Mechanistic Studies Of Long Non-coding RNA IVRPIE In The Anti-influenza Virus Response

Influenza virus poses a serious threat to human health,causing nearly one billion infections and hundreds of thousands of deaths each year.The high mutation rate and gene recombination in the genome of influenza virus lead to the continuous production of new strains,which evade the body's existing adaptive immunity and cause the partial failure of existing prevention and control measures.The influenza virus mainly invades the body's respiratory system,and the disease can develop into acute respiratory distress syndrome(ARDS),resulting in death.The innate immune response plays an important role in fighting influenza virus,but cytokine storm caused by the excessive immune response is an important cause of acute lung injury.Therefore,further in-depth study of the immune mechanism of influenza virus is crucial to more effective prevention and control of influenza virus.At present,most of the studies on the immune mechanism of influenza virus focus on protein molecules,but few on the role of RNA molecules.Long non-coding RNA(Lnc RNA)is a new type of non-coding RNA discovered in recent years,which plays an important role in various cellular physiological processes such as development and differentiation,cancer,cardiovascular diseases,immune response.Although a few lnc RNAs have been reported to participate in antiviral immunity,there are still a large number of lnc RNAs whose function is unknown,and their role in the immune response of influenza virus needs further study.Therefore,to explore the role and mechanism of lnc RNA in anti-influenza virus immunity will help to further understand the immune response of the body to influenza virus,and provide theoretical basis for the development of new anti-influenza virus methods and drugs.Based on the research background mentioned above,this project will study the role of lnc RNAs in innate immunity against influenza virus infection.This topic is divided into the following four parts:Part ?: lnc RNA IVRPIE is significantly highly expressed after IAV infection and inhibits IAV replicationThrough the analysis of transcriptome sequencing data of peripheral blood leukocytes in flu patients in the previous work of our laboratory,five upregulated lnc RNAs and five downregulated lnc RNAs were selected and their expressions were validated in A549 cells through the RT-q PCR method.These lnc RNAs were constructed into the eukaryotic expression vector pc DNA3.1(+)to study the effect of overexpression of lnc RNAs in A549 cells on influenza virus replication.This approach identified XLOC?026516(named as Inhibiting IAV Replication by Promoting IFN and ISGs Expression,IVRPIE)as the strongest inhibitor for influenza virus replication.Samples of A549 cells infected with influenza virus were collected at different times or A549 cells were infected with influenza virus at different doses.The expression of IVRPIE was determined by RT-q PCR,and it was found that the expression of IVRPIE was time-dependent and dose-dependent.Then,IVRPIE expression was detected in various cells infected with influenza virus,and it was found that IVRPIE was mainly highly expressed in human lung epithelial cell lines-A549 cells and BEAS-2B cells.A549 cells and BEAS-2B cells were inoculated with different viruses and preparations,and it was found that in addition to IAV,Se V VSV infection and polyinosinic-polycytidylic acid(poly I:C,TLR3 and MDA5 ligand)stimulation also induced significant high expression of IVRPIE.These results indicate that IVRPIE is related to replication of certain RNA viruses.However,stimulation of A549 cells with interferon-?(IFN-?)did not promote significantly high expression of IVRPIE,indicating that IVRPIE is not an interferon-stimulated gene.RACE experiments confirmed that IVRPIE was a 1316 nt transcript located in the promoter region of the TRAF family member associated NF-?B activator(TANK)gene.Experiments using inhibitors of cell signaling pathway indicate that the expression of IVRPIE is not regulated by NF-?B,TBK1 or IKK-?.Part II: IVRPIE plays an important role in antiviral immunity by promoting the expression of interferon ?1(IFN?1)and several critical ISGsIn order to further verify the effect of IVRPIE on influenza virus replication,after overexpression or knockdown