらの報告は、RNAポリメラーゼには、サブユニット間の情報伝達を通して様々なポリメラーゼ機能を調節する機構が存在することを示唆している。また、上記報告を考慮すると、PB1のVal 715は、PB1とPB2との間のシグナル伝達によりウイルス遺伝子の転写に寄与していると考えられる。この考えによれば、V715S変異により、PB1とPB2とは結合することはできるが、両者の間の適切な情報伝達は阻害されることが理解できる。
また、本発明者らは、PB2変異体を用いて追加の実験を実施した。その結果、種々の界面変異体は、mRNAの顕著な減少を示した。この結果は、上述したPB1-PB2複合体及びNi-NTAを用いたプルダウンアッセイの結果と一致する。
この結果は、相互作用の態様がわずかに変化しただけでポリメラーゼの効率において顕著な効果が現れることを示す。
1.PB1-PB2複合体のクローニング、発現及び精製
PA-PB1複合体について以前に報告したように(S1)、クローニングと精製を行った。インフルエンザA/Puerto Rico/8/34由来の配列を用いた(S2)。ヘキサヒスチジンタグ及びN末端のTEV切断部位とともに、残基1-37、1-86、37-174、252-490及び530-759をコードするPB2遺伝子の断片をpET28bにクローニングした。シャイン・ダルガノ配列を持つPB2遺伝子の下流にPB1-Cコーディング領域をクローニングした。得られた共発現プラスミドを大腸菌BL21(DE3)RILP codon-plus株にトランスフォームし、細胞を0.5 mM IPTGで誘導した後15℃で一晩培養した。Ni-NTAアガロース(Qiagen)を用いるクロマトグラフィー、次いでSP and Q(GE Healthcare)セファロースでPB1-PB2複合体を精製した。Ni-NTAクロマトグラフィー後ヒスチジンタグをTEVプロテアーゼ消化で除去し、その後精製複合体を結晶化のためにcentricon YM-3(Millipore)で5 mg/mlまで濃縮した。
以前に報告したのと同じ方法で(S1)、プルダウンアッセイを行った。複合体をnickel affinityカラムに結合させた後、複合体を500 mMイミダゾールで溶出した。溶出したタンパク質をSDS-acrylamideゲル電気泳動(15%)及びクーマシーブルー染色で分析した。
以前に報告したように、モデルウイルスRNPアッセイを準備した(S1、S14)。PA、PB1(野生型又は変異体のいずれか)、PB2(野生型又は変異体のいずれか)、NP、及びpHH21-vNS-Lucリポータープラスミドをコードするウイルスタンパク質発現プラスミドでHeLa細胞をトランスフェクトした。このリポータープラスミドは、インフルエンザウイルスセグメント8の23ヌクレオチド長の5’末端プロモーター配列と26ヌクレオチド長の3’末端プロモーター配列に挟まれた逆配向のルシフェラーゼ遺伝子を有している。ルシフェラーゼ遺伝子はヒトPol Iプロモーターの制御下に置かれている。16時間インキュベーションした後、ルシフェラーゼアッセイ(Promega)及びリアルタイムRT-PCRを行った。細胞から精製したRNAをオリゴ(dT)20で逆転写させ、ウイルスmRNAのレベルを測定した。ヌクレオチド配列351-380位のルシフェラーゼコード領域に相当する5’-TATGAACATTTCGCAGCCTACCGTAGTGTT-3’(配列番号13)とヌクレオチド配列681-700位のルシフェラーゼコード領域に相補的な5’-CCGGAATGATTTGATTGCCA-3’ (配列番号14)という2つの特異的プライマーを用いたリアルタイム定量PCRに合成一本鎖cDNAをかけた。発現プラスミドから転写されたNP mRNAは、内部コントロールとして使用した。
PB1-V715Sに関するセグメントをコードするウイルスゲノムを含む組換えウイルスを、Neumann et al.(S15)により報告されたプラスミドを用いた形質導入法(plasmid-based transfection method)により作製した。PB1-V715Sゲノムセグメント及び他の7つの野生型のゲノムセグメントは、細胞性RNAポリメラーゼIにより生成した。野生型PB1、PB2、PA、及びNPは、細胞性RNAポリメラーゼIIによりこれらのタンパク質をコードするプラスミドから生成した。細胞を形質導入から48時間インキュベートした後、細胞培養上清のアリコートをMDCK細胞におけるウイルス増幅に使用した。形質導入から48時間の時点で、培養液を回収し、使用するまで-80℃で保存した。
20℃の0.1 Mリン酸カリウム(pH 5.8)及び15% PEG 4,000を含有する結晶化バッファーに対するハンギングドロップ蒸気拡散法により、PB1-PB2複合体の結晶を成長させた。-180℃まで冷却した結晶から回折データを収集した。25%グリセロールを含有する結晶化バッファーを用いて、氷結を防いだ。日本のPhoton Factoryのビームライン17AでX線回折データを収集した。Selenomethionyl置換結晶を用いて、Se-K吸収端付近の3つの異なるX線エネルギーでデータセットを収集した。ADSC Quantum 270 CCD検出器を用いてデータを測定した。結晶は空間群P21に形成され、a = 44.27 Å、b = 61.48 Å、c = 45.47 Å、β = 103.4°であり、非対称ユニット中に2コピーの複合体が含まれていた。HKL2000及びSCALEPACK(S3)を用いて、回折データ積分、スケーリング及びマージングを行った。
