JP2007274988A - Type i interferon-non-responsible non-human model animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a picornavirus-specific type I interferon non-responsible non-human model animal deleting the response of type I interferon to the infection of viruses belonging to picornavirus virus family, to provide a method for identifying an anti-picornavirus agent with the type I interferon non-responsible non-human model animal. <P>SOLUTION: A non-human animal obtained by inactivating all or a part of an endogenous gene encoding MDA5 (Melanoma differentiation associated gene-5) with genetic abnormality such as destruction/deletion/substitution to lose a function for expressing the MDA5 is used as the type I interferon non-responsible non-human model animal deleting the response of the type I interferon to the infection of the viruses belonging to the picornavirus virus family. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a non-human animal in which the function of expressing MDA5 (Melanoma differentiation associated gene-5) has been lost, and the non-response to type I interferon in which the response of type I interferon to infection with a virus belonging to the picornavirus family has been deleted. The present invention relates to a method for use as a sex non-human model animal, a type I interferon non-responsive non-human model animal, and a method for identifying an anti-picornavirus agent using the type I interferon non-responsive non-human model animal.

  The innate immune response to microbial pathogens such as viruses and bacteria is characterized by Toll-like receptors (TLRs) and host pattern recognition receptors (PRRs) such as helicase family members that are specific for invading pathogens called pathogen-associated molecular patterns (PAMPs) It is triggered by recognizing the structure (see, for example, Non-Patent Document 1 and Non-Patent Document 2). Host pattern recognition receptors (PRRs) play an essential role in the recognition of virus-specific molecular patterns including DNA, single-stranded (ss) RNA, double-stranded (ds) RNA and glycoproteins. TLR3 localized in the endosomal membrane is known to recognize viral dsRNA in addition to polyinosine-polycytidylic acid (poly I: C), which is a synthetic dsRNA analog. RDA-I (Retinoic acid-inducible protein-I), which is a cytoplasmic protein, and MDA-5 (Melanoma differentiation associated gene-5), also known as helicard, have been identified as dsRNA detectors. RIG-I is a DExD / Hbox RNA helicase with two domain (CARD) -like modules that recruit caspases. The helicase domain is thought to be involved in dsRNA recognition and CARD has been reported to be essential for the initiation of downstream signaling leading to activation of IRF3, IRF7 and NF-κB ( For example, refer nonpatent literature 3). In addition, it has been reported that RIG-I-deficient mice do not consistently produce type I interferon (IFN) against vesicular stomatitis virus (VSV), Newcastle disease virus (NDV) and Sendai virus (SeV) infection. (For example, refer nonpatent literature 4). Furthermore, the activation of IRF3 and NF-κB in response to viral infection was impaired in RIG-I deficient cells. MDA-5 has a structure similar to RIG-I and has been reported to be involved in mediating antiviral responses (see, for example, Non-Patent Document 5 and Non-Patent Document 6).

  Although RIG-I and MDA5 are suggested to be involved in the recognition of viral dsRNA, in addition to the functional relationship between these dsRNA detectors in vivo, the functional role of MDA5 is unknown. Thus, since the clear function of the dsRNA detector has not been elucidated, interferon responses based on these actions could not be suppressed in mammals.

Annu Rev Immunol. 20, 197-216, 2002 Annu Rev Immunol. 21,335-376, 2003 See Nat Immunol. 5, 730-737, 2004. Immunity Vol.23 19-28 July 2005 Curr Biol. 12, 838-843, 2002 Proc Natl Acad Sci USA. 101, 17264-17269, 2004

  An object of the present invention is to provide a picornavirus-specific non-type I interferon-unresponsive non-human model animal in which the response of type I interferon to infection with a virus belonging to the picornavirus family has been deleted, and this type I interferon non-responsiveness An object is to provide a method for identifying an anti-picornavirus agent using a non-human model animal.

The inventors first established MDA5 ^{− / −} mice to investigate the functional role of MDA5 in vivo. These MDA5 ^{− / −} mouse-derived fibroblasts and dendritic cells lacked a type I interferon response to infection with encephalomyocarditis virus (EMCV) and Tyler virus (Theiler's virus) belonging to the picornavirus family. However, responsiveness to other RNA viruses was normal. Thus, it was found that MDA5 is a receptor that specifically recognizes a picornavirus genus and induces type I interferon production, thereby completing the present invention.

  That is, the present invention (1) inactivates all or part of the endogenous gene of non-human animal encoding MDA5 (Melanoma differentiation associated gene-5) by gene mutation such as disruption, deletion or substitution, and expresses MDA5 A non-human animal that is non-human type that does not respond to type I interferon response to infection with a virus belonging to the picornavirus family, and (2) a non-human model The non-human model animal described in (1) above, wherein the animal is a model mouse, or (3) the destruction or all of the endogenous gene encoding MDA5 (Melanoma differentiation associated gene-5) Non-human animals that have been inactivated by gene mutations such as deletion and substitution and have lost the function of expressing MDA5 belong to the Picornavirus family A method for use as a type I interferon non-responsive non-human model animal deficient in a type I interferon response to infection with a virus, and (4) the non-human model animal is a model mouse 3) The method described above.

  In addition, the present invention (5) inactivates all or part of the endogenous gene of non-human animal encoding MDA5 (Melanoma differentiation associated gene-5) by gene mutation such as disruption, deletion or substitution, and expresses MDA5 A non-human animal that has lost its function to be infected with a virus belonging to the picornavirus family, and a test substance is administered before, after, or at the same time as the virus infection, and the type I interferon, RANTES, or IL-6 in the non-human animal is administered. The production amount is measured, and when the production of type I interferon, RANTES or IL-6 is observed, the test substance is evaluated as an anti-picornavirus agent. , (6) Comparison and evaluation of type I interferon, RANTES or IL-6 production in wild-type animals of the same species as non-human animals And identification method of the above (5), wherein Rukoto, (7) a non-human animal, relates to a method of identifying the (5) or (6), wherein it is a mouse.

