CN116004565B - Application of avian influenza virus polymerase protein PB2 in preparation of JAK-STAT signal transduction inhibitor - Google Patents
Application of avian influenza virus polymerase protein PB2 in preparation of JAK-STAT signal transduction inhibitor Download PDFInfo
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Abstract
The invention belongs to the field of virus immunology, and relates to application of Avian Influenza Virus (AIV) polymerase protein PB2 in preparation of JAK-STAT signal transduction inhibitors. The present invention discovers that PB2 protein blocks IFNAR-JAK STAT signaling. The PB2 protein inhibits the function of the mammalian JAK1 protein in a dose-dependent manner, i.e., by interacting with the kinase-like and kinase domains of JAK1 and degrading JAK1 in a ubiquitin-proteasome manner, thereby inhibiting JAK 1-mediated antiviral gene expression. AIV carrying the PB2 gene that does not degrade JAK1 induces a stronger level of ISGs, reducing pathogenicity in mice. The invention provides new targets and theoretical support for prevention and control of transmission among avian influenza virus species and vaccine design.
Description
Technical Field
The invention belongs to the field of virus immunology, and particularly relates to application of avian influenza virus polymerase protein PB2 in preparation of JAK-STAT signal transduction inhibitors.
Background
Interferon (IFN) is the host's innate immunity line, protects against microbial infections such as viruses, and plays an important regulatory role in the initiation of host adaptive immunity. Interferon-based therapies have been successfully applied to infectious diseases such as Hepatitis B Virus (HBV). IFN is classified into type I, type II and type III according to the difference in IFN receptor. Type I and type III IFNs are regulated and expressed after the host cell pattern recognition receptor recognizes viral components, and type II IFNs are mainly produced by activated T cells and NK cells; the I-type interferon plays a main role in host antiviral reaction, the IFN antiviral effect is mainly mediated by ISGs, and the expression of the ISGs is mainly regulated and controlled by a classical JAK/STAT signal path downstream of an IFN receptor. This signaling pathway is essential in triggering antiviral immunity, including pro-inflammatory, antiproliferative, and T cell differentiation, among others. Type I and III IFNs activate JAK1 and TYK2 downstream of interferon receptor, and further phosphorylate STAT1 and STAT2, wherein phosphorylated STAT1 and STAT2 form heterotrimer ISGF3 with IRF9, ISGF3 binds to transcription element ISRE, and expression of ISGs such as Mx1, IFIT1, ISG15 is started. Type II IFNs activate receptors downstream JAK1 and JAK2 primarily through phosphorylation, thereby phosphorylating STAT1, and phosphorylated STAT1 forms homodimers, binding to the interferon gamma activating element (GAS), thereby up-regulating a large number of antiviral gene expression. To combat host defenses, viruses can inhibit the expression of ISGs by targeting the JAK/STAT signaling pathway downstream of IFN, promoting viral escape host antiviral response and enhancing host adaptation of the virus. For example, SARS-CoV-2 targets JAK1, tyk2 and IFNAR1, inhibiting IFN downstream signaling pathway transduction. ZiKV virus (ZIKV) takes JAK1 as a target to carry out proteasome degradation, inhibits a JAK/STAT signal pathway, and damages interferon-mediated antiviral response. Foot-and-mouth disease virus (FMDV) VP3 protein can promote JAK1 degradation, and further inhibit expression of ISGs downstream of IFN. However, the regulatory mechanism of the IFNAR-JAK1-STAT by the avian influenza virus (Avian influenza virus, AIV) PB2 protein is not yet known.
AIV is divided into 16 HA subtypes (H1-H16) and 9 NA subtypes (N1-N9) based on the difference in antigenicity of Hemagglutinin (HA) and Neuraminidase (NA), and viruses of each subtype can be isolated from wild birds. The high pathogenic avian influenza virus (highly pathogenic avian influenza viruses, HPAIV) and the low pathogenic avian influenza virus (low pathogenic avian influenza viruses, LPAIV) are classified according to their pathogenicity. Currently common HPAIVs include H5 and H7 subtypes. HPAIV is an important zoonotic disease, which seriously jeopardizes the development of the poultry industry and seriously threatens human health. Since the first report in 1997 that avian influenza virus H5N1 infects humans and is fatal, many events of avian influenza virus cross-host infection of humans have occurred so far. In order to control and eliminate influenza outbreaks and to better understand the interactions of viruses with the natural immune system, the role of viral proteins in natural immunity has yet to be elucidated further. The AIV genome encodes more than 18 viral proteins. The nonstructural protein (NS 1) is a multifunctional protein that inhibits the production of IFNs. NS1 may reduce RIG-I activation by interacting with RIG-I, by cleaving RNA helicase and its activating ligand, or by inhibiting TRIM 25-mediated ubiquitination of RIG-I. The PB2 protein inhibits its mediated IFN production by binding MAVS, thereby increasing viral replication and pathogenicity, which is only upstream of IFN production. It is not clear whether the AIV PB2 protein has an effect on the IFNAR-JAK-STAT signaling pathway and the mechanism of action thereof. The research of a new mechanism of a new function of the protein is important for effectively preventing and treating influenza.
Disclosure of Invention
The present invention found that AIV PB2 protein inhibited IFN-induced ISGs production and demonstrated that AIV PB2 protein blocked IFNAR-JAK-STAT signaling. Meanwhile, a novel function of PB2 protein is discovered, and the function of JAK1 protein is inhibited in a dose-dependent manner, namely AIV PB2 protein inhibits JAK1 mediated antiviral gene expression by interacting with kinase-like and kinase domains of JAK1 and degrading JAK1 in a ubiquitin-proteasome manner.
