CN109966497B - Application of substance taking IPAN or coding gene thereof as target point in preparation of influenza virus inhibitor - Google Patents

Abstract

The invention discloses application of a substance taking IPAN or an encoding gene thereof as a target point in preparation of an influenza virus inhibitor. The invention provides a substance for inhibiting the expression of an IPAN gene in a cell, a substance for inhibiting the activity of the IPAN gene in the cell and a substance for reducing the content of the IPAN gene in the cell, and application of the substances in preparing an influenza virus inhibitor or an influenza virus replication inhibitor. Experiments prove that the A-type influenza virus infection can be obviously inhibited after the IPAN gene in the human cell is silenced, so that the A-type virus yield can be maximally reduced by 90%. The substance for inhibiting the expression of the IPAN gene in the cell, the substance for inhibiting the activity of the IPAN in the cell and the substance for reducing the IPAN content in the cell can inhibit the infection of the influenza A virus, can be used for preparing an influenza virus inhibitor or an influenza virus replication inhibitor, and can be used for preparing a product for treating diseases caused by the infection of the influenza virus into the cell or/and preventing the diseases caused by the infection of the influenza virus into the cell.

Description

Application of substance taking IPAN or coding gene thereof as target point in preparation of influenza virus inhibitor

Technical Field

The invention relates to an application of a substance taking IPAN or a coding gene thereof as a target point in preparing an influenza virus inhibitor.

Background

Influenza virus can cause acute respiratory infectious diseases, is most threatened by influenza virus A (IAV), is easy to change, is highly infectious and highly pathogenic, and is susceptible to humans and various animals. Seasonal influenza and multiple influenza pandemics each year have caused serious threats and significant losses to human life health and economy for over a century. At present, the means for clinically preventing and treating influenza viruses mainly comprise vaccines and anti-influenza drugs, but because of extremely strong recombinant variation and antigen drift capability of the influenza viruses, the delay of newly-issued virus specific vaccines and the continuous emergence of drug-resistant virus strains are caused, so that the prevention and control situation of novel viruses is getting more and more serious.

Long non-coding RNAs (lncRNAs) generally refer to mRNA-like cellular endogenous RNAs located in the nucleus or cytoplasm and more than 200 nucleotides in length, which have little or no protein coding function. The expression of lncRNAs has tissue and cell specificity, regulates gene expression and protein synthesis in the form of RNA at various levels (epigenetic regulation, transcriptional regulation, post-transcriptional regulation, etc.), and participates in the regulation of various biological processes including genomic imprinting, dose compensation, cell growth and differentiation, apoptosis, immune response, tumorigenesis, etc., through interaction with protein, DNA or RNA.

Influenza PB1-Associated non-coding RNA (IPAN) gene maps to the positive human chromosome 1 strand, approximately 220kbp upstream of PKN2, and its gene ID numbers in the NONCODE database (database link: http:// www.noncode.org) are: NONHSAG 002002.3. IPAN has three non-coding transcripts, and the transcripts in the NONCODE database have ID numbers of NONHSAT225203.1, NONHSAT225202.1, and NONHSAT004318.2, respectively, and all have two exons.

Disclosure of Invention

The invention aims to solve the technical problem of inhibiting influenza virus from infecting cells.

In order to solve the technical problems, the invention develops the following purposes by taking IPAN or a coding gene thereof as a target:

1. use of a substance that inhibits expression of an IPAN gene in a cell in the preparation of an influenza virus inhibitor or an influenza virus replication inhibitor.

2. The application of the substance for inhibiting the expression of the IPAN gene in the cell in preparing products (such as medicines, vaccines, health products and/or foods) for treating diseases caused by the cell infected by the influenza virus or/and preventing the diseases caused by the cell infected by the influenza virus.

3. Use of a substance that inhibits the activity of IPAN in a cell in the preparation of an influenza virus inhibitor or an influenza virus replication inhibitor.

4. Application of substance for inhibiting IPAN activity in cell in preparing product (such as medicine, vaccine, health product and/or food) for treating diseases caused by influenza virus infection cell and/or preventing diseases caused by influenza virus infection cell.

5. Use of a substance that reduces the level of IPAN in a cell in the preparation of an influenza virus inhibitor or an influenza virus replication inhibitor.

6. Use of a substance that reduces the level of IPAN in a cell in the manufacture of a product (e.g., a medicament, a vaccine, a health product, and/or a food) for treating a disease caused by an influenza virus-infected cell or/and preventing a disease caused by an influenza virus-infected cell.

Use of IPAN in the manufacture of a product (e.g. a reagent) for promoting replication of influenza virus.

8. A method for inhibiting influenza virus infection in an animal comprising administering to a recipient animal an agent that inhibits expression of an IPAN gene in a cell to inhibit influenza virus infection in the animal.

9. A method for treating or/and preventing influenza, comprising administering to a recipient animal a substance that inhibits the expression of the IPAN gene in a cell to treat or/and prevent influenza.

In the above application, the disease caused by influenza virus infection of cells may be influenza. The product for treating diseases caused by influenza virus infected cells or/and preventing diseases caused by influenza virus infected cells can be an anti-influenza virus medicament.

In the above application, the influenza virus may be influenza a virus.

In the above application, the substance for inhibiting expression of IPAN gene in cells, the substance for inhibiting activity of IPAN in cells and the substance for reducing content of IPAN in cells can be any one of the following biological materials 1) to 7) that can inhibit or down-regulate expression of IPAN gene by RNA interference:

1)esiRNA，

2) siRNA or a chemical modification of said siRNA,

3) generating shRNA of siRNA or a chemical modifier of the shRNA,

4) an expression vector for expressing 3) the shRNA,

5) a recombinant microorganism expressing 3) the shRNA,

6) an expression vector for expressing the siRNA of 2),

7) a recombinant microorganism expressing 2) said siRNA.

In the application, the chemical modifier of the shRNA is a substance obtained by chemically modifying the shRNA. The chemical modification may include one or a combination of several selected from ribose modification, base modification, and phosphate backbone modification.

In the above application, the chemical modification of the siRNA is a substance obtained by chemically modifying the siRNA. The chemical modification may include one or a combination of several selected from ribose modification, base modification, and phosphate backbone modification.

In the above application, the esiRNA may be esiRNA targeting the following segment of the IPAN gene: the nucleotide sequence is DNA of sequence 1in the sequence table.

In the above application, the cell may be a mammalian cell, such as a human.

In the above applications, the product may contain the substance for inhibiting expression of an IPAN gene in a cell, the substance for inhibiting activity of IPAN in a cell, and/or the substance for reducing the amount of IPAN in a cell.

In the above application, the product may further comprise a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable carrier" should be compatible with the RNA molecule of the agent of the invention. The pharmaceutically acceptable carrier refers to an in vivo transfection reagent, such as Polyethyleneimine (PEI), linear polyethyleneimine (jetPEI), liposome, transferrin, folic acid, nanoemulsion, nanoparticle and the like. Other examples of substances which may serve as pharmaceutically acceptable carriers or components thereof are lyoprotectants sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; powdered gum tragacanth; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as glycerol, mannitol; alginic acid; emulsifiers, such as Tween; phospholipids, such as lecithin, soya lecithin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, stearamide; cholesterol; macromolecular polymers such as polyethyleneimine, chitosan, hyaluronic acid; wetting agents, such as sodium lauryl sulfate; a colorant; a flavoring agent; tabletting agents, stabilizers; an antioxidant; a preservative; pyrogen-free water; isotonic saline solution; and phosphate buffer and the like; physiological saline, glycerol and phosphate buffered saline.

The invention firstly utilizes an esiRNA library of targeting human lncRNAs to carry out high-throughput screening on functional lncRNAs and firstly discloses a long-chain non-coding RNA IPAN as a positive control factor to participate in the replication process of influenza viruses. The IAV replication can be obviously inhibited by IPAN gene silencing, and the replication of the IAV can be obviously inhibited by utilizing esiRNA to down-regulate the endogenous IPAN of the cell, but the replication of the IBV is not influenced. Overexpression of IPAN in cells is effective in promoting replication of IAV. IAV infection can promote expression and nuclear transport of IPAN. The IAV replicon system was used to demonstrate that esiRNA-mediated IPAN gene silencing can effectively inhibit the transcription and replication of viral RNA. IPAN influences the transcription and replication links of virus RNA by regulating the stability of the PB1 protein of the influenza virus, thereby participating in the replication of the influenza virus. In order to further clarify a specific link of promoting IAV RNA transcription and replication by IPAN, a replicon system of IAV and a detection technology of RNA-protein interaction are utilized, so that specific binding of IPAN and PB1 is found, and degradation of PB1 protein is remarkably promoted by IPAN gene silencing, and IAV infection is effectively inhibited. The invention discloses the biological function of promoting the replication of the IAV virus by the IPAN, plays an important role in the replication process of the influenza virus, lays a research foundation for finding a new antiviral target and developing a new disease prevention and control means, and provides a new basis for developing an antiviral drug target by using host lncRNA.