of IVRPIE in A549 cells,the virus titer were determined by HA assay and PFU assay,and the expression of IAV HA protein was detected by western blotting.The results further confirmed that IVRPIE could inhibit IAV replication.In order to understand the mechanism of IVRPIE inhibiting IAV replication,several experiments were carried out.Results of RT-q PCR indicate that IVRPIE does not affect the expression of adjacent TANK gene.The results of western blotting showed that IVRPIE did not affect the expression and phosphorylation of transcription factor IRF3 and NF-?B in the RIG-I antiviral signaling pathway.Through RT-q PCR and western blotting,we observed that IVRPIE promoted the expression of IFN?1 and several critical ISGs,including interferon regulatory factor 1(IRF1),interferon induced protein with tetratricopeptide repeats 1(IFIT1),interferon induced protein with tetratricopeptide repeats 3(IFIT3),myxovirus resistance protein 1(MX1),interferon-stimulated gene 15(ISG15)and interferon induced protein 44 like(IFI44L)to enhance host antiviral response.Part ?: IVRPIE promotes the expression of IFN?1 and ISGs,likely through regulating histone modifications of the ISGs by interacting with heterogeneous nuclear ribonucleoprotein U(hn RNP U)In an attempt to define the mechanism of IVRPIE regulating the expression of IFN?1 and ISGs,we determined the cellular localization of IVRPIE through subcellular fractionation.We found that IVRPIE was mainly distributed in the nucleus,so we hypothesized that IVRPIE might exert its function through regulation of gene transcription.Using the Ch IP-q PCR assay,we observed that IVRPIE significantly increased the level of histone 3 lysine 4 trimethylation(H3K4me3)and decreased the level of histone 3 lysine 27 trimethylation(H3K27me3)at the transcription start sites of IFN?1 and ISGs,thereby promoting the expression of these genes.To identify the protein partners of IVRPIE,we performed RNA pull-down.By analyzing the results of mass spectrometry(MS),we presumed that hn RNP U might be an interaction partner of IVRPIE.The interaction between hn RNP U and IVRPIE was further confirmed by western blotting and RIP experiments.In order to verify the role of hn RNP U in the expression of IFN?1and ISGs,we performed hn RNP U knockdown in IVRPIE-overexpressing cells,and the expression of IFN?1and ISGs was determined by RT-q PCR and western blotting.Compared with the control group without knocking down hn RNP U,the expression of IFN?1and ISG was significantly decreased.This indicates that hn RNP U and IVRPIE interact to regulate the expression of IFN?1 and ISGs.Part ?: IVRPIE inhibits virus replication in vivoIn order to verify the role of IVRPIE in vivo,IVRPIE was constructed into the nucleic acid vaccine expression vector p VAX1,which was delivered into the lungs of mice through nasal drip,to overexpress IVRPIE in the lungs of mice.The IVRPIE-overexpressing mice infected with IAV were found to have decreased mortality and weight loss compared with control mice.PFU assay was performed to detect the virus titers of mouse lungs,and we found that the virus titer was decreased after overexpression of IVRPIE in mouse lungs,indicating that IVRPIE had an inhibitory effect on IAV replication in vivo.Further wet/dry lung ratio test and pathological observation showed that IVRPIE reduced pulmonary edema and pulmonary inflammation in mice after IAV infection.The levels of inflammatory cytokines,including IL-1?,IL-6 and TNF-? in BALF were detected by ELISA,and it was found that overexpression of IVRPIE could reduce the levels of inflammatory cytokines in BALF,which further indicates that IVRPIE reduces the inflammatory response in the lungs of mice infected with influenza virus.In summary,we screened and obtained a brand new lnc RNA IVRPIE.Functional studies indicate that IVRPIE inhibits the replication of certain RNA viruses such as IAV and VSV by promoting the expression of IFN?1 and several critical ISGs including IRF1,IFIT1,IFIT3,MX1,ISG15 and IFI44 L.In terms of the molecular mechanism,IVRPIE regulates the expression of IFN 1 and ISG by modulating chromatin conformation through interaction with hn RNP U.Consistently,IVRPIE inhibited IAV replication in mice,and reduced lung inflammation and mortality in mice.This study provides a basis for the development of new antiviral agents.