  Further, the present invention provides (8) expression of MDA5 by inactivating all or part of the endogenous gene of non-human animal encoding MDA5 (Melanoma differentiation associated gene-5) by gene mutation such as disruption, deletion or substitution. A non-human animal that has lost its function to be infected is infected with a myocarditis virus (EMCV) belonging to the picornavirus family, and a test substance is administered before, after, or simultaneously with the EMCV infection, and the histology of the heart of the non-human animal When analysis is performed and the inhibition of focal necrosis of cardiomyocytes is observed, the test substance is evaluated as an anti-EMCV agent, and (9) the same kind as a non-human animal The identification method according to (8) above, characterized in that it is compared and evaluated with the degree of suppression of focal necrosis of cardiomyocytes in a wild type animal, and (10) the nonhuman animal is a mouse The identification method according to (8) or (9) above, or (11) destruction or deletion / replacement of all or part of an endogenous gene of a non-human animal encoding MDA5 (Melanoma differentiation associated gene-5) A cell derived from a non-human animal that has been inactivated by gene mutation and has lost the function of expressing MDA5 is infected with a virus belonging to the picornavirus family, and a test substance is contacted with the cell before, after, or simultaneously with the virus infection. When the amount of type I interferon, RANTES or IL-6 produced in the non-human animal-derived cells is measured and production of type I interferon, RANTES or IL-6 is observed, the test substance is treated with anti-picorna. A method for identifying an anti-picornavirus agent characterized by evaluation as a virus agent, and (12) a detailed method derived from a wild-type animal of the same species as a non-human animal. The method for identification according to (11) above, characterized in that it is compared and evaluated with the production amount of type I interferon, RANTES or IL-6 in (13), and (13) the nonhuman animal is a mouse The identification method according to (11) or (12), or (14) the identification method according to any one of (11) to (13) above, wherein the cell is an embryonic fibroblast or a dendritic cell. .

In the present invention, it was clarified that MDA5 specifically recognizes a virus of the picornavirus family. MDA5 is a helicase, and by adjusting this function, infection control of viruses belonging to this group, such as poliovirus, coxsackie virus, rhinovirus causing cold syndrome, etc. may be possible. In addition, MDA5 ^{− / −} mice are expected to be used as model mice for human viral infections by mating with gene mutant mice expressing human virus receptors. Furthermore, an agent that regulates the activity of MDA5 can be screened using MDA5-deficient (MDA5 ^{− / −} ) mice and control (wild-type) mice. It can be a model animal for virus infection susceptibility. This mouse can be used to screen for new drugs.

  As the non-human model animal of the present invention, all or part of the endogenous gene of the non-human animal encoding MDA5 was inactivated by gene mutation such as disruption, deletion, or substitution, and the function of expressing MDA5 was lost. The method is not particularly limited as long as it is a non-human animal that is non-human type non-responsive to type I interferon and lacks the type I interferon response to infection with a virus belonging to the picornavirus family. As a non-human animal in which all or part of the endogenous gene encoding MDA5 is inactivated by gene mutation such as disruption, deletion or substitution, and the function of expressing MDA5 is lost, it belongs to the picornavirus family As a non-human model animal that does not respond to type I interferon and lacks type I interferon response to viral infection It is not particularly limited as long as it is a method for use, as the type I interferons, can be specifically exemplified IFN-alpha or IFN-beta.

  In the present invention, type I interferon non-responsiveness means that the reactivity of type I interferon production in the living body or cells, tissues or organs constituting the living body against infection (stimulation) by a virus belonging to the picornavirus family has decreased, or It means almost lost. Therefore, in the present invention, the type I interferon non-responsive non-human animal is a type I interferon-producing reactivity of a living body or a cell, tissue or organ constituting the living body against infection with a virus belonging to the picornavirus family. A non-human animal such as a mouse, rat, or rabbit that is reduced or almost lost. Among these, a type I interferon non-responsive model mouse can be preferably exemplified. The type I interferon non-responsive non-human animal of the present invention is preferably non-responsive to RANTES and IL-6 in addition to non-type I interferon non-responsiveness.

  The virus belonging to the Picornaviridae family (Picornaviridae) is an RNA virus with a single plus-strand RNA as its genome. It has no envelope, an icosahedral capsid, and a small RNA (pico-rna). Viruses belonging to this picornavirus family named by meaning include viruses belonging to the Enterovirus genus such as poliovirus, coxsackie virus, echovirus, and rhinovirus genus such as human rhinovirus and mengovirus ( Rhinovirus), viruses belonging to Cardiovirus such as encephalomyocarditis virus (EMCV), viruses belonging to Aphthovirus such as foot-and-mouth disease virus, and hepatoviruses such as hepatitis A virus (Hepatovirus And viruses belonging to the genus Parechovirus such as Human parechovirus.

Examples of non-human animals that have lost the function of expressing MDA5 of the present invention include MDA5 ^{− / −} mice, as well as rodents such as MDA5 ^{− / −} rats that lack MDA5 gene function. As these non-human animals deficient in the function of the MDA5 gene, MDA5 ^{− / −} non-human animals born according to Mendel's law can be obtained together with wild-type littermates that can be used for accurate comparative experiments. Is preferable. A method for manufacturing animal functions deficient in such MDA5 gene, MDA5 ^{- / -} explained mice below for examples.