The invention utilizes AIV reverse genetic operating system to construct recombinant PB2 gene mutant strain, and discovers that the expression of JAK1 protein and the level of downstream stimulus gene ISGs in the mutant strain are related to virus pathogenicity, namely the pathogenicity of a recombinant virus infected animal containing PB2 incapable of degrading JAK1 is obviously reduced, and the interferon stimulus gene is obviously up-regulated after the inoculated animal is infected for 2 d.
The invention adopts the following technical scheme:
application of avian influenza polymerase PB2 in preparation of JAK-STAT signal transduction inhibitor, wherein the nucleotide sequence of avian influenza polymerase PB2 is shown in SEQ ID NO: 1.
Further, the avian influenza virus is subtype H5, H7, H9 and the like.
Further, the amino acid sequence of the expressed avian influenza polymerase protein PB2 is shown in SEQ ID NO: 2.
Further, PB2 protein inhibits JAK-STAT signaling by degrading the JAK1 protein.
Further, the PB2 protein degrades JAK1 in a ubiquitin-proteasome dependent manner by interacting with JAK1.
Further, the PB2 protein interacts with the kinase and kinase-like domains of JAK1.
An avian influenza polymerase protein PB2 mutant, said mutant constructed using the avian influenza polymerase protein PB2 of claim 1.
Further, the nucleotide sequence of the PB2 mutant is shown in SEQ ID NO: 3.
Further, the amino acid sequence of the PB2 mutant is shown in SEQ ID NO: 4.
The application of the recombinant avian influenza virus strain of the encoding avian influenza polymerase protein PB2 mutant gene in preparing influenza virus vaccines.
In a first aspect, the present invention utilizes reverse genetics techniques to construct recombinant H5 subtype avian influenza virus mutant rCZM containing PB2 which is incapable of degrading JAK1.
The recombinant H5 subtype avian influenza virus mutant rCZM is constructed as follows:
(1) Plasmid PHW2000-PB2 of rCZ virus rescue system was used as template (SEQ ID NO: 1), according to SEQ ID NO:3, the PB2 gene site-directed mutagenesis is carried out by using primers PB2M1-F (SEQ ID NO: 5) and PB2M1-R (SEQ ID NO: 6), and the correct sequence is named PHW2000-PB2M1 after sequencing identification.
(2) The PHW2000-PB2M1 is used as a template, the PB2M2-F (SEQ ID NO: 7) and PB2M2-R (SEQ ID NO: 8) are used for carrying out PB2M1 gene site-directed mutagenesis, and the correct one is named PHW2000-PB2M2 after sequencing identification.
(3) HEK293T and MDCK cells are mixed in equal amounts and spread into a 6-hole cell culture plate, and transfection is carried out when the cell coverage area reaches 80%. The transfection system contains 8 fragments of transcription/expression plasmid (PB 2 plasmid is PHW2000-PB2M 2).
(4) 48h after transfection, repeated freezing and thawing for 3 times, collecting transfection supernatant, and inoculating 10-day-old SPF chick embryo. Titers were determined using a Hemagglutination Assay (HA) to verify that the virus was successfully rescued.
(5) The positive chick embryo allantoic fluid extracts the total RNA of the virus, 8 fragments are amplified by PCR and sequenced, and the right identified chick embryo allantoic fluid is named rCZM.
The present invention finds that PB2 mutant rCZM has reduced degradation capacity and replication level for JAK1 at the cellular and tissue level compared to AIV rCZ.
The invention discovers that in animal bodies, compared with recombinant viruses with strong JAK1 degradation capability, recombinant strains with weak JAK1 degradation capability have lower pathogenicity when infected animals, and discovers that the interferon stimulation genes of vaccinated animals are obviously up-regulated after 2d infection. This suggests that after mutation of the PB2 gene, the PB2 protein loses the ability to disrupt the expression pathway of genes downstream of the interferon, which in turn, is expressed in large amounts, resulting in reduced viral replication in animals and reduced virulence of the virus to the infected animal.
In a second aspect, the PB2 protein of AIV degrades JAK1 by interacting with a specific region of JAK1 and in a proteasome-dependent manner, specifically identified as follows:
(1) The PB2 gene recombinant vector is respectively transfected with the JAK1 recombinant vector, or a plurality of inhibitors are added for treatment after transfection, and the action paths of the inhibitors are different.
(2) Judging the action mode of the PB2 protein for degrading intracellular components of the intracellular JAK1 pathway according to the action path of the inhibitor: PB2 degrades JAK1 via the proteasome pathway.
(3) The ubiquitination modification of PB2 to promote K48 ligation of JAK1 is demonstrated by ubiquitination experiments.
(4) The co-IP and confocal experiments demonstrate that PB2 and JAK1 interact and demonstrate the specific domains of their interaction.
(5) By comparing the differences between strains and mutating specific sites of PB2 protein, it was found that PB2 (SEQ ID NO: 4) carrying the JY strain pattern could not ubiquitously degrade JAK1.
The invention also discovers that the degradation function of AIV PB2 protein also exists in other different subtypes of AIV such as H7, H9 and the like.