Experiments prove that the A-type influenza virus infection can be obviously inhibited after the IPAN gene in the human cell is silenced, so that the A-type virus yield can be maximally reduced by 90%. The substance for inhibiting the expression of the IPAN gene in the cell, the substance for inhibiting the activity of the IPAN in the cell and the substance for reducing the IPAN content in the cell can inhibit the infection of the influenza A virus, can be used for preparing an influenza virus inhibitor or an influenza virus replication inhibitor, and can be used for preparing a product for treating diseases caused by the infection of the influenza virus into the cell or/and preventing the diseases caused by the infection of the influenza virus into the cell.

Drawings

FIG. 1 shows inhibition of influenza A virus infection by IPAN gene silencing.

A is IAV NP protein level of Western blot analysis.

B is the Gluc activity to determine the virus infection.

C is RT-qPCR to detect the intracellular IPAN RNA level.

D is TCID₅₀The method determines the virus titer of the supernatant filial generation.

E is CCK8 method to determine the effect of esiIPAN on cytotoxicity.

After 24h of transfection of esiIPAN or esiEGFP into 293T-Gluc cells, IAV and IBV strains (MOI ═ 0.5) were infected and virus infectivity was determined. In F, WSN/33 represents A/WSN/33(H1N1), Beijing/30/95 represents A/Beijing/30/95(H3N2), PR/8/34 represents A/PR/8/1934(H1N1), Beijinghaidian/1386/2013 represents B/Beijinghaidian/1386/2013(Victoria), and Massachusetts/02/2012 represents B/Massachusetts/02/2012 (Yama-gata).

In A-F, esiIPAN represents recombinant cell esiIPAN/293T-Gluc, and esiEGFP represents recombinant cell esiEGFP/293T-Gluc.

FIG. 2 is a graph showing that IPAN overexpression promotes IAV replication.

A is a schematic diagram of IPAN transcripts and mutants.

B, 293T-Gluc cells are transfected with different amounts of IPAN expression plasmids, and Gluc activity in cell supernatants is measured after WSN/33 (MOI: O.5) is infected for 24 h; IPAN-S, IPAN-M and IPAN-L represent the supernatants of recombinant cells transfected with pcDNA3.1(+) -IPAN-S, pcDNA3.1(+) -IPAN-M and pcDNA3.1(+) -IPAN-L, respectively.

C, detecting the IAV replication condition in the IPAN over-expression 293T-Gluc cells at different time points after WSN/33 infection (MOI is 1) by using RT-qPCR; EV, IPAN-S, IPAN-M, and IPAN-L represent recombinant cells transfected with pcDNA3.1(+), pcDNA3.1(+) -IPAN-S, pcDNA3.1(+) -IPAN-M, and pcDNA3.1(+) -IPAN-L, respectively.

D is 293T-Gluc cell infected with WSN/33 (MOI: O.5) after over-expressing IPAN, and RT-qPCR detects the mRNA, vRNA and cRNA levels of the virus NP; EV, IPAN-S, IPAN-M, and IPAN-L represent recombinant cells transfected with pcDNA3.1(+), pcDNA3.1(+) -IPAN-S, pcDNA3.1(+) -IPAN-M, and pcDNA3.1(+) -IPAN-L, respectively.

E and F are 293T-Gluc cells transfected with IPAN expression plasmid or empty vector, and TCID 24h after infection with WSN/33(MOI ═ O.5)₅₀The method determines the progeny virus titer (E) of the supernatant and the Gluc activity (F) in the cell supernatant. The empty vectors, IPAN-S, IPAN-M, IPAN-L, IPAN-IS1 and IPAN-IS2 represent the recombinant cell supernatants transfected with pcDNA3.1(+), pcDNA3.1(+) -IPAN-S, pcDNA3.1(+) -IPAN-M, pcDNA3.1(+) -IPAN-LpcDNA3.1(+) -IPAN-IS1 and pcDNA3.1(+) -IPAN-IS2, respectively.

FIG. 3 shows that IAV infection promotes IPAN expression and nuclear transport.

A is IAV of different MOI to infect A549 cells, and RT-qPCR is used for detecting the IPAN RNA level; in the figure, IAV is A/WSN/33(H1N 1).

B is after A549 cells are infected by various IAV strains and different viruses, RT-qPCR detects the IPAN RNA level.

C is RNA level of IPAN, MxA and ISG15 detected by RT-qPCR after IFN-alpha (1000U/ml) treatment of A549 cells.

D is the level of IPAN and IFN-beta detected by RT-qPCR after 293T-Gluc cells are transfected by different amounts of poly (I: C).

E is the positioning condition of IPAN before and after IAV infection of RNA-FISH analysis; in the figure, IAV is A/WSN/33(H1N 1).

F is a scatter plot of the IPAN fluorescence signal ratio (in-nucleus/total cells) in 50 cells, and IAV is A/WSN/33(H1N 1).

Fig. 4 shows the interaction of IPAN with PB1 protein.

A is 293T-Gluc cells transfected with PA, PB1, PB2 and NP expression plasmids respectively, and the binding condition of IPAN RNA and virus protein is detected by utilizing CL-RIP technology.

B is different transcripts of 293T-Gluc cell co-transfected PB1 and IPAN, and the binding condition of IPAN RNA and virus protein PB1 is detected by using a native RIP technology.

C is RNA pulldown experiment after A549 cell is infected with WSN/33 (MOI: O.5) for 36h, and the binding condition of IPAN RNA and a virus protein PB1 is detected.

Fig. 5 shows that IPAN promotes the stability of PB1 protein.

A is the Gluc activity at IPAN knockdown in viral infection and replication subsystems.

B293T-Gluc cells were transfected with esiIPAN or esiEGFP 24h, infected with WSN/33(MOI 0.5)24h, and then subjected to RT-qPCR to detect the levels of viral NP vRNA and mRNA in the cells.

C and D are the proteins of the transfection esiEGFP or esiIPAN in the replicon stable cell line A549-5Ps, Western blot analysis of protein levels of the viruses PA, PB1, PB2 and NP (C) RT-qPCR detection of virus PB1mRNA level (D).

E is A549-5Ps, esiEGFP or esiIPAN is transfected, after treatment with protein synthesis inhibitor CHX for different times, protein level of virus PB1 is analyzed by Western blot, and protein quantification is performed by Image J.

F is A549-5Ps, and after the transfection of esiEGFP or esiIPAN, cells are treated by DMSO or a proteasome inhibitor MG 132; western blot analysis of the protein levels of virus PB1 and quantification with Image J.

G is 293T-Gluc cell cotransformation esiIPAN and PB1 expression plasmid, protein level of virus PB1 was analyzed by Western blot 24h after infection with WSN/33(MOI O.5), and infectivity of the virus was quantified by measuring Gluc activity.

H is protein level of virus PB1 analyzed by Western blot 24H after 293T-Gluc cells are transfected with wild-type IPAN or IPAN mutant, and WSN/33 (MOI: O.5) is infected, and quantification is carried out by using Image J.

Detailed Description

The invention utilizes an IAV inducible reporter gene detection system to carry out high-throughput screening on an esiRNA library targeting known lncRNAs of human beings, and 50 targets of the esiRNA are screened to have obvious influence on influenza virus replication. Wherein, lncRNA IPAN is highly involved in influenza virus replication, and the function and action mechanism of the lncRNA IPAN in the influenza virus replication are researched by knocking down and over-expressing IPAN technology. The method comprises the following specific steps:

in a first aspect, lncRNA IPAN is used as a positive regulator in the replication of influenza virus

Expression levels of IPAN significantly affect replication of IAV. The research finds that the expression of NP protein can be obviously inhibited by IPAN gene silencing, and the Gluc activity and the virus titer in cell supernatant can be reduced along with the reduction of IPAN level. When IPAN gene-silenced cells were infected with IAVs of different MOI, the virus production could be downregulated by 90% at the maximum. Meanwhile, in order to evaluate the functional specificity of IPAN to the IAV replication process, IAV and IBV are selected for research respectively. The results show that IPAN is specifically involved in replication of IAV, but has no effect on replication of IBV.