  The MDA5 gene can be screened using a probe derived from the mouse MDA5 gene by amplifying a mouse gene library by the PCR method or the like. The screened MDA5 gene can be subcloned using a plasmid vector or the like, and can be identified by restriction enzyme mapping and DNA sequencing. Next, all or a part of the gene encoding MDA5 is replaced with a pMC1 neo gene cassette or the like, and a diphtheria toxin A fragment (DT-A) gene or herpes simplex virus thymidine kinase (HSV-tk) is placed on the 3 ′ end side. ) A target vector is prepared by introducing a gene such as a gene.

The prepared targeting vector is linearized, introduced into ES cells by electroporation (electroporation), etc., and homologous recombination is performed. Among the homologous recombinants, G418 and ganciclovia (GANC) ES cells that have undergone homologous recombination with antibiotics such as are selected. In addition, it is preferable to confirm whether the selected ES cell is the target recombinant by Southern blotting or the like. The confirmed ES cell clone is microinjected into a blastocyst of a mouse, and the blastocyst is returned to a temporary parent mouse to produce a chimeric mouse. When this chimeric mouse is crossed with a wild type mouse, a heterozygous mouse (F1 mouse; MDA5 ^+/− ) can be obtained, and by crossing this heterozygous mouse, the MDA5 ^{− / of the} present invention can be obtained. ^- it is possible to generate mice. In addition, as a method for confirming whether or not MDA5 occurs in MDA5 ^{− / −} mice, for example, RNA is isolated from the mouse obtained by the above method and examined by Northern blotting or the like. There is a method for examining the expression of MDA5 by Western blotting or the like.

In addition, the generated MDA5 ^{− / −} mice are unresponsive to type I interferon. For example, viruses belonging to the picornavirus family can be used to immunize macrophages, mononuclear cells, dendritic cells, etc. of MDA5 ^{− / −} mice. It can be confirmed by infecting cells or embryonic fibroblasts in vitro or in vivo, and measuring the production amount of type I interferon, IL-6, RANTES, etc. in such cells.

  The method for identifying the anti-picornavirus agent of the present invention includes a function of inactivating all or part of an endogenous gene of a non-human animal encoding MDA5 by gene mutation such as disruption, deletion or substitution, and expressing MDA5 A non-human animal that has lost the intestine, and is infected with a virus belonging to the picornavirus family, and a test substance is administered before, after, or simultaneously with the virus infection, and the amount of type I interferon, RANTES, or IL-6 produced in the non-human animal When the production of type I interferon, RANTES, or IL-6 is observed, the test substance is evaluated as an anti-picornavirus agent, or all of the endogenous genes of non-human animals encoding MDA5 Or non-human animals that have been partially inactivated by mutations, deletions, substitutions, etc., and have lost the function of expressing MDA5 The cell is infected with a virus belonging to the picornavirus family, and a test substance is contacted with the cell before or after the virus infection, and production of type I interferon, RANTES or IL-6 in the cell derived from the non-human animal When the amount is measured and production of type I interferon, RANTES or IL-6 is observed, the method is not particularly limited as long as the test substance is evaluated as an anti-picornavirus agent. By using a method for identifying a viral agent, an anti-picornavirus agent can also be screened. Such an embodiment of the identification method of the present invention will be described below with an example.

  Immune cells such as dendritic cells obtained from the type I interferon-nonresponsive human animal of the present invention and embryonic fibroblasts, a test substance, and a virus belonging to the picornavirus family are cultured together, and the cells A method for identifying and measuring the degree of production of type I interferon in the rat, or pre-administering a test substance to the type I interferon-unresponsive non-human model animal of the present invention, and then adding picorna to immune cells obtained from the non-human animal. An identification method for infecting a virus belonging to the virus family and measuring and evaluating the degree of production of type I interferon in the immune cells, and a virus belonging to the picornavirus family to the non-human model animal of the type I interferon non-responsive After pre-infection, the immune cells obtained from the non-human animal are present in the presence of the test substance. A method for identifying and measuring the degree of type I interferon production in the immune cells, or by infecting a non-human model animal of the present invention with a type I interferon-nonresponsive human infection with a virus belonging to the picornavirus family. Alternatively, an identification method for measuring and evaluating the degree of type I interferon production in immune cells obtained from the non-human model animal by administering a test substance before and after, or the type I interferon-unresponsive non-human model animal of the present invention An identification method in which a virus belonging to the picornavirus family is infected, a test substance is administered simultaneously with or before the infection, and the degree of type I interferon production in serum obtained from the non-human model animal is measured and evaluated. be able to. Instead of measuring the degree of production of type I interferon, the degree of production of RANTES or IL-6 can also be measured. In the evaluation, wild-type animals of the same species as non-human animals, particularly litters of the same litter are used. It is preferable to compare and evaluate the production amount of type I interferon, RANTES or IL-6.

  As an identification method of the anti-EMCV agent of the present invention, all or part of the endogenous gene of non-human animal encoding MDA5 (Melanoma differentiation associated gene-5) is inactivated by gene mutation such as disruption, deletion or substitution. A non-human animal that has lost the function of expressing MDA5 is infected with myocarditis virus (EMCV) belonging to the picornavirus family, and a test substance is administered before, after, or simultaneously with the EMCV infection, and the heart of the non-human animal When the suppression of focal necrosis of cardiomyocytes is observed, the identification method is not particularly limited as long as it is an identification method for evaluating the test substance as an anti-EMCV agent. Identification of the anti-EMCV agent of the present invention The method can also be used to screen for anti-EMCV agents. In the evaluation, it is preferable to compare and evaluate the degree of suppression of focal necrosis of cardiomyocytes in a wild-type animal of the same species as the non-human animal, particularly a litter.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Materials and methods]
(1) Production of MDA5 ^{− / −} mouse The MDA5 gene was isolated from genomic DNA extracted from ES cells (GSI-I) by PCR. A targeting vector was constructed by replacing the 4.3-kb fragment encoding MDA5ORF (including DExH box) with a neomycin resistance gene cassette (neo), and a simple perpesvirus thymidine kinase (HSV-) driven by the PGK promoter. TK) was inserted into the genomic fragment for negative selection. After transfecting the targeting vector into ES cells, double resistant colonies of G418 and gancyclovir were selected, screened by PCR, and confirmed by Southern blot. Homozygous recombinants were microinjected into female C57BL / 6 mice and heterozygous F1 progeny were intercrossed to obtain MDA5 ^{− / −} mice.

