Identification And Functional Analysis Of A Host Long Non-coding RNA Involved In Influenza Virus Infection And Replication

Influenza virus infection poses a significant threat to world health,but the mechanisms underlying IAV-host interaction are still elusive.Host anti-IAV innate immune response is initiated by the recognition of viral components by pathogen recognition receptors(PRRs),such as RIG-I,MDA5 and TLR3.Through the signaling cascade downstream the stimulated receptors,transcription factors including IRF3/7 and NF-?B are activated.Type ? and ? interferons(IFNs)are then rapidly produced,which induce the synthesis of hundreds of anti-viral proteins encoded by IFN-stimulated genes(ISGs).The influenza virus relies on the host factors and cellular machinery to complete its life cycle.In recent years,a growing number of studies show that host factors play an important role in the process of influenza virus infection and replication.So,understanding the role of the host factors in the process of influenza virus infection and replication will help us to develop more effective anti-viral drugs.Long non-coding RNAs(IncRNAs)are RNA polymerase ?(RNA Pol ?)or ?(RNA Pol III)transcripts that lack functional activity to encode functional proteins and polypeptides and are longer than 200 nucleotides.LncRNAs play an important role in cellular regulation by some mechanisms,including RNA-RNA,RNA-DNA or RNA-protein interaction,et al.LncRNAs have been recognized as an important new class of RNA regulators involved in various biological processes,such as chromatin modification,gene transcriptional regulation,RNA splicing and stability modulation.Here we have performed studies as described below:1.First,using the microarray and bioinformatics(ORF Finder and other softwares)we analysed and screened the long non-coding RNAs in IAV infected cells.RT-PCR analysis confirmed significantly differentially expressed lncRNAs in infected cells.We employed the plaque forming assay to identify a functional IncRNA termed NRAV(Negative Regulator of Anti-Viral response).2.The expressions of NRAV in many human cell lines were detected by RT-PCR.Its expression was dramatically reduced following IAV infection.RT-PCR confirmed that infection of IAV,Sev,MDRV and HSV down-regulated NRAV.Using pseudovirus,LPS and serum-free media to treat the cells,we found that expression level of NRAV was not changed by cellular stress.The results indicate that the reduction of NRAV level is associated with viral infection.3.The efficiency of NRAV overexpression and knockdown were examined in A549 and 293T cells.Plaque forming assay showed that overexpression of NRAV promoted IAV replication,while silence of NRAV inhibited virus replication.4.With three software RNAfold,Centroidfold and Genebee we predicted secondary structure of NRAV,subsequently designed eight truncated and/or deletion mutants and predicted their secondary structures,respectively.Their stabile expressing cell lines were constructed.Plaque forming assay showed that RNA sequences of stem-loops in NRAV except nt 618-872 might form a spatial structure that is essential for its function.5.We used various factors(such as virion genomic RNA,Viral RNA,Cell RNA,Poly(I:C),cytokines,silence of RNA-pattern recognition receptors and decitabine(DAC))to deal with infected or uninfected cells.Transfection with viral RNA caused notable reduction in NRAV expression.Virus-mediated down-regulation of NRAV is reversed by the DAC-mediated expression.Other treatments had no effect on NRAV expression.The data indicate that down-regulation of NRAV is caused by virus replication,protein synthesis and methylation.6.Functional experiments showed that NRAV promoted IAV replication.Many studies have shown that IFN-stimulated genes(ISGs)can inhibit virus replication.Here,qRT-PCR results confirmed that the expression levels of multiple critical interferon-stimulated genes(ISGs)were up-regulated,including IFIT2,IFIT3,IFITM3,OASL and MxA in NRAV kncokdown cells.Our data suggest that NRAV negatively regulates the transcription of multiple critical interferon-stimulated genes(ISGs),including IFIT2,IFIT3,IFITM3,OASL and MxA in human cells.These findings provide novel functional insights into the complex mechanisms underlying the regulation of anti-viral innate immune response.Host non-coding RNAs have critical functions in controlling IAV infection and replication.Therefore,this study has important scientific significance.