Advantageous effects
1. The invention further reveals a novel function of avian influenza virus PB2, and clarifies that AIV causes morbidity in animal organisms due to PB2 escaping IFNAR-JAK-STAT signaling.
2. The invention also provides and opens up a new view and approach for the research and development of the vaccine of the avian influenza virus focusing on natural immune response.
3. The invention provides new target and theoretical support for the prevention and control of avian influenza virus and vaccine design.
Drawings
FIG. 1 contains replication of recombinant H5 subtype avian influenza virus mutant rCZM in mammalian cells that is incapable of degrading PB2 of JAK1. (A) immunoblots of A549 cells infected with rCZ, rCZM or rJY. (B) Viral growth curves of rCZ, rCZM, or rJY (moi=0.01) infected a549 cells. * P <0.01.
FIG. 2 virulence assay of recombinant H5 subtype avian influenza virus mutant rCZM containing PB2 which is incapable of degrading JAK1 in mice. (a and B) BALB/c mice (n=10) were intranasally inoculated with rJY, rCZ or rCZM virus (10 6 EID50 /) and monitoring weight change (a) and survival (B). * Representing a significant difference between rCZ and rJY or the rCZM and rCZ groups. (C) mouse lungs at2 and 5dpi (n=5). The yellow frame marks severe pneumonia with diffuse solid changes. (D) 2 and 5dpi pulmonary virus titers were determined (n=5). (E-H) hematoxylin/eosin (HE) staining (E) and scoring (F), immunohistochemical (IHC) staining (G) and scoring (H) of lung sections. (n=5); scale bar: 200 μm. (I) immunoblotting of lung tissue of infected mice (n=3). (J and K) qPCR analysis ISG15 (J) and IFIT1 (K) mRNA levels (n=3) in lung tissue of infected mice. * P (P)<0.05，**P<0.01。
FIG. 3 AIV PB2 protein specifically degrades the JAK1 protein via the proteasome pathway. (A) immunoblotting of PB2 plasmid transfected HEK293T cells. (B) Density analysis results of Panel A. (C) After transfection of the PB2 plasmid into A549 cells, qPCR analysis of JAK1mRNA levels. (D) immunoblot analysis of AIV-infected A549 cells. (E) Density analysis results of Panel D. (F) Immunoblotting of HEK293T cells transfected with PB2 plasmid and treated with DMSO, MG132, NH4Cl, or Chloroquine (CQ). (G) Density analysis results of Panel F. The intensity of immunoblots from three independent experiments was quantified and normalized with Actin. ns P>0.05，*P<0.05，**P<0.01。
FIG. 4 AIV PB2 protein promotes K48 ubiquitination modification of JAK1. (A) Ni-NTA pull-Down analysis JAK1 ubiquitination in HEK293T cells transfected with JAK1-His, HA-ubiquitin (HA-Ub) and PB2 plasmids and treated with MG 132. (B) Ni-NTA pull-Down analysis of JAK1-His, HA-Ub and mutants thereof and PB2 plasmid transfected and MG132 treated HEK293T cells were subjected to JAK1 ubiquitination. (C) Co-immunoprecipitation (Co-IP) analysis JAK1 ubiquitination in AIV (moi=0.01) infected a549 cells.
FIG. 5 identification of the domain of the AIV PB2 protein interacting with JAK1. (A) Co-ip and Ni-NTA pull-Down analysis of PB2 and JAK1 interactions with JAK1 following transfection of HEK293T cells with plasmids PB2 and JAK1. (B) Co-ip and Ni-NTA pull-Down analysis PB2 interacted with JAK1 in AIV-infected HEK293T cells. (C) JAK1 (green) and AIV PB2 (red) co-localize in AIV infected a549 cells. Nuclei were stained with DAPI (blue). Scale bar: 10 μm. Fluorescence intensity at the designated location was scanned with LAS X software. Schematic of deletion mutants of JAK1. (E) The interaction of PB2 with JAK1 and its truncated mutants in HEK293T cells was analyzed by Ni-NTA pull-down.
FIG. 6 identification of PB2 and JAK1 interactions of JY strain patterns. (A) Immunoblots of HEK293T cells transfected with JAK1 and PB2-CZ, PB2M-CZ or PB2-JY plasmids (left). The intensity of immunoblots from three independent experiments was quantified and normalized with Actin (right). (B) Ni-NTA pull-down assay PB2 and its mutant plasmid transfected HEK293T cells were ubiquitinated with JAK1. (C) Co-IP analysis of PB2 and its mutants interacted with JAK1 in HEK293T cells. (D) JAK1 (green) and PB2 (red) co-localize in rJY or rCZM infected a549 cells. Nuclei were stained with DAPI (blue). Scale bar: 10 μm. Fluorescence intensity at the designated location was scanned with LAS X software. ns P>0.05，**P<0.01。
FIG. 7 shows that degradation of PB2 of JAK1 inhibits its mediated production of ISGs and inhibits IFNs-induced STAT1/STAT2 phosphorylation levels. (A, B) qPCR analysis of A549 cells in PB2 or its mutant plasmid transfection, and IFN beta (A) or IFN alpha (B) treatment after treatment, IFIT1 and ISG15mRNA level. (C, D) double luciferase activity detection of IFN beta (C) or IFN alpha (D) treatment, transfection of PB2 plasmid HEK293T cells in STAT1-luc or ISRE-luc activity. (E, F) transfection of PB2 or its mutant plasmid, with IFN beta (E) or IFN alpha (F) treatment of HEK293T cells immunoblotting, (upper). Densitometric analysis of phosphorylated STAT/total STAT ratio on immunoblots (below). The intensity of immunoblots bands from three independent experiments was quantified and normalized with total STAT. * P <0.05, P <0.01.