(2) MyD88 ^{- / -} mice, TRIF ^{- / - mice, TLR3 - / -, TRIF -} / - mice and ^{RIG-I - / -} Preparation of Mouse MyD88 ^{- / -} mice, TLR3 ^{- / -} mice, TRIF ^{- / -} the mice literature Sceience 301, was prepared by the method described in 640-3 (2003). Moreover, IFNα / βR ^{− / −} mice were prepared by the method described in Int Immunol 14 1225-31 (2002). Furthermore, RIG-I ^{− / −} adult mice were produced by crossing with ICR mice.

^{(3) RIG-I - /} - MDA5 - / - when mating and mouse produced RIG-I heterozygotes ^{(RIG-I +/-)} mice and MDA5 heterozygous ^{(MDA5 +/-)} mice, that child RIG-I ^{+ / +} MDA5 ^{+ / +} , RIG-I ^+/- MDA5 ^{+ / +} , RIG-I ^{+ / +} MDA5 ^+/- , RIG-I ^+/- MDA5 ^+/- . This genotype can be identified by using PCR or Southern blotting using genomic DNA extracted from the mouse tail. Of ^these, by mating with each other between the RIG-I ^{+/- MDA5} +/- genotype ^{mice, RIG-I + / + MDA5} + / +, RIG-I +/- MDA5 + / +, RIG- I ^{+ / +} MDA5 ^+/− , RIG-I ^+/− MDA5 ^+/− , RIG-I ^{− / −} MDA5 ^{+ / +} , RIG-I ^{− / −} MDA5 ^+/− , RIG-I ^{+ / +} MDA5 ^{− / −} , RIG-I ^+/− MDA5 ^{− / −} , RIG-I ^{− / −} MDA5 ^{− / −} . Among these, RIG-I ^{− / −} MDA5 ^{− / −} mice were identified by PCR or Southern blotting.

(4) cells, reagents and virus ^{RIG-I -} / ^- for Preparation of MEF and bone marrow-derived GMCSF or Flt3L-DC is Document Immunity 23, to produce 19-28 (2005) as described. PDC and cDC were isolated from Flt3L-DC by MACS using anti-B220 and CD11c microbeads manufactured by Miltenyi Biotech as described above. Poly (I: C) was purchased from Amersham Biosciences. An anti-MDA5 polyclonal antibody was produced corresponding to amino acids 1005-1019 of mouse MDA5. NCV, VSV, influenza virus, ΔNS1, JEV, EMCV, Tyler virus, and Mengo virus were each prepared by methods described in the literature. As SeV (V-) and (Cm) mutants, those provided by Dr. A. Kato were used.

(5) Northern blot PEC was treated for 8 hours with or without 1000 U / ml IFN-β, and total RNA was extracted with TRIzol reagent (Invitrogen). RNA was electrophoresed, transferred to a nylon membrane, and then hybridized with the indicated cDNA probe. To detect the expression of MDA5 mRNA, a 308 bp fragment (777-1084) was used as a probe. The same membrane was rehybridized with β-actin probe.

(6) Western blot MEF was treated with 1000 U / ml IFN-β for 8 hours. The cells were then lysed in a lysis buffer containing 1.0% Nonidet-P40, 150 mM NaCl, 20 mM Tris-Cl (pH 7.5), 1 mM EDTA and protease inhibitor cocktail (Roche). . The cell lysate was lysed by SDS-PAGE and transferred to a PVDF membrane. Such membranes were blotted with a specific antibody of MDA5 protein and visualized with an enhanced chemiluminescence system (Perkinermer).

(7) Histological analysis Hearts were collected from EMCV-infected mice and fixed with 3.7% formaldehyde. Cross sections (5 μm) were cut across the heart and stained with hematoxylin and eosin.

(8) Plaque assay The hearts of EMCV-infected mice were homogenized in PBS. Virus titration in PBS-containing virus was confirmed with a standard plaque assay as described above. Prepared HeLa cells were infected with serial dilutions. DMEM containing 1% low melting point agarose was overlaid on the cells and incubated for 48 hours. Thereafter, plaques were counted.

(9) Luciferase Assay MEFs obtained from wild-type or RIG-I ^{− / −} MDA5 ^{− / −} embryos on embryonic day 11.5, together with empty vector (control), RIG-I or MDA5 expression vector, IFN-β promoter Was transiently transfected with a reporter construct containing. As an internal control, Renilla luciferase construct was transfected. Transfected cells were subjected to mock treatment (Mock) or infected with EMCV with increasing moi or with SVc- (moi = 20) for 24 hours. Cells were lysed and subjected to luciferase assay using a dual luciferase reporter assay system (Promega).

(10) Measurement of production of IFN-α, IL-12p40, IL-6 and Rantes Culture supernatant is collected and production of IFN-α, IL-6 or IL-12p40 by enzyme immunosorbent assay (ELISA) analyzed. ELISA kits for mouse IFN-α were purchased from PBL Biomedical Laboratories, and ELISA kits for IL-6, IL-12p40, and Rantes were obtained from R & D Systems.

(11) Flow cytometry 200 μg of poly I: C was injected into a mouse vein, and the mouse was disposed 24 hours after the injection. As described above, splenocytes were prepared from mice using collagenase and DNaseI. Spleen cells were stained with CD86-FITC (BD Biosciences Pharmingen), B220-PE, and CD11c-APC and analyzed with FACS Calibur (BD Bioscience).