SHELXC及びSHELXD(S4、S16)を用いて、14の可能なSe-Met部位のうち12のセレンの位置を見つけた。SOLVE(S5)を用いて、phase determinationを行った。溶媒をフラット化した後、RESOLVE(S6)を用いて、解像度2.1オングストロームの高品質電子密度マップを得た。電子密度を解析し、COOT(S7)を用いてトレースし、REFMAC(S8)でモデルを精密化した。球状の電子密度ピークが|2Fo-Fc|マップの1.3σより上及び|Fo-Fc|マップの3.0σより上に見つかり、かつ立体化学的に合理的な水素結合が許される位置に溶媒分子を置いた。PROCHECK(S9)を用いてPB1-PB2複合体の最終モデルの構造評価を行ったところ、残基の94%がラマチャンドランプロットの最も好ましい領域に存在し、”許されない”領域に存在する残基はなかった。最終モデルは配列中の117残基のうち109残基を含んでおり、PB1の残基678-684及びPB2の残基36-37は観察されなかった。データ収集及び精密化統計のまとめを表1に示す。複合体の原子配位及び構造因子はタンパク質データバンク(Protein Data Bank)にアクセッションコード2ZTTで登録されている。
(引用文献)
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配列番号1は、インフルエンザA/Puerto Rico/8/34 H1N1型のRNAポリメラーゼPB1サブユニットの678-757をコードするDNAのヌクレオチド配列を示す。
<配列番号2>
配列番号2は、インフルエンザA/Puerto Rico/8/34 H1N1型のRNAポリメラーゼPB1サブユニットの678-757のアミノ酸配列を示す。
<配列番号3>
配列番号3は、インフルエンザA/Puerto Rico/8/34 H1N1型のRNAポリメラーゼPB2サブユニットの1-37をコードするDNAのヌクレオチド配列を示す。
<配列番号4>
配列番号4は、インフルエンザA/Puerto Rico/8/34 H1N1型のRNAポリメラーゼPB2サブユニットの1-37のアミノ酸配列を示す。
<配列番号5>
配列番号5は、インフルエンザAウイルス(A/Duck/Hong Kong/2986.1/2000(H5N1))のRNAポリメラーゼPB1サブユニットの678-751をコードするDNAのヌクレオチド配列を示す。
<配列番号6>
配列番号6は、インフルエンザAウイルス(A/Duck/Hong Kong/2986.1/2000(H5N1))のRNAポリメラーゼPB1サブユニットの678-751のアミノ酸配列を示す。
<配列番号7>
配列番号7は、インフルエンザAウイルス(A/Duck/Hong Kong/2986.1/2000(H5N1))のRNAポリメラーゼPB2サブユニットの1-37をコードするDNAのヌクレオチド配列を示す。
<配列番号8>
配列番号8は、インフルエンザAウイルス(A/Duck/Hong Kong/2986.1/2000(H5N1))のRNAポリメラーゼPB2サブユニットの1-37のアミノ酸配列を示す。
<配列番号9>
配列番号9は、インフルエンザAウイルス(A/Equine/London/1416/1973(H7N7))のRNAポリメラーゼPB1サブユニットの678-757をコードするDNAのヌクレオチド配列を示す。
<配列番号10>
配列番号10は、インフルエンザAウイルス(A/Equine/London/1416/1973(H7N7))のRNAポリメラーゼPB1サブユニットの 678-757のアミノ酸配列を示す。
<配列番号11>
配列番号11は、インフルエンザAウイルス(A/Equine/London/1416/1973(H7N7))のRNAポリメラーゼPB2サブユニットの1-37をコードするDNAのヌクレオチド配列を示す。
<配列番号12>
配列番号12は、インフルエンザAウイルス(A/Equine/London/1416/1973(H7N7))のRNAポリメラーゼPB2サブユニットの1-37のアミノ酸配列を示す。
<配列番号13>
配列番号13は、ヌクレオチド配列351-380位のルシフェラーゼコード領域に相当する特異的プライマーのヌクレオチド配列を示す。
<配列番号14>
配列番号14は、ヌクレオチド配列681-700位のルシフェラーゼコード領域に相補的な特異的プライマーのヌクレオチド配列を示す。
<配列番号15>
配列番号15は、インフルエンザA/Puerto Rico/8/34 H1N1型のRNAポリメラーゼPB1サブユニットの全長をコードするDNAのヌクレオチド配列を示す。
<配列番号16>
配列番号16は、インフルエンザA/Puerto Rico/8/34 H1N1型のRNAポリメラーゼPB1サブユニットの全長アミノ酸配列を示す。
<配列番号17>
配列番号17は、インフルエンザA/Puerto Rico/8/34 H1N1型のRNAポリメラーゼPB2サブユニットの全長をコードするDNAのヌクレオチド配列を示す。
<配列番号18>
配列番号18は、インフルエンザA/Puerto Rico/8/34 H1N1型のRNAポリメラーゼPB2サブユニットの全長アミノ酸配列を示す。