FIG. 8 PB2 protein from different subtypes of AIV promotes the degradation of JAK1. Immunoblots of HEK293T cells transfected with different subtypes of AIV PB2 plasmid (left). The intensity of immunoblots from three independent experiments was quantified and normalized with Actin (right). * P <0.01.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings and specific embodiments thereof, which are only for explaining the present invention and not limiting the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; materials, reagents and the like used, unless otherwise indicated, are all commercially available.
Virus strain
Two strains of HPAI H5N8 virus A/goose/easter China/CZ/2013 (CZ) and A/dock/easter China/JY/2014 (JY) were identified and stored by the laboratory. The classical eight-plasmid reverse genetics was used to rescue recombinant viruses, and 8 fragments of CZ and JY strains were cloned into the bidirectional reverse genetics plasmid pHW2000 to rescue recombinant viruses rCZ and rJY.
Example 1: construction of recombinant H5 subtype avian influenza virus containing PB2 which does not degrade JAK1
(1) The plasmid PHW2000-PB2 of rCZ virus rescue system is used as a template (SEQ ID NO: 1), primers are designed according to JY virus sequence (SEQ ID NO: 3), and the primers PB2M1-F (SEQ ID NO: 5) and PB2M1-R (SEQ ID NO: 6), PB2M2-F (SEQ ID NO: 7) and PB2M2-R (SEQ ID NO: 8) are used for carrying out PB2 gene site-directed mutagenesis in sequence, and the correct sequence is identified as PHW2000-PB2M2.
(2) HEK293T and MDCK cells are mixed in equal quantity and spread into a 6-hole cell culture plate, and when the cell coverage area reaches 80%, 8 fragments of transcription/expression plasmid cotransfection (PB 2 plasmid is PHW2000-PB2M 2) is carried out.
(3) 48h after transfection, repeated freezing and thawing for 3 times, collecting transfection supernatant, and inoculating 10-day-old SPF chick embryo. The HA positive chick embryo allantoic fluid extracts the total RNA of the virus, 8 fragments are amplified by PCR and sequenced, and the identified correct one is named rCZM.
Example 2: replication capacity assay of recombinant H5 subtype avian influenza virus containing PB2 which does not degrade JAK1 on A549 cells
A549 cells were infected with recombinant AIV. The expression levels of viral proteins PB2 and NP were significantly reduced in the rCZM virus-infected cells compared to rCZ-infected cells (fig. 1A). Meanwhile, the rCZM infection caused less JAK1 degradation than rCZ infection, and rJY infection of cells did not cause significant changes in JAK1 (fig. 1A). Further, the growth curves of the rCZ, rJY and rCZM viruses in a549 cells were compared. In a549 cells, the viral titer of rCZ was higher than that of rCZM and rJY at 12hpi, and the growth rate was also higher than that of rCZM and rJY, indicating that mutation to JY mode PB2 reduced the replication capacity of rCZ (fig. 1B). These data indicate that AIV PB2 is not conducive to viral replication in mammalian cells after degradation of JAK1.
Example 3: determination of virulence of recombinant H5 subtype avian influenza virus containing PB2 which does not degrade JAK1 in mice
Female BALB/c mice of 4-6 weeks of age were selected and infected with AIV by nasal drip (10) 6 EID 50/mouse), mice survival and weight changes were observed. Observation period 14d. As shown in fig. 2a,2b, the body weight of the rCZ infected mice was significantly reduced and mortality was significantly increased compared to the rCZM and rJY infected groups. Then, the lung tissue of the mice was taken 2d and 5d after infection, respectively, and virus titer, HE, IHC, western-blotting and RT-qPCR were detected. Mice vaccinated with rCZ virus showed severe pneumonia, manifested as solid changes, bleeding and edema, 5d after infection (fig. 2C). In contrast, at 2dpi, little or no actual changes occurred in the lungs of rJY and rCZM virus infected mice (fig. 2C). The intracavitary viral titers were lower in the rCZM group compared to the rCZ infected group (fig. 2D). Furthermore, histopathological evaluation of the lungs of infected mice showed that at 2dpi, rcz, caused severe bronchiolitis and significant inflammatory cell infiltration; at 5dpi, bronchopneumonia was progressed (fig. 2e, f). rCZM infected mice showed moderate pathogenic changes at 5dpi (FIGS. 2E, F). Viral protein NP stained more strongly in lung sections of rCZ-infected mice than corresponding rJY or rCZM-infected mice (fig. 2g, h).
Infected mice lung tissue was further examined to determine the relationship of viral infection to JAK1. JAK1 expression was lower in the pulmonary infection rCZ groups of mice than in the rJY or rCZM groups, respectively (fig. 2I). 2dpi, mice infected rJY or rCZM mice had higher levels of ISG15 and IFIT1mRNA than the rCZ infected groups (FIGS. 2J, K). Taken together, these results indicate that inhibition of AIV PB2 degradation of JAK1 significantly reduces virulence of AIV in mice.