Furthermore, experiments show that the excessive expression of IPAN-M and IPAN-L can obviously improve the Gluc level and has dose dependence. Time addition experiments showed that IPAN-M promotes viral RNA expression more early in infection (12 h post infection) than late in infection (24 h post infection). RT-qPCR and virus titer determination results show that IPAN overexpression can increase the level of virus transcripts (v/c/mRNA) to different degrees and improve the yield of infectious viruses, while two truncations of IPAN, namely IPAN-IS1 and IS2 (two homologous sequences of three transcripts of IPAN) can not promote IAV replication. These results demonstrate that IPAN is able to regulate IAV replication in the forward direction.

Meanwhile, qRT-PCR and RNA-FISH results show that IAV infection can up-regulate IPAN level by more than 5 times and has the specificity of influenza A virus infection. Expression of IPAN was not affected by treatment with interferon alpha and poly (I: C). Through counting the IPAN fluorescence signals in the nucleus and cytoplasm, the IAV infection is found to promote the nuclear transport of IPAN. These results demonstrate that IAV infection not only promotes expression of IPAN, but also recruits IPAN into the nucleus to facilitate viral replication.

In a second aspect, IPAN is involved in the influenza virus replication process by stabilizing the viral PB1 protein to regulate the transcriptional replication machinery of viral RNA.

Experiments show that IPAN gene silencing can reduce Gluc activity by about 75% in IAV infection and IAV replication subsystems. At the same time, the mRNA and vRNA levels of the viral NP gene were found to be dose-dependent with esiIPAN. Taken together, esiIPAN-mediated silencing of IPAN genes could inhibit transcription and replication of viral RNA, suggesting that IPAN promotes IAV replication by regulating transcriptional replication of viral RNA.

Further, it was found through experiments that IPAN can bind to PB1 and silencing IPAN gene can reduce PB1 protein level, but not due to PB1mRNA level reduction. Experiments with the protein synthesis inhibitor CHX and the proteasome inhibitor MG132 showed that the PB1 protein degradation was promoted by IPAN gene silencing and was accomplished via the proteasome pathway. Further, the replication capacity of the virus was also significantly increased when PB1 protein was recruited in IPAN-knockdown cells, indicating that IPAN is involved in IAV replication by modulating PB1 protein levels.

The present invention is further described in detail below with reference to specific examples, which are given only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The cell culture solution used in the following examples was DMEM medium containing 10% FBS.

ZIKV (strain FSS13025) (Alexander Sze et al Sophoraflavone G restricts Dengue and Zika virus infection via RNA polymerase interface. viruses 2017,9,287) was gifted by professor Mark Wainberg, university of Megill, Canada. Enterovirus 71(EV71) strains EV71-HP and VSV-G packaged pseudovirus HIV-1pNL4-3Luc (R-E-) for specific information reference (Yong-Xin Zhang et al A high level conserved amino acid in VP1 regulations for analysis of infectious virus 71.PLOS Patholons | https:// doi.org/10.1371/j ournal. p.p.1006625September 22,2017; Zeyun Mi et al A small molecule composition B-LA inhibitors HIV-1infection by a preceding viral Vpu anti-viral infection virus strain BST-2.Scientific Reports | 1845: DOb 99/10.1038).

The human lung cancer cell lines A549 and A545-5Ps (Zhen Wang et al. assessment of a high-through put assay to monitor in fluorescence A virus RNA transcription and reproduction. PLoS ONE 2015; 10(7) e0133558), the human kidney epithelial cell lines 293T and 293T-Gluc, canine kidney cells (MDCK cells), A/WSN/33(H1N1), A/PR/8/1934(H1N1), B/Beijjiadian/1386/2013 (Victoria), B/Massachusetts/02/2012(Yama-gata) (Qian Gao scientific. A cell-base high-through put assay to positive infection inhibition of biological samples A306. the biological material obtained from this research institute by Biotech, Sapon, Sapons, Sapon, it is not usable for other purposes.

A/Beijing/30/95(H3N2) (Jingning Wang et al host Long non-coding RNA lncRNA-PAAN regulations of nonfluenza A viruses 2018,10,330) in the following examples can obtain the biomaterial from the research institute of medical biotechnology of Chinese medical science institute, and the biomaterial is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

Example 1 inhibition of influenza A Virus infection by IPAN Gene silencing

This example uses two esirnas, named esiIPAN and esiEGFP, respectively. esiIPAN is esiRNA used to silence the human IPAN gene. esiIPAN targets the human IPAN gene and three transcripts of human IPAN (NONCODE TRANSCRIPT ID NONHSAT225203.1 (hereinafter abbreviated to IPAN-S), NONCODE TRANSCRIPT ID NONHSAT225202.1 (hereinafter abbreviated to IPAN-M), and NONCODE TRANSCRIPT ID NONHSAT004318.2 (hereinafter abbreviated to IPAN-L)). The target sequence of esiIPAN in the human IPAN gene (esiIPAN cDNA target sequence) is sequence 1in the sequence listing. esiIPAN is a product from Sigma, having a product number EHNC007351(https:// www.sigmaaldrich.com/catalog/product/Sigma/EHNC007351lang ═ en & region ═ HK).

esiEGFP is esiRNA as a negative control of esiIPAN, which targets EGFP, and is esiRNA for silencing EGFP gene. esiEGFP is a product of Sigma, Cat # EHUEGFP

(https://www.sigmaaldrich.com/catalog/product/sigma/ehuegfplang＝en&region＝HK)。

1. Significant reduction of viral NP protein levels following silencing of IPAN Gene by esiIPAN

In six well plates, the density of independent reverse transfections with esiIPAN and esiEGFP was 3X 10, respectively, according to the Lipofectamine RNAiMAX transfection reagent instructions (Invitrogen)⁵And (3) 293T-Gluc cells per ml, wherein the transfection concentration of esiRNA is set to be 20nM, and recombinant cells transfected with esiIPAN alone (named esiIPAN/293T-Gluc) and esiEGFP alone (named esiEGFP/293T-Gluc) are obtained. At 37 ℃ with 5% CO₂Culturing in an incubator. After 48H the cells were infected with influenza a/WSN/33(H1N1) at MOI 0.5, after 24H the cells were washed once with 1mL of pre-cooled PBS, washed and 80 μ L RIPA (50mM Tris-HCl (pH 7.4),150mM NaCl, 1% NP-40, 0.1% SDS, Roche protease inhibitor cocktail) was added, scraped with cells and collected in 1.5mL EP tubes. Adding 20 μ L of 5 xSDS protein loading buffer, vortex, shaking, and lysing, and performing metal bath at 100 deg.C for about 20min to obtain cell total protein sample. Protein samples were directly subjected to routine immunoblot (Western blots) detection or stored at-20 ℃ or-80 ℃. In this example, Western Blot was used to detect the expression of the NP protein from the virus. Western Blot procedure: protein samples were separated by 10% SDS-PAGE. And (3) after the PVDF membrane is used for membrane conversion for 1 hour by a 70V constant-pressure wet method, sealing the 5% skimmed milk for 1 hour at room temperature. And (3) incubating with a primary antibody and a secondary antibody, finally carrying out ECL (electrogenerated chemiluminescence) color development, and carrying out quantitative analysis on protein bands by using Image J software. Antibodies were used at concentrations of beta-actin (1:2000, Santa Cruz), NP (1:5000, Genetex), goat anti-mouse (1:5000, Miyaojin) and goat anti-rabbit (1:5000, Miyaojin). The experiment was performed in triplicate and all data were processed statistically using an independent sample t-test using SPSS12.0(SPSS inc., USA) statistical software.

The results show that silencing esiIPAN gene significantly reduces viral NP protein levels in cells (a in figure 1).