(12) Preparation of in vitro transcribed dsRNA The cDNA sequence of mouse Lamin A / C was amplified by PCR, cloned with pT7 blue T vector (Novagen) and sequenced. The T7 RNA polymerase promoter was tagged to the ends of the 200 and 400 bp DNA sequences corresponding to mouse Lamin A / C by another PCR reaction using primers tagged with the following T7 sequences;
7T Lamin. Forward, TAATACGACTCACTATAGGacttgtgtggctgcgcaggc
7T Lamin. 200 reverse, TAATACGACTCACTATAGGccactcgcctcagcatctcat
7T Lamin. 400 Reverse, TAATACGACTCACTATAGGctgtcccctgggtcccatg
The PCT product was gel purified and used for in vitro transcription and production of dsRNA using the T7 RiboMAX ^™ Express RNAi System (Promega) as per manufacturer's instructions.

(13) Echocardiography Ultrasonic examination (SONOS-5500, equipped with a 15-MHz linear transducer, manufactured by Philips Medical Systems) on mice anesthetized with 2.5% avertin (8 μl / g) Echocardiography was performed using The heart was projected as a two-dimensional parasternal short-axis image, and an M-mode echocardiogram of the midventricle was recorded at the papillary muscle level. LV heart rate, anterior and posterior wall thickness, and end-diastolic and end-systolic inner diameters were obtained from M-mode images.

[result]
To investigate the functional role of MDA5 in vivo, MDA5 ^{− / −} mice were established and the contribution of MDA5 to virus recognition was analyzed (see FIGS. 1a and 1b). MDA5 mRNA expression was not detected in MDA5 ^{− / −} cells as in the case of MDA5 protein (see FIGS. 1c and 1d). In contrast to RIG-I ^{− / −} mice, many of which die at the embryonic stage, MDA5 ^{− / −} mice are born and grow healthy according to the Mendelian ratio, and macroscopic developmental abnormalities are observed up to 24 weeks of age. I couldn't. Flow cytometric analysis of CD3, B220, CD11c in the spleen and lymph nodes confirmed that there was no change in the number of lymphocytes and dendritic cells between wild type and MDA5 ^{− / −} mice.

TLR3, RIG-I and MDA5 have been implicated in poly I: C recognition and subsequent induction of antiviral responses. However, the in vivo contribution of these molecules in response to dsRNA stimulation has not yet been clarified. Therefore, we next examined the in vivo response to poly I: C in mice lacking RIG-I, MDA-5, TRIF, or both MDA-5 and TRIF. Surprisingly, administration of poly I: C rapidly induced IFN-α, IL-6 and IL-12 in both sera of wild-type and RIG-I ^{− / −} mice, and RIG-I was in vivo It was shown not to be essential for the response via poly I: C (see FIG. 2a). In sharp contrast, MDA5 ^{− / −} mice did not produce IFN-α in response to poly I: C, and IL-6 and IL-12p40 production was also significantly reduced. TRIF ^{− / −} mice produced normal amounts of IFN-α, but IL-12p40 production was significantly reduced and IL-6 production was also significantly reduced. Furthermore, MDA5 ^{− / −} TRIF ^{− / −} mice did not induce IFN-α, IL-6 and IL-12p40 in response to poly I: C (see FIG. 2b). These results indicate that MDA5 is essential for poly I: C-induced IFN-α production and TLR3 signaling is important for IL-12 production, while both MDA5 and TLR3 produce IL-6. Indicates to adjust. In addition to cytokine induction, poly I: C treatment of wild-type mice also increased the surface expression of costimulatory molecules such as CD86 in conventional spleen DC (see FIG. 2c). Deficiency of either MDA5 or TRIF did not affect CD86 expression, but double deficiency resulted in inhibition of CD86 upregulation. This indicates that both MDA5 signaling and TLR3 signaling are involved in poly I: C responsive cDC activation in vivo. When bone marrow derived DCs (GMCSF-DC) made with GM-CSF were stimulated with poly I: C, either wild type or RIG-I ^{− / −} mice cells responded to poly I: C. Produces IFN-α, IL-6 and IL-12p40 in a dose dependent manner. In contrast, MDA5 ^{− / −} GMCSF-DC had severely reduced production of these cytokines (see FIG. 2d). Therefore, it was confirmed that in the cytoplasm, MDA5, not RIG-I, mainly recognizes poly I: C.

However, since inosinic acid is not used in the virus replication process in the natural state, the present inventors synthesized long-chain dsRNA by in vitro transcription, and embryonic fibroblasts (MEF) of mice lacking MDA5 or RIG-I. ) IFN response was examined. MEF was stimulated with two dsRNAs of different length (200 bp and 400 bp) corresponding to the sequence of mouse Lamin A / C forming a complex with the lipofection reagent. Unlike poly I: C, wild type and MDA5 ^{− / −} MEF produced equivalent amounts of IFN-β (see FIG. 2e). RIG-I ^{− / −} MEF did not produce any detectable amount of IFN-β. This indicates that RIG-I is essential for the detection of in vitro transcribed dsRNA. In addition, in vitro transcribed dsRNA corresponding to several other sequences induced type I IFN via RIG-I rather than MDA5. Overall, this provides evidence that MDA5 and RIG-I are differentially involved in the detection of poly I: C and in vitro transcribed dsRNA, respectively.

