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Construction of expression system for RNA polymerase derived from influenza virus, crystallization of the RNA polymerase, and screening method for Anti-influenza agent

a technology of rna polymerase and expression system, which is applied in the field of construction of an expression system for rna polymerase, crystallization of rna polymerase, and screening method of anti-influenza agent, can solve the problems of drug use in children limited, drug useless against many strains, and catastrophic loss of li

Inactive Publication Date: 2011-05-05
UNIV OF TSUKUBA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0045]The present invention enables the large-scale expression of an RNA polymerase PA-PB1 complex. The present invention also enables the provision of a crystal of the RNA polymerase PA-PB1 complex for use in analysis of protein three-dimensional structure. The present invention further provides a method for screening a substance capable of serving as an active ingredient in anti-influenza drugs. Since the interaction site between PA and PB1 locates in a region where amino acid sequence is highly conserved, this site can be used as a target for development of anti-influenza drugs, regardless of the phenotypes of influenza viruses or mutations in their genome.

Problems solved by technology

Influenza A virus is a major human and animal pathogen, which periodically causes a global pandemic and may result in a catastrophic loss of life.
However, oseltamivir-resistant influenza is already emerging, and this drug is of limited use in children due to its side effects.
However, in the case of these drugs targeting the M2 protein, a single residue mutation in M2 is sufficient to confer resistance to the virus, which may render the drugs useless against many strains.
However, none of the current medications targets the viral RNA polymerase.
In addition, none of the current reports mentions techniques for large-scale expression of the RNA polymerase, which are the key to RNA polymerase studies.

Method used

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  • Construction of expression system for RNA polymerase derived from influenza virus, crystallization of the RNA polymerase, and screening method for Anti-influenza agent
  • Construction of expression system for RNA polymerase derived from influenza virus, crystallization of the RNA polymerase, and screening method for Anti-influenza agent
  • Construction of expression system for RNA polymerase derived from influenza virus, crystallization of the RNA polymerase, and screening method for Anti-influenza agent

Examples

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example 1

1. Materials and Methods

(1) Cloning, Expression and Purification of PA-PB1 Complex

[0197]To construct a protein expression system for use in this study, the influenza A / Puerto Rico / 8 / 1934 (also referred to as “A / PR / 8 / 34”) H1N1 gene (cDNA (influenza A / PR / 8 / 34, H1N1) in pET14b) was used. A gene encoding amino acids 239-716 of the RNA polymerase PA subunit was amplified by PCR, digested with restriction enzymes BamHI and NotI, and then integrated into a protein expression vector, pET28b (Novagen), which had been digested with the same enzymes. In this case, a histidine tag was integrated N-terminal to PA in order to facilitate protein purification, and a TEV protease cleavage sequence was integrated between the histidine tag and PA in order to remove this tag after purification (pET28HisTEV-PA(239-716)). Separately, a gene encoding amino acids 1-81 of the PB1 subunit was amplified by PCR, digested with restriction enzymes NdeI and NotI, and then integrated into a protein expression vect...

example 2

1. Materials and Methods

(1) Reporter Assay for Measurement of Production Levels of Various Viral RNAs

[0222]Viral transcription and replication were reconstituted in 293T cells by transfection with a plasmid carrying a fragment of the Luciferase gene whose 5′- and 3′-terminal ends were linked respectively to cDNAs of the viral genomic 3′- and 5′-terminal promoter sequences (26 and 23 nucleotides from the 3′- and 5′-terminal ends, respectively) under the control of the host DNA-dependent RNA polymerase I promoter and with a plasmid carrying viral genes (PB1, PB2, PA, NP) essential for transcription and replication of the viral genome under the control of the DNA-dependent RNA polymerase II promoter.

[0223]In this assay, the PA genes used were the wild-type PA gene, as well as PA variant genes encoding PA amino acid sequences with a mutation from Val 636 to Ser, from Leu 640 to Glu, from Leu 666 to Glu, or from Trp 706 to Ala in the wild-type PA gene. At 16 hours after transfection with...

example 3

1. Materials and Methods

(1) In Silico Search for Candidate Compounds

[0226]Based on the results of X-ray crystal structure analysis on the binding site between PA and PB1, the inventors of the present invention conducted an in silico search for candidate compounds capable of inhibiting the interaction between PA and PB1.

[0227]More specifically, two million compounds were divided into 200 groups of ten thousand each and calculated in parallel to search the compounds.

[0228]As a result, 5559 compounds were hit as candidate compounds through the in silico analysis.

[0229]Among the above 5559 compounds, 1298 compounds were determined to have a structure unsuitable for use as a drug. These compounds were excluded and the remaining 4261 compounds were able to be obtained as the final results of screening.

[0230]The selected compounds were purchased from Namiki Shoji Co., Ltd., Japan.

(2) Screening of Candidate Compounds

[0231]In this example, six compounds, i.e., Compounds A to F were used as c...

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Abstract

The present invention aims to express influenza virus RNA polymerase on a large scale, to crystallize the influenza virus RNA polymerase, and to provide a method for screening a substance capable of serving as an active ingredient in anti-influenza drugs which target a protein highly conserved among influenza virus species.The present invention provides a complex comprising a polypeptide consisting of an amino acid sequence at positions 239-716 of the RNA polymerase PA subunit in influenza A / Puerto Rico / 8 / 1934 H1N1 and a polypeptide consisting of an amino acid sequence at positions 1-81 of the RNA polymerase PB1 subunit in influenza A / Puerto Rico / 8 / 1934 H1N1, as well as a method for screening a substance capable of serving as an active ingredient in anti-influenza drugs, which comprises the step of selecting a substance which inhibits the interaction between α-subunit and β-subunit 1, each constituting influenza virus RNA polymerase, in the presence of a candidate substance.

Description

TECHNICAL FIELD[0001]The present invention relates to the construction of an expression system for influenza virus RNA polymerase, crystallization of the influenza virus RNA polymerase, and a method for screening a substance capable of serving as an active ingredient in anti-influenza drugs.BACKGROUND ART[0002]Influenza virus is an RNA virus having negative-strand RNA as its genome. Frequent mutations occur in the phenotype or genomic nucleotide sequences of influenza virus, and hence the virus occasionally give rises inter-specie infection.[0003]Influenza A virus is a major human and animal pathogen, which periodically causes a global pandemic and may result in a catastrophic loss of life. The recent emergence in Asia of avian influenza related to highly pathogenic forms of the human virus has highlighted the urgent need for new effective treatments (Non-patent Document 1).[0004]Most current influenza drugs target either hemagglutinin (HA) or neuraminidase (NA) on the virus surface...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/53C07K14/00C12N15/63C12N5/10C12P21/02C07H21/00
CPCC07K14/005C12N2760/16122C12N9/127C07K2299/00
Inventor NAGATA, KYOSUKEKAWAGUCHI, ATSUSHIPARK, SAM-YONGOBAYASHI, EIJI
Owner UNIV OF TSUKUBA
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