<配列番号19>
配列番号19は、インフルエンザA/Puerto Rico/8/34 H1N1型のRNAポリメラーゼPB2サブユニットの1-86をコードするDNAのヌクレオチド配列を示す。
<配列番号20>
配列番号20は、インフルエンザA/Puerto Rico/8/34 H1N1型のRNAポリメラーゼPB2サブユニットの1-86のアミノ酸配列を示す。
Claims (35)
- 下記の(a1)、(a2)又は(a3)のいずれかのポリペプチドと、下記の(b1)、(b2)又は(b3)のいずれかのポリペプチドとを含む複合体。
(a1)配列番号2のアミノ酸配列からなるポリペプチド
(a2)配列番号2のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(a3)配列番号1のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b1)配列番号4のアミノ酸配列からなるポリペプチド
(b2)配列番号4のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b3)配列番号3のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド - 下記の(a1)、(a2)又は(a3)のいずれかのポリペプチドと、下記の(b4)、(b5)又は(b6)のいずれかのポリペプチドとを含む複合体。
(a1)配列番号2のアミノ酸配列からなるポリペプチド
(a2)配列番号2のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(a3)配列番号1のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b4)配列番号20のアミノ酸配列からなるポリペプチド
(b5)配列番号20のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b6)配列番号19のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド - (a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b1)、(b2)又は(b3)のいずれかのポリペプチドをコードするDNAとを含有する組換えベクター。
- (a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b4)、(b5)又は(b6)のいずれかのポリペプチドをコードするDNAとを含有する組換えベクター。
- (a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b1)、(b2)又は(b3)のいずれかのポリペプチドをコードするDNAとを導入した形質転換細胞。
- (a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b4)、(b5)又は(b6)のいずれかのポリペプチドをコードするDNAとを導入した形質転換細胞。
- (a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b1)、(b2)又は(b3)のいずれかのポリペプチドをコードするDNAとを導入した形質転換細胞を培養し、培養物から請求項1記載の複合体を採取することを含む、請求項1記載の複合体の製造方法。
- (a1)、(a2)又は(a3)のいずれかのポリペプチドをコードするDNAと、(b4)、(b5)又は(b6)のいずれかのポリペプチドをコードするDNAとを導入した形質転換細胞を培養し、培養物から請求項2記載の複合体を採取することを含む、請求項2記載の複合体の製造方法。
- 請求項1記載の複合体の結晶。
- 請求項2記載の複合体の結晶。
- 空間群がP21である請求項9記載の結晶。
- 単位格子が、a=41.12±50Å、b=61.37±50Å、c=45.36±50Åの大きさとβ=103.5±30°の角度を持つ請求項11記載の結晶。
- 請求項1又は2記載の複合体を沈殿剤の存在下に結晶化させることを含む、請求項1又は2記載の複合体の結晶の製造方法。
- 沈殿剤がリン酸カリウム及びPEG 4000である請求項13記載の方法。
- 下記の(a1)、(a2)又は(a3)のいずれかのポリペプチド。
(a1)配列番号2のアミノ酸配列からなるポリペプチド
(a2)配列番号2のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(a3)配列番号1のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(a1)のポリペプチドと同様の生物学的活性を有するポリペプチド - 請求項15記載のポリペプチドをコードするDNA。
- 請求項16記載のDNAを含有する組換えベクター。
- 請求項15記載のポリペプチドをコードするDNAを導入した形質転換細胞。
- 請求項15記載のポリペプチドをコードするDNAを導入した形質転換細胞を培養し、培養物から請求項15記載のポリペプチドを採取することを含む、請求項15記載のポリペプチドの製造方法。
- 下記の(b1)、(b2)又は(b3)のいずれかのポリペプチド。