From these findings, we hypothesized that RIG-I and MDA5 are involved in the detection of different RNA viruses. RNA viruses are classified into minus-strand ssRNA viruses, plus-strand ssRNA viruses, and dsRNA viruses based on genomic structure. We have previously shown that a series of negative strand RNA viruses are recognized by RIG-I. We first examined the production of IFN-α in MDA5 ^{− / −} MEFs in response to a series of negative strand ssRNA viruses including NDV, Sev, VSV, and influenza virus. Since infection with most wild-type viruses other than NDV does not induce IFN-α in MEF because the IFN response is suppressed by viral proteins (data not shown), we have inhibited virus. A mutant virus lacking the protein was also used. Such viruses include (V-) deficient (V-) or (Cm) SeV having a mutated C protein, VSV (NCP) deficient in a mutant of M protein, and influenza virus deficient in NS1 protein (ΔNS1) Is included. As shown in FIG. 3a, IFN-α production is severely impaired in RIG-I ^{− / −} MEF compared to wild-type cells, whereas MDA5 is responsible for producing IFN-α in response to these viruses. It was not essential. Paramyxovirus V proteins such as Sev have been shown to cooperate with MDA5 to suppress dsRNA-induced IFN production. Wild-type and SeVV (-) are both control and ^{MDA5 -} / - to induce the expression of IP-10 gene and type I IFN in MEF (FIG. 2a and FIG. 6). Moreover, RIG-I ^{− / −} MEF did not respond to either wild type or SeVV (−). This indicates that MDA5 does not contribute to SeV recognition regardless of the presence of virus inhibitory proteins. Japanese encephalitis virus (JEV), which is a plus strand ssRNA virus belonging to flavivirus, also required RIG-I for production of IFN-β, but did not require MDA5 (FIG. 3b).

Next, the IFN response of MEF to encephalomyocarditis virus (EMCV), which is a positive strand ssRNA virus belonging to the picornavirus family, was examined. Surprisingly, EMCV-induced IFN-β production was inhibited in MDA5 ^{− / −} MEF (FIG. 3c). In contrast, wild-type and RIG-I ^{− / −} MEF produced equivalent amounts of IFN-β, indicating that EMCV was specifically recognized by MDA5 (FIG. 3c). . In addition, the production of IFN-α and IL-6 in response to EMCV is inhibited by MDA5 ^{− / −} GMCSF-DC, which indicates that MDA5, not RIG-I, is an EMCV in MEF and conventional DC. It is essential for the recognition of (Figs. 3d and 3e). Furthermore, other viruses belonging to the picornavirus family, Tyler virus and Mengo virus, also induced IFN-α via MDA5 (FIG. 7).

Next, RIG-I ^{− / −} MDA5 ^{− / −} double-deficient MEFs that are completely inhibited from induction of IFN-β in response to exposure to SeVCm or EMCV are transfected with RIG-I or MDA5 expression vectors. Thus, a reconstruction experiment was performed (FIG. 3f). Local expression of human RIG-I but not MDA5 resulted in SeVCm-responsive IFN-β promoter activation. Conversely, cells expressing human MDA5 but not RIG-I activated the IFN-β promoter in response to EMCV in a dose-dependent manner (FIG. 3f). These results indicate that human RIG-I and MDA5 recognize different RNA viruses.

Previous studies have shown that pDC primarily uses the TLR system instead of RIG-I in the recognition of several RNA viruses. MyD88 is an adapter protein involved in signaling of various TLRs excluding TLR3. pDC highly expresses TLR7 and TLR9 and generates type I IFN in a MyD88-dependent manner upon exposure to the virus. To address whether MDA5 is involved in virus detection in pDC, we exposed B220 ⁺ pDC purified from Flt3L-constructed BM-derived (Flt3L-DC) DC to EMCV to produce IFN-α. It was measured. PDCs derived from MyD88 ^{− / −} mice but not from MDA5 ^{− / −} mice showed severe defects in the production of IFN-α. This indicates that neither RIG-I nor MDA5 plays an important role in pDC (FIG. 8). Conversely, B220 purified from Flt3L-DC ^- The production of IFN-alpha in the conventional DC of, it is necessary MDA5 instead MyD88 (FIG. 8). These results indicate that neither MDA5 nor RIG-I is essential for viral induction of IFN-α in pDC.

We also investigated the in vivo role of MDA5 or RIG-I in host defense against viral infection. Most of RIG-I ^{− / −} mice die during the embryonic stage, but by mating with ICR mice, the present inventors were able to efficiently obtain living adult mice. Adult RIG-I ^{− / −} mice did not show any macroscopic developmental abnormalities and survived after 20 weeks of age. When mice were infected with JEV, serum IFN-α levels were significantly reduced in RIG-I ^{− / −} mice compared to littermate control mice. In contrast, MDA5 ^{− / −} mice did not show a major defect for JEV-induced systemic IFN-α production (FIG. 4a). It was RIG-I ^{− / −} mice, but not MDA5 ^{− / −} mice, that were more susceptible to JEV compared to control mice, which is consistent with the above findings (FIG. 4b). Also, it was RIG-I ^{− / −} mice but not MDA5 ^{− / −} mice that died from VSV infection, which is consistent with inhibition of the IFN response (FIG. 4c). Thus, recognition of a series of viruses through RIG-I plays an important role in antiviral host defense in vivo.