2. Inhibition of influenza a virus infection following silencing of IPAN gene by esiIPAN

Further, to confirm that the inhibition of viral replication was not due to an esiRNA-mediated non-specific antiviral state, the method of step 1 was referenced for cell reverse transfection and viral infection, with esiRNA concentrations set at 10nM, 20nM and 40nM, respectively, and real-time quantitative fluorescent PCR (RT-qPCR) was used to detect the knockdown effect of esiRNA on endogenous IPAN in the cells, while measuring Gluc activity and TCID in the cell supernatant₅₀The value is obtained. The specific method comprises the following steps:

293T-Gluc cells were separately counter-transfected with esiIPAN and esiEGFP, respectively (cell density 3X 10) in six-well plates according to Lipofectamine RNAiMAX transfection reagent instructions (Invitrogen)⁵Individual/ml), transfection concentrations of esirnas were made 10nM, 20nM and 40nM, respectively, to give recombinant cells transfected with 10nM esiIPAN alone (named 10nM-esiIPAN/293T-Gluc), recombinant cells transfected with 20nM esiIPAN alone (named 20nM-esiIPAN/293T-Gluc), recombinant cells transfected with 40nM esiIPAN alone (named 40nM-esiIPAN/293T-Gluc), recombinant cells transfected with 10nM esiEGFP alone (named 10nM-esiEGFP/293T-Gluc), recombinant cells transfected with 20nM esiEGFP alone (named 20nM-esiEGFP/293T-Gluc), and recombinant cells transfected with 40nM esiEGFP alone (named 40 nM-esiEGFP/293T-Gluc). At 37 ℃ with 5% CO₂Culturing in an incubator. After 48H, the cells were infected with influenza A/WSN/33(H1N1) with an MOI of 0.5, and after 24H the cells and supernatant were collected, 6 cells of 10nM-esiIPAN/293T-Gluc infected with influenza A/WSN/33(H1N1), 20nM-esiIPAN/293T-Gluc infected with influenza A/WSN/33(H1N1), 40nM-esiIPAN/293T-Gluc infected with influenza A/WSN/33(H1N1), 10nM-esiEGFP/293T-Gluc infected with influenza A/WSN/33(H1N1), 20nM-esiEGFP/293T-Gluc infected with influenza A/WSN/33(H1N1), and 40nM-esiEGFP/293T-Gluc infected with influenza A/WSN/33(H1N1) and supernatants thereof were obtained, respectively. The cells were subjected to RT-qPCR of 2.1 described below to detect the IPAN content of each cell, and the supernatant was subjected to Gluc activity detection of 2.2 described below and TCID of 2.3₅₀The method detects the infectivity of influenza virus A/WSN/33(H1N1) to each cell.

2.1esiIPAN significantly reduced IPAN levels in human cells

Specifically, the RT-qPCR technical process comprises the following steps: total cellular RNA was extracted according to the Invitrogen TRIZOL instructions. After the concentration was measured, 2. mu.g of total RNA was taken, and the RNA was reverse-transcribed into cDNA using Primescript RT Master kit (Takara). The cDNA amplification reaction was performed using SYBR premix Ex Taq II kit (Takara). The method comprises the following steps: SYBR premix Ex Taq II (Tli RNaseH Plus) 10. mu.l, forward primer (10. mu.M) 0.8. mu.l, reverse primer (10. mu.M) 0.8. mu.l, template cDNA 1. mu.l, RNase-free water 7.4. mu.l. The reaction conditions were 95 ℃ for 30 sec; then 40 cycles, each cycle at 95 ℃ for 5 sec; 60 ℃,20 sec; 72 ℃ for 30 sec; the melting curve is 60-95 ℃, and the process is continuous. The housekeeping gene gapdh was used as the internal reference 2^-ΔΔCtThe method can relatively quantify the RNA level of the gene to be detected. The experiment was performed in triplicate and all data were processed statistically using an independent sample t-test using SPSS12.0(SPSS inc., USA) statistical software.

Wherein, the primer of the IPAN is IPAN-F (sequence 5'-GAATGAACTACTGGAAGGACGG-3') and IPAN-R (sequence 5'-CTGAGCAGATGAAGCACCGT-3'). IPAN-F and IPAN-R correspond to the consensus sequences of the three non-coding transcripts of IPAN as follows: NONCODE TRANSCRIPT ID NONHSAT225203.1 (hereinafter referred to as IPAN-S), NONCODE TRANSCRIPT ID NONHSAT225202.1 (hereinafter referred to as IPAN-M) and NONCODE TRANSCRIPT ID NONHSAT004318.2 (hereinafter referred to as IPAN-L). The length of the amplification products of IPAN-F and IPAN-R is 140 bp.

Primer sequence for gapdh: f, 5'-GTCCACTGGCGTCTTCACCA-3';

R，5’-GTGGCAGTGATGGCATGGAC-3’。

the RT-qPCR technology detects the low knock-out effect of esiRNA on the cell endogenous IPAN gene, and shows that the IPAN content of 10nM-esiEGFP/293T-Gluc infected with influenza virus A/WSN/33(H1N1) is 1.0, and the relative IPAN content of 10nM-esiIPAN/293T-Gluc infected with influenza virus A/WSN/33(H1N1) is 0.92 +/-0.01, and the difference between the two is not significant (p is more than 0.05); the IPAN content of 20nM-esiEGFP/293T-Gluc infected by influenza virus A/WSN/33(H1N1) is 1.0, and the relative IPAN content of 20nM-esiIPAN/293T-Gluc infected by influenza virus A/WSN/33(H1N1) is 0.56 +/-0.02, which are obviously different (p is less than 0.05); the IPAN content of 40nM-esiEGFP/293T-Gluc infected by influenza virus A/WSN/33(H1N1) is 1.0, and the relative IPAN content of 40nM-esiIPAN/293T-Gluc infected by influenza virus A/WSN/33(H1N1) is 0.35 +/-0.00, which are very different (p < 0.01). Indicating that esiIPAN can significantly reduce IPAN levels in human cells (C in fig. 1).

2.2Gluc Activity assay showed that esiIPAN significantly reduced the infectivity of influenza A/WSN/33(H1N1) to human cells

The Gluc activity detection method comprises the following steps: first, coelenterazine-h was dissolved in PBS solution to prepare a substrate of coelenterazine-h at a concentration of 16.7. mu.M, and incubated at room temperature in the dark for 30 min. At the same time, 10. mu.L of the supernatant to be assayed was placed in a white opaque flat-bottomed 96-well plate, and 30. mu.L of PBS solution was added. Fluorescence intensity measurements were performed using Bertholdtech sciences Centro XS3LB 960. In the detection process, because the half-life period of the reaction product is extremely short, a sample injector is used for adding and collecting signals of substrates incubated in a dark state by a hole according to the sample amount of 60 mu L per hole, and the relative activity of Gluc is determined. The experiment was performed in triplicate and all data were processed statistically using an independent sample t-test using SPSS12.0(SPSS inc., USA) statistical software.

Taking the fluorescence intensity (reporter gene activity (namely influenza virus infectivity)) of 10nM-esiEGFP/293T-Gluc supernatant infected with influenza virus A/WSN/33(H1N1) as 1.0, and the relative fluorescence intensity (reporter gene activity (namely influenza virus infectivity)) of 10nM-esiIPAN/293T-Gluc supernatant infected with influenza virus A/WSN/33(H1N1) as 0.54 +/-0.10, wherein the fluorescence intensity and the relative fluorescence intensity are significantly different (p is less than 0.05); taking the fluorescence intensity (reporter gene activity (namely influenza virus infectivity)) of 20nM-esiEGFP/293T-Gluc supernatant infected with influenza virus A/WSN/33(H1N1) as 1.0, and the relative fluorescence intensity (reporter gene activity (namely influenza virus infectivity)) of 20nM-esiIPAN/293T-Gluc supernatant infected with influenza virus A/WSN/33(H1N1) as 0.26 +/-0.10, wherein the fluorescence intensity and the reporter gene activity (namely influenza virus infectivity) are very different (p is less than 0.01); the relative fluorescence intensity (reporter activity (i.e. influenza virus infectivity)) of 40nM-esiEGFP/293T-Gluc supernatant infected with influenza virus A/WSN/33(H1N1) was 0.18 + -0.13, with a very significant difference (p < 0.01) between the reporter activity (i.e. influenza virus infectivity) of 40 nM-esiEGAN/293T-Gluc supernatant infected with influenza virus A/WSN/33(H1N 1). Indicating that esiIPAN can significantly reduce the infectivity of influenza a/WSN/33(H1N1) to human cells (fig. 1B).