EXAMPLE 4 construction of PB2 and JAK1 expression vectors
All plasmid constructions were performed according to standard molecular biology procedures. Full-length cDNA encoding JAK1 was cloned into pCDNA3.1-His. To obtain various truncated forms of JAK1 plasmid, DNA fragments were amplified from JAK 1-His. Several deletion forms of JAK1 plasmid were designed: JAK1 (301-1154 aa) -His, JAK1 (1-435 aa) -His, JAK1 (1-559 aa) -His, JAK1 (560-1154 aa) -His, JAK1 (1-850 aa) -His, and JAK1 (851-1154 aa) -His. The viral genes were amplified from the AIV genome by RT-PCR and cloned into 3xFlag-CMV-10 or pHW 2000. PB2-H9N2/TX was cloned from A/chicken/Taixing/10/2010 (H9N 2/TX), PB2-H7N9/GD was cloned from A/chicken/Guangdong/4/2017 (H7N 9/GD), PB2-H5N1/YZ was cloned from A/chicken/Yangzhou/11/2016 (YZ/H5N 1), PB2-H5N1/DT was cloned from A/chicken/Jiangsu/DT1/2016 (H5N 1/DT), PB2-CZ was cloned from CZ and PB2-JY was cloned from JY. PB2 site-directed mutants were constructed using standard molecular biology techniques. All constructed vectors were verified by sequencing.
Example 5: AIV PB2 protein degradation of mammalian JAK1
First, the A549 cells were transfected with PB2 expression plasmid, and after 36 hours, western-blotting or RT-qPCR detection was performed. As shown in fig. 3a,3b, AIV PB2 protein specifically inhibited JAK1 protein expression without affecting IFNAR1, STAT1 expression; RT-qPCR assay showed that AIV PB2 protein did not affect the transcript levels of JAK1 (FIG. 3C). Subsequently, AIV infected A549 cells were collected at 0,6, 12, and 24 hours after infection, respectively, and Western-blotting detection was performed. As shown in fig. 3d,3e, AIV infection can inhibit JAK1 expression. To further explore the pathway of action of PB2 to inhibit JAK1, HEK293T cells were seeded in 12-well plates, transfected with PB2 expression plasmid at a concentration gradient, and after 24h, cells were lysed after 12h treatment with DMSO, MG132, NH4Cl, or Chloroquine (CQ) for Western-blotting detection. As shown in fig. 3f,3g, only proteasome inhibitor MG132 could inhibit the degradation of JAK1 by PB2, indicating that PB2 degrades JAK through the proteasome pathway.
Example 6: AIV PB2 protein promotes ubiquitination of JAK1
First, HEK293T cells were inoculated into 6-well plates, transfected with JAK1-His, HA-Ub, and PB2 expression plasmid at a concentration gradient, and after 24 hours, cells were lysed after 12 hours of treatment with MG132, and a pull-down experiment was performed. As shown in fig. 4A, PB2 promotes ubiquitination of JAK1 protein in a dose-dependent manner. In addition, HEK293T cells were transfected with JAK1-His, HA-Ub-K48 or HA-Ub-K63, and PB2 expression plasmids, and 24 hours later, cells were lysed after 12 hours of treatment with MG132, and pull-down experiments were performed. As shown in fig. 4B, AIV PB2 promotes K48 ubiquitination of JAK1 protein. Subsequently, AIV infected a549 cells, and samples were collected after 0,6,9 and 12 hours, respectively, and the cells were lysed, and an immunoprecipitation experiment was performed. As shown in fig. 4C, AIV infection promotes K48 ubiquitination of JAK1 protein.
Example 7: interaction of AIV PB2 protein with JAK1 protein
HEK293T transfected JAK1 and PB2 expression plasmids, 36h after transfection, cells were collected and pull-down and co-IP experiments were performed. As shown in fig. 5A, there is an interaction between PB2 and JAK1 in the case of transfection.
HEK293T cells were then seeded into 6-well plates, transfected with JAK1-His, infected with IAV, and cells were collected 24h after infection and subjected to pull-down and co-IP experiments. As shown in fig. 5B, in the case of infection, PB2 and JAK1 interact. Next, a549 cells were inoculated into confocal dishes, infected with AIV, and after 12h infection, the cells were collected for confocal microscopy analysis. As shown in fig. 5C, PB2 and JAK1 present a distinct co-localization phenomenon. Constructing a deletion mutation vector of the JAK1 according to the functional domain of the JAK1, co-transfecting cells with PB2, collecting the cells after 36h, and performing a pull-down experiment. As shown in fig. 5d,5e, PB2 binds to the kinase-like and kinase domains of JAK1.
Example 8: PB2 of JY strain mode cannot degrade JAK1
Western-blotting was performed after 36h with different modes PB2 and JAK1 co-transferred cells. As shown in fig. 6a,6b, PB2 carrying the JY strain pattern failed to ubiquitously degrade JAK1. Further co-IP (FIG. 6C) and confocal experiments (FIG. 6D) analysis, PB2 and JAK1 carrying the JY strain pattern did not interact. These experiments indicate that PB2 from JY strain mode could not target down JAK1.
Example 9: AIV PB2 protein inhibits JAK1-STAT signaling by degrading JAK1
Whereas JAK1 protein levels affect cellular sensitivity to IFNs, the ability of the PB2 protein of H5AIV to degrade JAK1 is different, exploring the regulatory role of PB2 of different H5 AIVs in IFNs-mediated signaling pathways. First, PB2-CZ significantly inhibited IFIT1 and ISG15mRNA transcription triggered by IFNs by qPCR analysis (fig. 7a,7 b). Second, PB2-CZ expression significantly inhibited the relative activity of ISRE and STAT1 promoters after IFNs treatment, but PB2M-CZ and PB2-JY expression did not (FIGS. 7C, 7D).