Furthermore, mice infected with EMCV as a virus model recognized by MDA5 were verified. In the sera of MDA5 ^{− / −} mice, the induction of IFN-α, RANTES and IL-6 was severely reduced. This indicates that MDA5 is essential for the induction of an in vivo humoral response upon EMCV infection (FIG. 5a). Therefore, the susceptibility of MDA5 ^{− / −} mice to EMCV infection was further investigated and compared with that of IFN-α / βR ^{− / −} , MDA5 ^{− / −} , RIG ^{− / −} , and TLR3 ^{− / −} mice. As shown in FIG. 5b, MDA5 ^{- / -} and IFN-α ^/ βR ^- / ^- mice, as compared to littermate mice as a control, were more sensitive to EMCV infection. In contrast, neither RIG-I or TLR3 deficiency affected the survival of EMCV-infected mice. EMCV is known to selectively infect cardiomyocytes and cause myocarditis. Compared to control mice, MDA5 ^{− / −} mice had significantly higher viral titers in the heart, consistent with increased sensitivity to EMCV (FIG. 5c). Histological analysis of the heart 2 days after EMCV infection revealed that focal necrosis of cardiomyocytes had developed in MDA5 ^{− / −} mice, but the heart of wild type mice was histologically No abnormalities were shown (FIG. 5d). Of note, at this point no immune cell infiltration was observed in either wild type or MDA5 ^{− / −} heart sections. To assess changes in cardiac function in response to EMCV infection, cardiac work was analyzed by echocardiography 2 days after infection (FIG. 5e). Cardiac contractility as judged by shortening rate (FS) was severely reduced in MDA5 ^{− / −} mice (wild type 48.2 ± 4.9% vs. MDA5 ^{− / −} 21.2 ± 5.8%). This suggests that MDA5 ^{− / −} mice had severe heart failure due to virus-induced cardiomyopathy (FIG. 5f). Thus, recognition of EMCV via MDA5 is a prerequisite not only for preventing myocardial dysfunction but also for causing an antiviral response. Taken together, these results clearly show that RIG-I and MDA5 play an essential role in the recognition of different groups of RNA viruses and the subsequent generation of type I IFN and inflammatory cytokines. ing.

FIG. 1a is a diagram showing the structures of a mouse MDA5 gene, a targeting vector, and a predicted disrupted gene. ■ indicates coding exon. B: BamHI is shown. FIG. 1b is a diagram showing the results of Southern blot analysis of offspring by heterozygous intercrossing. Genomic DNA was extracted from the mouse tail, digested with BamHI, separated by electrophoresis, and hybridized with the radiolabeled probe shown in a. By Southern blot, wild type (+ / +) mice had a single band of 9.4 kb, homozygous (− / −) mice had a 4.6 kb band, both heterozygous (+/−) mice had Band was obtained. FIG. 1c is a diagram showing the results of Northern blot analysis of peritoneal exudate cells (PEC). Wild type (WT) and MDA5 ^{− / −} PEC total RNA treated with 1000 U / ml IFN-β for 8 hours was extracted, and Northern blot analysis was performed for expression of MDA5 mRNA. The same membrane was rehybridized with β-actin probe. FIG. 1d shows the results of Western blot analysis of MDA5 expression. WT and MDA5 ^{− / −} MEFs were treated with 1000 U / ml IFN-β for 8 hours and whole cell lysates were immunoblotted with MDA5 antibody. 2a and 2b show that 200 μg of poly I: C was injected into the vein of the indicated mouse for the indicated time, and the production of IFN-α, IL-6 and IL-12p40 in the serum was performed by ELISA. It is a figure which shows the measurement result. Data are expressed as mean ± SEM of serum samples. D. It is. FIG. 2c is a diagram showing the results of analyzing the surface expression of CD86 by flow cytometry on spleen CD11c ⁺ B220 ^- cDC of mice injected with PBS or poly I: C. FIG. 2d shows the results of stimulation of WT, RIG-I ^{− / −} , and MDA5 ^{− / −} GMCSF-DC with poly I: C with increasing concentrations for 24 hours. Production of IFN-α, IL-6 and IL-12p40 in the culture supernatant was measured by ELISA. FIG. 2e shows the results of treatment of WT, RIG-I ^{− / −} , and MDA5 ^{− / −} MEF with the indicated length of in vitro transcribed dsRNA complexed with Lipofectamine 2000 for 24 hours. It is. The production of IFN-β in the culture supernatant was measured by ELISA. The error bars for d and e are 3 times ± S. D. Indicates. FIG. 3a is a diagram showing the results of infecting WT, RIG-I ^{− / −} , and MDA5 ^{− / −} MEF with the displayed minus-strand ssRNA virus. The production of IFN-α in the culture supernatant was measured by ELISA. B and c in FIG. ^{3, WT, RIG-I - /} -, and ^{MDA5 -} / - a MEF, the indicated plus strand ssRNA viruses, a diagram showing the results infected with JEV (b) and EMCV (c) is there. The production of IFN-β in the culture supernatant was measured by ELISA. FIGS. 3 d and e are diagrams showing the results of infecting WT, RIG-I ^{− / −} , and MDA5 ^{− / −} GMCSF-DC with EMCV with gradually increasing moi. The production of IFN-α (d) and IL-6 (e) in the culture supernatant was measured by ELISA. FIG. 3 f and g are transiently transfected with WT and RIG-I ^{− / −} MDA5 ^{− / −} MEF with a reporter construct containing the IFN-β promoter and with or without the indicated expression vector; Thereafter, the cells are infected with EMCV or SeVCm and are subjected to luciferase assay. Error bars for a, b, c, d, and e are ± S. D. Indicates. FIG. 4a shows 2 × 10 ⁷ pfu of JEV injected intravenously into RIG-I ^+/− , RIG-I ^{− / −} , MDA5 ^+/− and MDA5 ^{− / −} mice for 24 hours. Thereafter, serum is collected from the mouse, and the production level of IFN-α is measured by ELISA. Bars show average values. FIG. 4b shows the results of monitoring the survival of display mice (6 weeks old) infected with 2 × 10 ⁷ pfu of JEV via veins over 15 days. FIG. 4c shows the results of monitoring the survival of mice (3 weeks old) infected intranasally with 4 × 10 ⁶ pfu of VSV over 9 days. FIG. 5a shows that 10 ⁷ pfu of EMCV was intravenously inoculated into MDA5 ^+/− and MDA5 ^{− / −} mice (n = 5), and sera were collected 4 hours after injection, and IFN-α, Rantes and IL It is a figure which shows the result of having confirmed the production | generation level of -6 by ELISA. FIG. 5b shows the results of monitoring the survival of display mice (6 weeks of age) intraperitoneally infected with 10 ² pfu of EMCV every 6 hours for 6 days. FIG. 5c shows that MDA5 ^+/− and MDA5 ^{− / −} mice were infected intraperitoneally with 10 ² pfu of EMCV. After 48 hours, the mice were discarded and the virus titer in the heart was confirmed by plaque assay. It is a figure which shows the result. FIG. 5d shows the results of evaluating histological changes by staining with hematoxylin and eosin for the hearts of MDA5 ^+/− and MDA5 ^{− / −} mice two days after infection. Arrows indicate focal necrosis of cardiomyocytes. E and f of Drawing 5 are figures showing the result of having evaluated the cardiac function 48 hours after EMCV infection by echocardiography. FIG. 5e is a translucent view of transthoracic echocardiography in M mode of MDA5 ^+/− and MDA5 ^{− / −} mice 48 hours after EMCV infection. FIG. 5f is a diagram showing the shortening rate (FS) after infection confirmed by echocardiography. FIG. 6 shows the results of Northern blot analysis of MEFs. WT (wild type), RIG-I ^{− / −} , and MDA5 ^{− / −} MEFs were infected with SeV V (−) with moi = 10, and the extracted total RNA was subjected to Northern blot analysis, and IFN-β and IP10 The expression of mRNA was examined. Bands of 28S and 18S ribosomal RNA on gels stained with ethidium bromide were used as RNA loading controls. FIG. 7 shows that WT (wild type) and MDA5 ^{− / −} GMCSF-DCs were infected with moi-increased Tyler virus or Mengovirus for 24 hours and IFN-α in the culture supernatant was It is a figure which shows the result of having measured the production amount by ELISA. FIG. 8 shows that B220 ⁺ and B220 ⁻ CD11c ⁺ cells isolated from MyD88 ^{− / −} , MDA5 ^{− / −} , and MDA5 ^{− / −} Flt3L-DCs by MACS were infected with EMCV with moi = 1 for 24 hours. It is a figure which shows the result of having measured the production amount of IFN- (alpha) in the supernatant of ELISA by ELISA.