2.3TCID₅₀The detection method shows that esiIPAN obviously reduces the infectivity of influenza virus A/WSN/33(H1N1) to mammalian cells

Virus TCID₅₀The measuring method comprises the following steps: one day prior to influenza A/WSN/33(H1N1) infection, MDCK was inoculated into 96-well plates in columns 1-11 and cultured in DMEM (2% FBS). Inoculation of 10 per well⁴Individual cells, 100 μ L system; the cells were plated for about 24h, 11 rows of 96-well plates were used, and 180. mu.L of cell maintenance solution was added to each well. The first 10 columns are used as gradient dilution virus, and the 11 th column is normal culture solution; inoculating 20 μ L of influenza A virus/WSN/33 (H1N1) virus stock solution into each well of the first row, mixing, adding 20 μ L of each well into the corresponding well of the next row, and repeating the steps until the solution is continuously diluted by 10 times to reach the 10 th row; the medium in a 96-well culture plate in which MDCK was cultured was aspirated, and the diluted virus was inoculated to the plate in a well-to-well manner, with 100 μ L of virus solution per well. Column 11 is a normal cell control group; culturing in an incubator at 37 ℃, observing day by day and recording results, wherein the observation is generally carried out for 5-7 days; the results were calculated according to the Reed-Muench two-handed method or the Karber method. The experiment was performed in triplicate and all data were processed statistically using an independent sample t-test using SPSS12.0(SPSS inc., USA) statistical software.

The results show that log of the supernatant of 10nM-esiEGFP/293T-Gluc infected with influenza A/WSN/33(H1N1)₁₀Viral Titer (TCID)₅₀/mL) was 0, relative log of 10nM-esiIPAN/293T-Gluc infected with influenza A/WSN/33(H1N1)₁₀Viral Titer (TCID)₅₀/mL) was-0.43. + -. 0.29, with no significant difference (p > 0.05); log of 20nM-esiEGFP/293T-Gluc infected with influenza A/WSN/33(H1N1)₁₀Viral Titer (TCID)₅₀/mL) was 0, relative log of 20nM-esiIPAN/293T-Gluc infected with influenza A/WSN/33(H1N1)₁₀Viral Titer (TCID)₅₀/mL) is-1.86 +/-0.08, and the two have significant difference (p < 0.05); log of 40nM-esiEGFP/293T-Gluc infected with influenza A/WSN/33(H1N1)₁₀Viral Titer (TCID)₅₀/mL) was 0, relative log of 40nM-esiIPAN/293T-Gluc infected with influenza A/WSN/33(H1N1)₁₀Viral Titer (TCID)₅₀/mL) was-1.80. + -. 0.05 with significant differences (p < 0.05). Indicating that esiIPAN can significantly reduce the infectivity of influenza a/WSN/33(H1N1) to human cells (fig. 1, D).

The results of 2.1, 2.2 and 2.3 show that both Gluc activity and virus titer in the cell supernatant decreased with decreasing levels of IPAN.

2.4 antiviral Activity of esiIPAN independent of its cytotoxicity

To exclude non-specific antiviral activity due to the cytotoxicity of esiIPAN, cell reverse transfection of esiRNA was performed using the same method as above, and the effect of esiIPAN on cell activity was verified according to CCK8 kit instructions (petunia). During detection, cell supernatant was replaced with cell culture medium containing 10% CCK-8 reagent, and cells were incubated at 37 deg.C and 5% CO₂Culturing for 1h in an incubator, and detecting the absorbance at 450nm by using an EnSpire 2300 multifunctional microplate reader. The results showed no significant difference in the absorbance of esiIPAN versus the control esiEGFP, indicating that the antiviral activity of esiIPAN was not related to its cytotoxicity (E in fig. 1). The specific experimental methods and experimental results are as follows:

293T-Gluc cells were separately counter-transfected with esiIPAN and esiEGFP, respectively (cell density 3X 10) in six-well plates according to Lipofectamine RNAiMAX transfection reagent instructions (Invitrogen)⁵One/ml), transfection concentrations of esiRNAs were 10nM, 20nM and 40nM, respectively, to obtain recombinant cells transfected with 10nM esiIPAN alone (named 10nM-esiIPAN/293T-Gluc), recombinant cells transfected with 20nM esiIPAN alone (named 20nM-esiIPAN/293T-Gluc), recombinant cells transfected with 40nM esiIPAN alone (named 40nM-esiIPAN/293T-Gluc), recombinant cells transfected with 10nM esiEGFP alone (named 10nM-esiEGFP/293T-Gluc), recombinant cells transfected with 20nM esiEGFP alone (named 20nM-esiEGFP/293T-Gluc), and recombinant cells transfected with 40nM esiEGFP alone (named 40nM-esiEGFP/293T-Gluc), at 37 ℃ and 5% CO₂Culturing in an incubator. After 48h, the 6 cells were tested for proliferation activity using the CCK8 kitAnd (6) measuring. The experiment was performed in triplicate and all data were processed statistically using an independent sample t-test using SPSS12.0(SPSS inc., USA) statistical software.

The results showed no significant difference in cell activity (expressed as OD 450) between 10nM-esiEGFP/293T-Gluc and 10nM-esiIPAN/293T-Gluc (p > 0.05); the cellular activities (expressed as OD 450) of 20nM-esiEGFP/293T-Gluc and 20nM-esiIPAN/293T-Gluc were not significantly different (p > 0.05); there was no significant difference in cell activity (expressed as OD 450) between 40nM-esiEGFP/293T-Gluc and 40nM-esiIPAN/293T-Gluc (p > 0.05). Indicating that the antiviral activity of esiIPAN was not associated with its cytotoxicity (E in figure 1).

3. Inhibition of influenza A virus infection and inhibition of influenza B virus infection after silencing of IPAN gene by esiIPAN

After knocking down IPAN using esiIPAN in the same manner as described above, IAV and IBV strains (MOI ═ 0.5) were infected and the virus infectivity was measured, and it was shown that esiIPAN could significantly reduce the infectivity of influenza a virus a/WSN/33(H1N1) to human cells, but could not reduce the infectivity of influenza B virus to human cells. The specific experimental methods and experimental results are as follows:

293T-Gluc cells were reverse transfected individually with esiIPAN and esiEGFP in six well plates according to the Lipofectamine RNAImax transfection reagent instructions (Invitrogen) to achieve a transfection concentration of esiRNA of 20 nM. Recombinant cells transfected with 20nM esiIPAN alone (designated 20nM-esiIPAN/293T-Gluc) and 20nM esiEGFP alone (designated 20nM-esiEGFP/293T-Gluc) were obtained. And 5% CO at 37 ℃₂Culturing in an incubator. After 48H, the cells were infected with influenza viruses (any of 5 influenza viruses, A/WSN/33(H1N1), A/PR/8/1934(H1N1), A/Beijing/30/95(H3N2), B/Beijinghaidian/1386/2013(Victoria) and B/Massachusetts/02/2012 (Yama-gata)), at an MOI of 0.5, and after 24H the supernatants were collected to give 20nM-esiIPAN/293T-Gluc supernatants, respectively, infected with the corresponding influenza viruses and 20nM-esiEGFP/293T-Gluc supernatants, infected with the corresponding influenza viruses. The supernatant was assayed for Gluc activity of 2.2 as described above. The experiment was performed in triplicate and all data were processed statistically using an independent sample t-test using SPSS12.0(SPSS inc., USA) statistical software.