Next, we studied activation of STAT1/STAT2 in IFN response at PB2 expression. The addition of exogenous IFNs may cause activation of STAT1, which may be evidenced by an increase in STAT1 phosphorylation levels. However, PB2-CZ inhibited IFNs induced pSTAT1/pSTAT2, whereas PB2M-CZ and PB2-JY expression did not inhibit it (FIGS. 7E, 7F). In summary, AIV PB2 protein inhibits JAK1-STAT signaling by degrading JAK1.
Example 10: PB2 proteins of different subtypes of AIV promote degradation of JAK1
HEK293T transfected JAK1 and different subtypes of AV PB2 (PB 2-H9N2/TX, PB2-H7N9/GD, PB2-H5N1/YZ, PB2-H5N1/DT and PB 2-CZ) expression plasmids, cells were harvested 36H after transfection and Western-blotting was performed. As shown in FIG. 8, PB2 proteins from different subtypes of AIV promoted degradation of JAK1 to varying degrees.
Finally, it should also be noted that the above list is merely a few specific embodiments of the present invention. Obviously, the invention is not limited to the above embodiments, but many variations are possible. All modifications directly derived or suggested to one skilled in the art from the present disclosure should be considered as being within the scope of the present invention.
SEQ ID NO：1
A/goose/Eastern China/CZ/2013PB2
ATGGAGAGAATAAAAGAACTAAGAGATTTGATGTCGCAGTCTCGCACTCGCGAGATACTGACAAAAACCACTGTGGACCATATGGCCATAATCAAGAAGTATACGTCAGGAAGACAGGAGAAGAATCCTGCTCTTAGGATGAAATGGATGATGGCAATGAAATACCCGATTACAGCAGACAAAAGGATAATGGAGATGATCCCTGAGAGAAATGAGCAAGGTCAGACTCTTTGGAGCAAAACGAATGATGCTGGATCAGACAGAGTGATGGTATCACCTCTTGCTGTGACGTGGTGGAACAGAAATGGACCAACGACAAGTACAGTCCATTATCCAAAGGTTTACAAAACCTACTTTGAGAAGGTCGAAAGATTAAAGCATGGAACCTTCGGTCCCGTTCACTTTCGAAATCAGGTCAAAATACGTCGCAGGGTTGACATAAACCCGGGTCACGCAGATCTTAGTGCTAAAGAAGCACAAGACGTCATAATGGAGGTCGTTTTCCCAAACGAAGTAGGAGCCAGGATATTGACATCAGAGTCACAGTTAACAATAACAAAAGAAAAGAAGGAAGAGCTCCAGGACTGTAAGATCGCTCCTTTGATGGTGGCATACATGTTGGAAAGGGAACTAGTTCGCAAAACCAGATTCCTACCAGTAGCTGGCGGGACAAGCAGCGTGTATATCGAGGTGTTGCACTTGACCCAAGGGACCTGCTGGGAACAAATGTACACACCGGGAGGGGAGGTGAGAAATGATGATGTTGATCAGAGTTTAATTATTGCTGCTAGAAATATTGTCAGGAGAGCAACAGTATCAGCAGACCCGTTGGCTTCGCTCTTGGAAATGTGCCATAGTACACAAATTGGCGGAATAAGGATGGTAGACATCCTTAGACAAAATCCAACAGAAGAGCAAGCTGTGGATATATGCAAAGCAGCAATGGGTCTAAGGATCAGTTCATCCTTCAGCTTTGGAGGTTTCACTTTCAAAAGGACAAGTGGGTCATCTGTCAAAAGAGAAGAAGAAGTGCTCACAGGCAATCTCCAGACATTGAAAATAAGAGTGCATGAAGGATATGAGGAATTCACAATGGTCGGGCGAAGAGCAACAGCCATTCTAAGGAAAGCAACCAGAAGGCTGATCCAATTGATAGTGAGTGGAAGAGACGAGCAGTCAATTGCCGAAGCAATCATAGTGGCAATGGTGTTCTCACAAGAGGATTGCATGATAAAAGCAGTGCGAGGTGATTTGAATTTTGTCAACAGAGCGAACCAGCGGCTAAATCCCATGCATCAACTTCTGAGGCATTTCCAGAAGGATGCAAAGGTGCTGTTTCAAAACTGGGGAGTTGAACCCATTGACAATGTCATGGGAATGATCGGGATATTACCTGACATGACCCCCAACACAGAGATGTCACTAAGGGGAGTGAGAGTCAGTAAAATGGGAGTGGATGAATATTCCAGTACTGAGAGAGTGGTCGTGAGCATTGATCGTTTCTTGAGGGTCCGAGACCAGAGGGGAAATGTGCTCTTGTCTCCTGAAGAGGTTAGTGAAACACAGGGAACAGAAAGGCTGACGATAACATATTCATCGTCCATGATGTGGGAAATCAATGGCCCGGAATCAGTGTTAGTCAACACATATCAATGGATCATTAGAAACTGGGAAACTGTGAAGATCCAGTGGTCCCAAGACCCTACAATGCTATACAATAAGATGGAATTTGAGCCCTTCCAATCCTTGGTGCCTAAGGCTGCCAGAGGCCAGTACAGCGGCTTTGTGAGGACGCTATTCCAGCAGATGCGTGATGTGCTGGGGACATTTGACACTGTCCAGATAATAAAGCTGCTACCATTTGCAGCAGCCCCACCAGAACAGAGTAGAATGCAGTTCTCTTCTCTAACTGTGAACGTAAGGGGTTCAGGAATGAGAATACTTGTGAGAGGCAATTCCCCTGTGTTCAATTATAACAAGGCAACCAAGAGGCTCACAGTCCTTGGAAAGGATGCAGGTGCATTGACAGAAGATCCAGATGAGGGAACAGCAGGAGTGGAATCTGCGGTATTAAGAGGGTTTCTAATTCTGGGCAAAGAAGACAAAAGATATGGACCAGCATTGAGCATCAACGAATTGAGCAATCTTGCGAAAGGGGAGAAGGCTAATGTGTTGATAGGGCAAGGAGACGTGGTGTTGGTAATGAAACGGAAACGGGACTCTAGCATACTTACTGACAGCCAGACAGCGACCAAAAGAATTCGGATGGCCATCAATTAG
SEQ ID NO：2
A/goose/Eastern China/CZ/2013 PB2
MERIKELRDLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPALRMKWMMAMKYPITADKRIMEMIPERNEQGQTLWSKTNDAGSDRVMVSPLAVTWWNRNGPTTSTVHYPKVYKTYFEKVERLKHGTFGPVHFRNQVKIRRRVDINPGHADLSAKEAQDVIMEVVFPNEVGARILTSESQLTITKEKKEELQDCKIAPLMVAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEVRNDDVDQSLIIAARNIVRRATVSADPLASLLEMCHSTQIGGIRMVDILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSVKREEEVLTGNLQTLKIRVHEGYEEFTMVGRRATAILRKATRRLIQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNFVNRANQRLNPMHQLLRHFQKDAKVLFQNWGVEPIDNVMGMIGILPDMTPNTEMSLRGVRVSKMGVDEYSSTERVVVSIDRFLRVRDQRGNVLLSPEEVSETQGTERLTITYSSSMMWEINGPESVLVNTYQWIIRNWETVKIQWSQDPTMLYNKMEFEPFQSLVPKAARGQYSGFVRTLFQQMRDVLGTFDTVQIIKLLPFAAAPPEQSRMQFSSLTVNVRGSGMRILVRGNSPVFNYNKATKRLTVLGKDAGALTEDPDEGTAGVESAVLRGFLILGKEDKRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAIN-
SEQ ID NO：3
A/duck/Eastern China/JY/2014(JY)PB2
ATGGAGAGAATAAAAGAATTAAGAGATTTGATGTCGCAGTCTCGCACTCGCGAGATACTGACAAAAACCACTGTGGACCATATGGCCATAATCAAGAAGTATACGTCAGGAAGACAGGAGAAGAATCCTGCTCTTAGGATGAAATGGATGATGGCAATGAAATACCCGATTACAGCAGACAAGAGGATAATGGAGATGATCCCTGAGAGAAATGAGCAAGGTCAGACTCTTTGGAGCAAAACGAATGATGCTGGATCAGACAGAGTGATGGTATCACCTCTTGCTGTGACGTGGTGGAACAGAAATGGACCAACGACAAGTACAGTCCATTATCCAAAGGTTTACAAAACCTACTTTGAGAAGGTCGAAAGATTAAAGCATGGAACCTTCGGTCCCGTTCACTTTCGAAATCAGGTCAAAATACGTCGCAGGGTTGACATAAACCCGGGTCACGCAGATCTTCGTGCTAAAGAAGCACAAGACGTCATAATGGAGGTCGTTTTCCCAAACGAAGTAGGAGCCAGGATATTGACATCAGAGTCACAGTTAACAATAACAAAAGAAAAGAAGGAAGAGCTCCAGGGCTGTAAGATCGCTCCTTTGATGGTGGCATACATGTTGGAAAGGGAACTAGTTCGCAAAACCAGATTCCTACCAGTAGCTGGCGGGACAAGCAGCGTGTATATCGAGGTGTTGCACTTGACCCAAGGGACCTGCTGGGAACAAATGTACACACCAGGAGGGGAGGTGAGAAATGATGATGTTGATCAGAGTTTAATTATTGCTGCTAGAAATATTGTCAGGAGAGCAACAGTATCAGCAGACCCGTTGGCTTCGCTCTTGGAAATATGCCATAGTACACAAATTGGCGGAATAAGGATGGTAGACATCCTTAGACAAAATCCAACAGAAGAGCAAGCTGTGGATATATGCAAAGCAGCAATGGGTCTAAGGATCAGTCCATCCTTCAGCTTTGGAGGTTTCACTTTCAAAAGGACAAGTGGGTCATCTGTCAAAAGAGAAGAAGAAGTGCTCACAGGCAATCTCCAGACATTGAAAATAAGAGTGCACGAAGGATATGAGGAATTCACAATGGTCGGGCGAAGAGCAACAGCCATTCTAAGGAAAGCAACCAGAAGGCTGATCCAATTGATAGTGAGTGGAAGAGACGAGCAGTCAATCGCCGAAGCAATCATAGTGGCAATGGTGTTCTCACAAGAGGATTGCATGATAAAAGCAGTGCGAGGTGATTTGAATTTTGTCAACAGAGCGAACCAGCGGCTAAATCCCATGCATCAACTTCTGAGGCATTTCCAGAAGGATGCAAAGGTGCTGTTTCAAAACTGGGGAGTTGAACCCATTGACAATGTCATGGGAATGATCGGGATATTACCTGACATGACCCCCAACACAGAGATGTCACTAAGGGGAGTGAGAGTCAGTAAAATGGGAGTGGATGAATATTCCAGTACGGAGAGAGTGGTCGTGAGCATTGATCGTTTCTTGAGGGTCCGAGACCAGAGGGGAAATGTGCTCTTGTCTCCTGAAGAGGTTAGTGAAACACAGGGAACAGAAAAGCTGACGGTAACATATTCATCGTCCATGATGTGGGAAATCAATGGCCCGGAATCAGTGTTAGTCAACACATATCAATGGATCATTAGAAACTGGGAAACTGTGAAGATACAGTGGTCCCAAGACCCTACAATGCTATACAATAAGATGGAATTTGAGCCCTTCCAATCCTTGGTGCCTAAGGCTGCCAGAGGCCAGTACAGCGGCTTTGTGAGGACGCTATTCCAGCAGATGCGTGATGTGCTGGGGACATTTGACACTGTCCAGATAATAAAGCTGCTACCATTTGCAGCAGCCCCACCAGAACAGAGTAGAATGCAGTTCTCTTCTCTAACTGTGAATGTAAGGGGTTCAGGAATGAGAATACTTGTGAGAGGCAATTCCCCTGTGTTCAATTATAACAAGGCAACCAAGAGGCTCACAGTCCTTGGAAAGGATGCAGGTGCATTGACAGAAGATCCAGATGAGGGAACAGCAGGAGTGGAATCTGCGGTATTAAGAGGGTTTCTAATTCTGGGCAAAGAAGACAAAAGATATGGACCAGCATTGAGCATCAACGAACTGAGCAATCTTGCGAAAGGGGAGAAGGCTAATGTGTTGATAGGGCAAGGAGACGTGGTGTTGGTAATGAAACGGAAACGGGACTCTAGCATACTTACTGACAGCCAGACAGCGACCAAAAGAATTCGGATGGCCATCAATTAG
SEQ ID NO：4
A/duck/Eastern China/JY/2014(JY)PB2
MERIKELRDLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPALRMKWMMAMKYPITADKRIMEMIPERNEQGQTLWSKTNDAGSDRVMVSPLAVTWWNRNGPTTSTVHYPKVYKTYFEKVERLKHGTFGPVHFRNQVKIRRRVDINPGHADLRAKEAQDVIMEVVFPNEVGARILTSESQLTITKEKKEELQGCKIAPLMVAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEVRNDDVDQSLIIAARNIVRRATVSADPLASLLEICHSTQIGGIRMVDILRQNPTEEQAVDICKAAMGLRISPSFSFGGFTFKRTSGSSVKREEEVLTGNLQTLKIRVHEGYEEFTMVGRRATAILRKATRRLIQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNFVNRANQRLNPMHQLLRHFQKDAKVLFQNWGVEPIDNVMGMIGILPDMTPNTEMSLRGVRVSKMGVDEYSSTERVVVSIDRFLRVRDQRGNVLLSPEEVSETQGTEKLTVTYSSSMMWEINGPESVLVNTYQWIIRNWETVKIQWSQDPTMLYNKMEFEPFQSLVPKAARGQYSGFVRTLFQQMRDVLGTFDTVQIIKLLPFAAAPPEQSRMQFSSLTVNVRGSGMRILVRGNSPVFNYNKATKRLTVLGKDAGALTEDPDEGTAGVESAVLRGFLILGKEDKRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAIN-
SEQ ID NO：5
PB2M1-F：GGCTTCGCTCTTGGAAATATGCCATAGTAC
SEQ ID NO：6
PB2M1-R：TATTTCCAAGAGCGAAGCCAACGGGTCT
SEQ ID NO：7
PB2M2-F：ACACAGGGAACAGAAAAGCTGACGATAAC
SEQ ID NO：8
PB2M2-R：TTTTCTGTTCCCTGTGTTTCACTAACCTCT。
Claims (6)
1. The application of the avian influenza polymerase protein PB2 in preparing JAK-STAT signal transduction inhibitors is characterized in that the nucleotide sequence of the avian influenza polymerase protein PB2 is shown in SEQ ID NO: 1.
2. The use of avian influenza polymerase protein PB2 of claim 1 for the preparation of JAK-STAT signal transduction inhibitor, wherein the avian influenza virus is subtype H5.
3. The use of avian influenza polymerase protein PB2 of claim 1 in the preparation of JAK-STAT signal transduction inhibitor, wherein the amino acid sequence of the expressed avian influenza polymerase protein PB2 is as set forth in SEQ ID NO: 2.
4. Use of the avian influenza polymerase protein PB2 of claim 1 for the preparation of JAK-STAT signal transduction inhibitors, wherein the PB2 protein inhibits JAK-STAT signal transduction by degrading the JAK1 protein.
5. The use of the avian influenza polymerase protein PB2 of claim 4 for the preparation of JAK-STAT signal transduction inhibitors, wherein the PB2 protein degrades JAK1 in a ubiquitin-proteasome dependent manner by interacting with JAK1.
6. The use of avian influenza polymerase protein PB2 of claim 5 for the preparation of JAK-STAT signal transduction inhibitors, wherein the PB2 protein interacts with the kinase and kinase-like domain of JAK1.
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