Claims (14)

Non-human in which all or part of the endogenous gene of non-human animal encoding MDA5 (Melanoma differentiation associated gene-5) is inactivated by gene mutation such as disruption, deletion or substitution, and the function of expressing MDA5 is lost A type I interferon non-responding non-human model animal that lacks a type I interferon response to infection with a virus belonging to the picornavirus family. The non-human model animal according to claim 1, wherein the non-human model animal is a model mouse. A non-human animal in which all or part of an endogenous gene encoding MDA5 (Melanoma differentiation associated gene-5) is inactivated by gene mutation such as disruption, deletion or substitution, and the function of expressing MDA5 is lost is pico A method for use as a type I interferon-unresponsive non-human animal animal that lacks the type I interferon response to infection with a virus belonging to the Luna virus family. The method according to claim 3, wherein the non-human model animal is a model mouse. Non-human in which all or part of the endogenous gene of non-human animal encoding MDA5 (Melanoma differentiation associated gene-5) is inactivated by gene mutation such as disruption, deletion or substitution, and the function of expressing MDA5 is lost An animal is infected with a virus belonging to the picornavirus family, a test substance is administered before, after, or simultaneously with the virus infection, and the amount of type I interferon, RANTES or IL-6 produced in the non-human animal is measured. A method for identifying an anti-picornavirus agent, comprising evaluating the test substance as an anti-picornavirus agent when production of interferon, RANTES, or IL-6 is observed. 6. The identification method according to claim 5, wherein the method is compared and evaluated with the production amount of type I interferon, RANTES or IL-6 in a wild-type animal of the same species as the non-human animal. The identification method according to claim 5 or 6, wherein the non-human animal is a mouse. Non-human in which all or part of the endogenous gene of non-human animal encoding MDA5 (Melanoma differentiation associated gene-5) is inactivated by gene mutation such as disruption, deletion or substitution, and the function of expressing MDA5 is lost The animal is infected with a myocarditis virus (EMCV) belonging to the picornavirus family, and a test substance is administered before, after, or simultaneously with the EMCV infection, and the histological analysis of the heart of the non-human animal is performed to determine the lesion of the cardiomyocytes. A method for identifying an anti-EMCV agent, wherein when the necrosis is suppressed, the test substance is evaluated as an anti-EMCV agent. 9. The identification method according to claim 8, wherein the method is compared and evaluated with the degree of suppression of focal necrosis of cardiomyocytes in a wild-type animal of the same species as the non-human animal. The identification method according to claim 8 or 9, wherein the non-human animal is a mouse. Non-human in which all or part of the endogenous gene of non-human animal encoding MDA5 (Melanoma differentiation associated gene-5) is inactivated by gene mutation such as disruption, deletion or substitution, and the function of expressing MDA5 is lost A cell derived from an animal is infected with a virus belonging to the picornavirus family, and a test substance is contacted with the cell before, after or simultaneously with the virus infection, and the interferon type I, RANTES or IL-6 in the cell derived from the non-human animal is contacted. A method for identifying an anti-picornavirus agent, wherein the test substance is evaluated as an anti-picornavirus agent when the amount of production of IFN is measured and production of type I interferon, RANTES or IL-6 is observed . The identification method according to claim 11, wherein the method is compared and evaluated with the production amount of type I interferon, RANTES, or IL-6 in a cell derived from a wild-type animal of the same species as the non-human animal. The identification method according to claim 11 or 12, wherein the non-human animal is a mouse. The identification method according to any one of claims 11 to 13, wherein the cells are embryonic fibroblasts or dendritic cells.