The results showed that the relative fluorescence intensity (reporter activity (i.e., influenza infectivity)) of the supernatant of 20nM-esiEGFP/293T-Gluc infected with influenza A/WSN/33(H1N1) was 0.25 + -0.09 with a very significant difference (p < 0.01) between the fluorescence intensity of the supernatant of 20nM-esiEGFP/293T-Gluc infected with influenza A/WSN/33(H1N1) (reporter activity (i.e., influenza infectivity)) of 1.0; taking the fluorescence intensity (reporter gene activity (namely influenza virus infectivity)) of the supernatant of 20nM-esiEGFP/293T-Gluc infected with influenza virus A/PR/8/1934(H1N1) as 1.0, and the relative fluorescence intensity (reporter gene activity (namely influenza virus infectivity)) of the supernatant of 20nM-esiIPAN/293T-Gluc infected with influenza virus A/PR/8/1934(H1N1) as 0.07 +/-0.00, wherein the fluorescence intensity and the reporter gene activity (namely influenza virus infectivity) are very different (p is less than 0.01); taking the fluorescence intensity (reporter gene activity (namely influenza virus infectivity)) of the supernatant of 20nM-esiEGFP/293T-Gluc infected with influenza virus A/Beijing/30/95(H3N2) as 1.0, and the relative fluorescence intensity (reporter gene activity (namely influenza virus infectivity)) of the supernatant of 20nM-esiIPAN/293T-Gluc infected with influenza virus A/Beijing/30/95(H3N2) as 0.17 +/-0.02, the two have very significant difference (p is less than 0.01); the relative fluorescence intensity (reporter activity (i.e., influenza virus infectivity)) of the supernatant of 20nM-esiEGFP/293T-Glu infected with influenza B/Beijinghaidian/1386/2013(Victoria) was 0.79. + -. 0.08, with no significant difference (p > 0.05), given that the fluorescence intensity (reporter activity (i.e., influenza virus infectivity)) of the supernatant of 20nM-esiEGFP/293T-Glu infected with influenza B/Beijinghaidian/1386/2013(Victoria) was 1.0; when the reporter gene activity (namely, influenza virus infectivity) of the supernatant infected with 20nM-esiEGFP/293T-Gluc of influenza virus B/Massachusetts/02/2012(Yama-gata) is 1.0, and the relative fluorescence intensity (reporter gene activity (namely, influenza virus infectivity)) of 20nM-esiIPAN/293T-Gluc of the supernatant infected with influenza virus B/Massachusetts/02/2012(Yama-gata) is 0.74 +/-0.11, the two are not significantly different (p is more than 0.05). Indicating that esiIPAN can significantly reduce the infectivity of influenza a virus a/WSN/33(H1N1) to human cells, but cannot reduce the infectivity of influenza B virus to human cells (F in fig. 1).

Example 2 IPAN overexpression promotes IAV replication

1. Construction of expression vectors

The structures of the IPAN gene, three transcripts of IPAN (NONCODE TRANSCRIPT ID NONHSAT225203.1 (hereinafter referred to as IPAN-S), NONCODE TRANSCRIPT ID NONHSAT225202.1 (hereinafter referred to as IPAN-M) and NONCODE TRANSCRIPT ID NONHSAT004318.2 (hereinafter referred to as IPAN-L)) and two truncations of IPAN-IS1 and IPAN-IS2 are shown in FIG. 2A. A recombinant expression vectors capable of expressing the three transcripts of IPAN, IPAN-S, IPAN-M and IPAN-L, and recombinant expression vectors of the two truncations of IPAN-IS1 and IPAN-IS2 of IPAN are constructed:

the DNA with the nucleotide sequence of 1 st-1316 th site of SEQ ID No.2 is used to replace the segment between the BamH I and XhoI recognition sites of pcDNA3.1(+), and the other sequences of pcDNA3.1(+) are kept unchanged to obtain the IPAN-S gene recombinant expression vector named pcDNA3.1(+) -IPAN-S. pcDNA3.1(+) -IPAN-S contains DNA molecule with the nucleotide sequence of SEQ ID No.2 and expresses IPAN-S.

The DNA with the nucleotide sequence of 1-2179 th site of SEQ ID No.3 is used to replace the fragment between the BamH I and XhoI recognition sites of pcDNA3.1(+), and the other sequences of pcDNA3.1(+) are kept unchanged to obtain the IPAN-M gene recombinant expression vector which is named pcDNA3.1(+) -IPAN-M. pcDNA3.1(+) -IPAN-M contains DNA molecule with the nucleotide sequence of SEQ ID No.3 and expresses IPAN-M.

The DNA with the nucleotide sequence of 1-2232 of SEQ ID No.4 is used to replace the segment between the BamH I and XhoI recognition sites of pcDNA3.1(+), and the other sequences of pcDNA3.1(+) are maintained to obtain the recombinant expression vector of IPAN-L gene, named pcDNA3.1(+) -IPAN-L. pcDNA3.1(+) -IPAN-L contains DNA molecule with the nucleotide sequence of SEQ ID No.4 and expresses IPAN-L.

The DNA whose nucleotide sequence IS the 1 st to 234 th sites of SEQ ID No.2 IS used to replace the fragment between the BamH I and XhoI recognition sites of pcDNA3.1(+), and the other sequences of pcDNA3.1(+) are kept unchanged to obtain the IPAN-IS1 gene recombinant expression vector which IS named pcDNA3.1(+) -IPAN-IS 1. pcDNA3.1(+) -IPAN-IS1 contains DNA molecule whose nucleotide sequence IS the 1 st to 234 th positions of SEQ ID No.2, and expresses IPAN-IS1, and IPAN-IS1 IS a homologous fragment of three transcripts of IPAN.

The DNA with the 296-1255 th site of SEQ ID No.2 IS used to replace the fragment between the BamH I and XhoI recognition sites of pcDNA3.1(+), and the other sequences of pcDNA3.1(+) are kept unchanged to obtain the IPAN-IS2 gene recombinant expression vector named pcDNA3.1(+) -IPAN-IS 2. pcDNA3.1(+) -IPAN-IS2 contains DNA molecule with the nucleotide sequence of position 296-1255 of SEQ ID No.2, and expresses IPAN-IS2, and IPAN-IS2 IS another homologous fragment of three transcripts of IPAN.

IPAN overexpression significantly increases Gluc activity and promotes virus replication

Different amounts (0, 25, 50 and 250ng/mL) of IPAN plasmid solutions (any one of pcDNA3.1(+) -IPAN-S, pcDNA3.1(+) -IPAN-M and pcDNA3.1(+) -IPAN-L) were transfected into 293T-Gluc cells, and 24H later were infected with A/WSN/33(H1N1) (MOI ═ 0.5). After 48h Gluc activity was measured according to the method 2.2 in example 1. The results show that overexpression of IPAN-M and IPAN-L significantly increases Gluc activity, facilitating viral replication (B in FIG. 2).

Further, 293T-Gluc cells were transfected with 250ng/mL plasmid solutions (any one of pcDNA3.1(+) -IPAN-S, pcDNA3.1(+) -IPAN-M, pcDNA3.1(+) -IPAN-L, pcDNA3.1(+) -IPAN-IS1, pcDNA3.1(+) -IPAN-IS2 and empty vector pcDNA3.1(+), respectively, and after 24 hours, A/WSN/33(H1N1) (MOI ═ 1) infection was performed, and after 12, 15, 18, 21 and 24 hours after infection, the cells were collected, respectively, and the effect of IPAN overexpression on virus replication was determined by detecting the level of virus NP mRNA at different times after virus infection according to the RT-qPCR method of 2.1 of example 1. Similarly, RT-qPCR was used to measure the levels of three different RNA products (v/c/mRNA) during the transcriptional replication of influenza and the viral Titer (TCID) in the cell supernatant was determined by MDCK cells using the same method as described above₅₀Measurement). The results show that IPAN-M can promote the expression of viral RNA more in the early infection stage (12 h after infection) than in the late infection stage (24 h after infection), IPAN can increase the level of viral transcripts (v/c/mRNA) to different degrees and improve the yield of infectious viruses, and the results of Gluc activity detection show that two truncations of IPAN, IPAN-IS1 and IS2 (two homologous sequences of three transcripts of IPAN) can not promote IAV replication (FIG. 2).

Primer sequences for detecting NP mRNA in D in fig. 2: f, 5'-GGGTCAGTTGCTCACAAGTCC-3'; and R, 5'-TTGAAGCAGTCTGAAAGGGTCT-3'.

Specific reverse transcription primers and qPCR primer sequences for detection of three different viral RNA products (v/c/mRNA) in E in FIG. 2 (from Kawakami et al, Strand-specific real-time RT-PCRfor distingizing influenza vRNA, cRNA, and mRNA. J Virol methods.2011April; 173(1): 1-6):

the NP vRNA is a reverse transcription primer,

5’-GGCCGTCATGGTGGCGAATGAATGGACGGAGAACAAGGATTGC-3’,F，

5'-CTCAATATGAGTGCAGACCGTGCT-3' and R, 5'-GGCCGTCATGGTGGCGAAT-3';

NP cRNA reverse transcription primer, 5' -GCTAGCTTCAGCTAGGCATC

AGTAGAAACAAGGGTATTTTTCTTT-3 ', F, 5'-CGATCGTGCCCTCCTTTG-3' and R,

5’-GCTAGCTTCAGCTAGGCATC-3’；

the NP mRNA is a reverse transcription primer which is a reverse transcription primer,

5’-CCAGATCGTTCGAGTCGTTTTTTTTTTTTTTTTTCTTTAATTGTC-3’,F，

5'-CGATCGTGCCCTCCTTTG-3' and R, 5'-CCAGATCGTTCGAGTCGT-3'.