(b1)配列番号4のアミノ酸配列からなるポリペプチド
(b2)配列番号4のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b3)配列番号3のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b1)のポリペプチドと同様の生物学的活性を有するポリペプチド - 下記の(b4)、(b5)又は(b6)のいずれかのポリペプチド。
(b4)配列番号20のアミノ酸配列からなるポリペプチド
(b5)配列番号20のアミノ酸配列において、1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド
(b6)配列番号19のヌクレオチド配列からなるDNAに相補的なDNAとストリンジェントな条件下でハイブリダイズするDNAによりコードされるポリペプチドであり、かつ(b4)のポリペプチドと同様の生物学的活性を有するポリペプチド - 請求項20又は21記載のポリペプチドをコードするDNA。
- 請求項22記載のDNAを含有する組換えベクター。
- 請求項20又は21記載のポリペプチドをコードするDNAを導入した形質転換細胞。
- 請求項20又は21記載のポリペプチドをコードするDNAを導入した形質転換細胞を培養し、培養物から請求項20又は21記載のポリペプチドを採取することを含む、請求項20又は21記載のポリペプチドの製造方法。
- 候補物質の存在下で、インフルエンザウイルスのRNAポリメラーゼを構成するPB1サブユニット又はその部分断片とPB2サブユニット又はその部分断片とを接触させ、前記PB1サブユニット又はその部分断片と前記PB2サブユニット又はその部分断片との相互作用を阻害する物質を選択する工程を含む、抗インフルエンザ薬の有効成分となり得る物質のスクリーニング方法。
- PB1サブユニットが以下の(a4)又は(a5)のポリペプチドからなるものである請求項26に記載の方法。
(a4) 配列番号16のアミノ酸配列からなるポリペプチド
(a5) 配列番号16のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、インフルエンザウイルスのRNAポリメラーゼのPB1サブユニット活性を有するポリペプチド - PB1サブユニットの部分断片が請求項15に記載のポリペプチドからなるものである請求項26に記載の方法。
- PB2サブユニットが以下の(b7)又は(b8)のポリペプチドからなるものである請求項26に記載の方法。
(b7) 配列番号18のアミノ酸配列からなるポリペプチド
(b8) 配列番号18のアミノ酸配列において1若しくは数個のアミノ酸が欠失、置換若しくは付加されたアミノ酸配列からなり、かつ、インフルエンザウイルスのRNAポリメラーゼのPB2サブユニット活性を有するポリペプチド - PB2サブユニットの部分断片が請求項20又は21に記載のポリペプチドからなるものである請求項26に記載の方法。
- PB1サブユニットの相互作用部位のアミノ酸残基が、配列番号16のアミノ酸配列におけるLeu 695、Lys 698、Phe 699、Val 715、Asp 725、Ile746及びIle 750のアミノ酸残基並びに配列番号2のアミノ酸配列における前記アミノ酸残基に対応する残基からなる群から選択される少なくとも1つのアミノ酸残基である請求項26~30のいずれか1項に記載の方法。
- PB2サブユニットの相互作用部位のアミノ酸残基が、配列番号4、18又は20のアミノ酸配列におけるGlu 2、Arg 3、Ile 4、Lys 5、Glu 6、Leu 7、Arg 8、Asn 9及びLeu 10のアミノ酸残基ならびにからなる群から選択される少なくとも1つのアミノ酸残基である、請求項26~30のいずれか1項に記載の方法。
- PB1サブユニットの相互作用部位のアミノ酸残基が、配列番号16のアミノ酸配列におけるLeu 695、Phe 699、Val 715、Ile746及びIle 750のアミノ酸残基並びに配列番号2のアミノ酸配列における前記アミノ酸残基に対応する残基からなる群から選択される少なくとも1つのアミノ酸残基である請求項26~30に記載の方法。
- PB2サブユニットの相互作用部位のアミノ酸残基が、配列番号4、18又は20のアミノ酸配列におけるGlu 2、Ile 4、Leu 7及びLeu 10からなる群から選択される少なくとも1つのアミノ酸残基である、請求項26~30に記載の方法。
- 候補物質が、化合物及びその塩、ペプチド、抗体並びに核酸からなる群から選択される少なくとも一種である、請求項26~34のいずれか1項に記載の方法。
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JPWO2010044468A1 (ja) | 2012-03-15 |
US8569016B2 (en) | 2013-10-29 |
US20110262944A1 (en) | 2011-10-27 |
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EP2366784B1 (en) | 2015-09-23 |
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