Example 3 IAV infection promotes expression and Nuclear transport of IPAN

To examine the effect of IAV infection on IPAN expression and localization, A549 or 293T-Gluc cells were cultured at 2X 10⁵Cells were seeded at individual/ml density in six-well plates or glass dishes and 24h later were infected with IAV at different MOI or with various other viruses (negative controls were uninfected and inactivated WSN-infected cells), or treated with IFN- α (1000U/ml, Biovision) for A549 and varying amounts of poly (I: C) (Sigma-Aldrich) were transfected into 293T-Gluc cells. Expression levels of IPAN were determined using the same RT-qPCR method described above. The subcellular localization of IPAN was examined and the fluorescence signal statistically analyzed according to the QuantiGene ViewRNA ISHcell assay kit instructions (Affymetrix). The results show that WSN infection can up-regulate IPAN levels by more than 5-fold and is virus dose-dependent, while IFN-alpha and poly (I: C) have little effect on it. WSN infection can facilitate nuclear transport of IPAN.

Example 4 interaction of IPAN with viral PB1 protein

293T-Gluc cells are respectively transfected with PA, PB1, PB2 and NP expression plasmids, and the combination condition of endogenous IPAN and virus protein is detected by utilizing an RNA binding protein immunoprecipitation (RIP) technology 48 hours later, wherein the specific flow is as follows: after washing the cells twice with PBS, the cells were fixed with PBS containing 1% formaldehyde for 10min, lysed with lysis buffer (50mM HEPES, pH7.5,400mM NaCl,1mM EDTA,1mM DTT, 0.5% Triton X-100, 10% Glycerol) and sonicated (10 sec/pulse, 10 pulses). Magnetic beads Protein G (Invitrogen) were mixed with IgG or the corresponding antibody at room temperature for 2 hours, added to the cell lysate and incubated overnight at 4 ℃. The magnetic beads were washed five times with lysis buffer, after de-cross-linking at 70 ℃ RNA was extracted with Trizol LS (Invitrogen), and IPAN and GAPDH levels were detected using RT-qPCR. The results show that PB1 protein can significantly enrich IPAN compared to viral PA, PB2 and NP proteins (fig. 4).

293T cells were cotransfected with three different transcripts of PB1 and IPAN, and Native RIP technology was used to detect binding of exogenous IPAN to the viral protein PB 1. The specific process of Native RIP is as follows: the collected cells were lysed with lysis buffer (25mM Tris, pH 7.4,150mM NaCl, 1% NP-40,1mM EDTA, 5% glycerol,100U/ml RNase inhibitor and protease inhibitor cocktail reagent) for 1h at 4 ℃. Magnetic beads Protein G (Invitrogen) were mixed with IgG or the corresponding antibody at room temperature for 2 hours, added to the cell lysate and incubated overnight at 4 ℃. The beads were washed five times with PBST (0.02% Tween20) and RNA was extracted with Trizol LS (Invitrogen) and IPAN and GAPDH levels were detected by RT-qPCR. The results showed that the three transcripts of IPAN were all enriched by a factor of 4-6 compared to the negative control IgG group, and IPAN-IS2 was not enriched, indicating that IPAN was able to specifically bind to PB1 (FIG. 4).

A549 cells were infected with A/WSN/33(H1N1) (MOI 0.5) for 36H, and then binding of IPAN RNA to the viral Protein PB1 was detected using the Magnetic RNA-Protein Pull-Down Kit (Pierce). The results show that, in addition to the M2 protein, each of the components PB1, PA, PB2 and NP of the viral RNP complex can be co-precipitated, indicating that IPAN binds to RdRp via PB1 during IAV infection and thus performs its function (fig. 4).

Example 5 silencing of IPAN inhibits transcriptional replication of influenza RNA, and IPAN stabilizes the PB1 protein of IAV

In order to determine the action link of the IPAN participating in the IAV replication, the influence of the IPAN down-regulation on the virus replication is detected by using IAV A/WSN/33(H1N1) infection (293T-Gluc) and an IAV replication subsystem (A549-5Ps) at the same time. The results show that IPAN silencing down-regulates Gluc activity to a similar extent in both systems, around 75%. At the same time, the mRNA and vRNA levels of the viral NP gene were found to be dose-dependent with esiIPAN. Taken together, esiIPAN-mediated silencing of IPAN was concluded to inhibit transcription and replication of viral RNA, suggesting that IPAN promotes IAV replication by regulating transcriptional replication of viral RNA (fig. 5).

Further, the effect of IPAN silencing on the expression of the relevant viral proteins was examined by Western Blot using the above-described transcriptional replicon system. As a result, ipa silencing was found to significantly reduce PB1 protein levels. And the mRNA level of PB1 was detected by RT-qPCR, demonstrating that IPAN silencing mediated down-regulation of PB1 protein levels is not the result of PB1 self mRNA transcriptional inhibition. We therefore speculate that this may be due to IPAN affecting the stability of the PB1 protein at the post-translational level. To verify this hypothesis, the levels of PB1 protein were monitored at different time points (0h, 4h, and 8h) using the protein synthesis inhibitor CHX (100mg/ml, Sigma-Aldrich) to block intracellular protein synthesis after transfection of esiIPAN and esiEGFP by A549-5Ps cells. The Western Blot results showed a faster rate of PB1 protein degradation in esiIPAN transfected cells compared to esiEGFP controls, indicating that ipa silencing promotes PB1 protein degradation. To further determine the degradation pathway of PB1, the PB1 protein level was detected by Western Blot after treating cells with proteasome inhibitor MG132(10 μ M, Sigma-Aldrich) for 8 h. The results found that MG132 treatment of cells almost completely blocked the effect of IPAN silencing on PB1 protein levels, indicating that esiIPAN-mediated PB1 protein degradation is accomplished via the proteasomal pathway (fig. 5).

Since IPAN silencing can induce PB1 protein degradation, we will further investigate whether IPAN is involved in IAV replication by modulating PB1 protein levels. To this end, we transfected esiIPAN into 293T-Gluc cells while overexpressing PB1 plasmid, analyzed protein levels of virus PB1 by Western blot 24H after infection with a/WSN/33(H1N1) (MOI ═ 0.5), and quantified infectivity of the virus by measuring Gluc activity. The results show that the replication capacity of the virus is significantly increased when PB1 protein is recruited in IPAN-knockdown cells, indicating that IPAN is involved in IAV replication by modulating PB1 protein levels (fig. 5).

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

Sequence listing

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<211> 2179

<212> DNA

<213> human (Homo sapiens)

<400> 3

tgggattctg ggcagttggg actttctttt caggaaagga ggtgagaggt gccggggaag 60

cccacacgag cagccactct tggagaaatg gggaatggcg ctgtaaaagc gcaggccctc 120

ctcctcctcc tttctcccca tcacacaaaa aggatgggcc ttgaaaggga tgagtttatt 180

atgcactttt aaatgcctat tcagaaaaga acaggaggca aaaataaaga tgcttccacc 240

tccacaatgg tttttgttta aaataaaggt catgattgtt tcccattatt acagaatgga 300

aaccgatcct agacatctgt gaaacgtatc ttaaaaattc cactactgag ttgggaaaag 360

cagagagttc atgtttactt cattggcgaa gtccagttga atctacttaa ctcgaccagc 420

tttaatccaa tctaataaac catgagcttg ttcctgtgtc aaaaaaaaaa aaaaaaaaaa 480

aagccaatat gctgccctga gcctgtgaaa gttgtcagaa acaaaatgga gtcacttgtg 540

ttaaaaaatt aattaattaa ttaattaaaa gaaccctgac aaatatagct gcagaaggct 600

atgaaaaaaa aggttctcat gcataaatgc ctgataacaa aaactatcac aaaagaccct 660

gcaaaaacca cagtccttgc tcaaaaatga ttacaacctt acacaaaaaa tacttcagca 720

agaacatccg cccagtgact gcctatccaa ccttcgactg gcatcaccct tgctattgtt 780

atgggtcttc atagccaagg ataattatct caaaacaatg atataatcct catttttcct 840

ttaaaagcct ttgtctttct ttacctccct gcatgcctgt agtttactat ggcatgtgta 900

ttcccattcc aatgctctgt tcccaaataa atgtaatttc cttttagaca gcctctctct 960

gttagttact taggctgata gccccaaatt ccatctttcg cattgccatg tcatcaaata 1020

aacctgtgat tttcttgttt ttgctaagca aatggcttgt gttgtggtta gtgtctctag 1080

tgtaacaatc actttgtgtt tccctgactc tcctaagtac aaagaaatag gaacccccgc 1140

actttatgtg ggaaaaaaac agagactgaa aaattaatta actgcccact ctcacatggg 1200

tctaccacca tcttccaggt ctttctttcc agacctgtgg tctctcccca cctccagcct 1260

cttatggaga gcaaaggaag gaaatgggaa caggactcaa cagcaacatt ccaggaaaat 1320

gaatagaatg tgggaacggt tcgaaatgat aaatccaggt gatagaacga gtttcaaaga 1380

gcaagagcct gatggtaata gggaagccag gggaacggca gaggttgtgc agaagacgac 1440

tctggcaaaa taaggactgt ggagagaaag taagtggctt ttaagatctt aaacatgggg 1500

tcaaaattat accatgagaa gaaagaaaaa gtccctggga gtgtttgtag acagaccaaa 1560

ggacagaacg tgaagaaggg cctatgagag aattggtatc cggaagattg tttatgtgca 1620

acacttctga ggaataaagg gtgataaact gtctcgtgag aatgaactac tggaaggacg 1680

gagagacaca aggaatggct gcagtcaagg tgaaatgtag gccgttgtga gagtttcatc 1740

ccagggcatg atgagaatgc cagggcctga tgcgtgtcaa cggtgcttca tctgctcaga 1800

gagtgtccta aaaccagtca gaccctagat gtagaagtat gccacccaat gtcacctagc 1860

agttcaatgc cagggagaga aaattcaggc tttcttatcc cccttctagt gcaagcccat 1920

ggaatatgcc actttcctag ttaggatttg gaattggtca ttgggaaggg ctccgaagag 1980

aaactctgag aattgttgat ttccatttca aatcccccta atgcataaaa taagaatgtg 2040

ttctgcggtg ttcctcagct tattaacttt tataaaagaa aggaatgatc tttgttgtta 2100

cttgaaaaca gccacatttg gaaaggtcta cgctacccaa atgtaataaa aataaatttt 2160

agttcaattg gtcagtctt 2179

<210> 4

<211> 2232

<212> DNA

<213> human (Homo sapiens)

<400> 4

gcagctgccg ctggggctgc cgcgggcgcc gctcagccgg agcgcgcctc cgcccggccc 60

gcccgcgccc cgcagcagcg cgctcgctcc ttaaccactt cgccgctgcc tttcgtcggg 120

gccgcgagcg gggcgacggc cgcccagcgc ccctggcacc cctgatctcc agccggggct 180

cccggcccgc gccttggtgg ctggaggccc aggatgagcc tcgatggcga gaggcccagg 240

atgcgccacc aggccgggct cacttctttg acgccgaata aactgacaaa acaattagct 300

ggccggctgg ggaaggtggt gggtgactca aatgccagtc ggctgcgtgc ccccgcgctt 360

agatgagtct gggattctgg gcagttggga ctttcttttc aggaaaggag gtgagaggtg 420

ccggggaagc ccacacgagc agccactctt ggagaaatgg ggaatggcgc tgtaaaagcg 480

caggccctcc tcctcctcct ttctccccat cacacaaaaa ggatgggcct tgaaagggat 540

gagtttatta tgcactttta aatgcctatt cagaaaagaa caggaggcaa aaataaagat 600

gcttcccacc ccaccaattc ccttccctcg aagaaaaaag gaaagagaaa gcaacccaca 660

aaggtctttc tttccagacc tgtggtctct ccccacctcc agcctcttat ggagagcaaa 720

ggaaggaaat gggaacagga ctcaacagca acattccagg aaaatgaata gaatgtggga 780

acggttcgaa atgataaatc caggtgatag aacgagtttc aaagagcaag agcctgatgg 840

taatagggaa gccaggggaa cggcagaggt tgtgcagaag acgactctgg caaaataagg 900

actgtggaga gaaagtaagt ggcttttaag atcttaaaca tggggtcaaa attataccat 960

gagaagaaag aaaaagtccc tgggagtgtt tgtagacaga ccaaaggaca gaacgtgaag 1020

aagggcctat gagagaattg gtatccggaa gattgtttat gtgcaacact tctgaggaat 1080

aaagggtgat aaactgtctc gtgagaatga actactggaa ggacggagag acacaaggaa 1140

tggctgcagt caaggtgaaa tgtaggccgt tgtgagagtt tcatcccagg gcatgatgag 1200

aatgccaggg cctgatgcgt gtcaacggtg cttcatctgc tcagagagtg tcctaaaacc 1260

agtcagaccc tagatgtaga agtatgccac ccaatgtcac ctagcagttc aatgccaggg 1320

agagaaaatt caggctttct tatccccctt ctagtgcaag cccatggaat atgccacttt 1380

cctagttagg atttggaatt ggtcattggg aagggctccg aagagaaact ctgagaattg 1440

ttgatttcca tttcaaatcc ccctaatgca taaaataaga atgtgttctg cggtgttcct 1500

cagcttatta acttttataa aagaaaggaa tgatctttgt tgttacttga aaacagccac 1560

atttggaaag gtctacgcta cccaaatgta ataaaaataa attttagttc aattggtcag 1620

tcttgaaagc taattgaaca gtcatgcttt gttctaatta ttcctggcta ccattaaagt 1680

gatggaaaag taagtacgtt tttattgcca actccttggg tttttttctc tttttttaat 1740

ttgtttgttt tgtttttaaa taagactaac cagtttgggc aaggacctaa tgctctctca 1800

gcaggtaaat tttgcaacaa tgtttctgaa accctgggtt ctgggatgcc gtgtgggtct 1860

tcatcacttg caaaagctgt actcatggag cctgaaatct agctgaattc tgcatgggta 1920

caaaatatgg tgttctggtt gactggatac catgattaac tgatggtgac aacacacatc 1980

ataaaccatt gtctgattct taaaaaattc aaagatcgtt aaaggagagt tagatgccag 2040

ctcatcttac acagagcatc aggcagaaat tgttggtaat ctggcaaatg aaactactca 2100

tatcctatga tgttcggttt gtttatagac tgaatttgca cataatcgag agttatttcc 2160

tcacaaataa tttatctcta atgaataatg atgtctttgt tgtgagctca tggtccaaat 2220

agcaagtttc cc 2232

Claims (7)

1. The application of a substance for inhibiting the expression of the IPAN gene in a cell in preparing an influenza virus inhibitor is characterized in that the substance for inhibiting the expression of the IPAN gene in the cell is esiRNA, and the influenza virus is influenza A virus.

2. Use according to claim 1, characterized in that: the influenza virus inhibitor is an influenza virus replication inhibitor.

3. The application of a substance for inhibiting the expression of the IPAN gene in a cell in preparing a product for treating influenza virus infection or/and preventing the influenza virus infection, wherein the substance for inhibiting the expression of the IPAN gene in the cell is esiRNA, and the influenza virus is A-type influenza virus.

4. The application of a substance for inhibiting the activity of IPAN in cells in preparing an influenza virus inhibitor is characterized in that the substance for inhibiting the activity of IPAN in cells is esiRNA, and the influenza virus is influenza A virus.

5. The application of a substance for inhibiting the activity of IPAN in cells in preparing a product for treating influenza virus infection or/and preventing the influenza virus infection is disclosed, wherein the substance for inhibiting the activity of IPAN in cells is esiRNA, and the influenza virus is A-type influenza virus.

6. The application of a substance for reducing the content of IPAN in cells in preparing an influenza virus inhibitor is characterized in that the substance for reducing the content of IPAN in cells is esiRNA, and the influenza virus is influenza A virus.

7. The application of the substance for reducing the IPAN content in the cells in the preparation of products for treating influenza virus infection or/and preventing the influenza virus infection is disclosed, wherein the substance for reducing the IPAN content in the cells is esiRNA, and the influenza virus is influenza A virus.

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