Long Noncoding RNA TSPOAP1-AS1 Facilitates Influenza A Virus Replication By Negatively Modulates Type Ⅰ IFN Antiviral Innate Immune Response

Influenza A Virus(IAV)is an orthomyxoviridae virus that also seriously endangers the health and safety of humans and susceptible animals.The innate immunity provides the body’s first line of defense against the invasion of pathogens.Innate immune cells have complex signaling pathways for sensing pathogens and initiating antiviral innate immune responses.Long non-coding RNA(lncRNA)is a type of RNA transcript that does not encode proteins.Viruses can use the host lncRNA to manipulate the cellular gene expression and reshape the intracellular environment to suit their needs,including those facilitating viral replication and antagonizing host antiviral defense mechanisms.Preliminary data on transcriptome profiling of IAV-infected A549 cells suggest that IAV infection significantly induces the expression of host lncRNA TSPOAP1-AS1.To investigate the effects of TSPOAP1-AS1 on IAV infection and its mechanism,we conducted the following research work.Firstly,we infected A549 cells with the different IAV strains and the different MOIs,and detected the expression of host TSPOAP1-AS1,Ifnb1 and IAV M gene at different time points.The results showed that the expression of TSPOAP1-AS1 was significantly induced by IAV infection,which was accompanied by increased expression of IAV M and Ifnb1 mRNA.We then detected the expression of TSPOAP1-AS1 in the selected human cell lines from different organs or tissue types.The results showed that TSPOAP1-AS1 was expressed in all examined human cell lines.Next,we investigated the subcellular localization of TSPOAP1-AS1.The results confirmed that TSPOAP1-AS1 existed in both nuclear and cytoplasmic fractions,and an increased nuclear/cytoplasmic ratio of TSPOAP1-AS1 was present in A549 cells infected with IAV.Secondly,we investigated whether viral RNA was a critical factor responsible for the induction of TSPOAP1-AS1.We tested TSPOAP1-AS1 expression in A549 cells response to polyinosinic: polycytidylic acid(poly I:C),lipopolysaccharides(LPS),cisplatin and serum withdrawal.The results showed that TSPOAP1-AS1 expression was strongly induced by poly(I:C)in a dose-dependent manner.By contrast,TSPOAP1-AS1 levels were not affected by LPS stimulation,cisplatin treatment,or serum withdrawal.These data suggest that increment of TSPOAP1 ‐ AS1 level is associated with viral RNA accumulation during replication.Next,we used pharmacological(pathway inhibitor)and genetic(overexpression plasmids and siRNAs)approaches to confirm that TSPOAP1-AS1 induction by IAV is mediated by the activation of NF-κB pathway.Finally,we explored the role of TSPOAP1-AS1 in IAV infection and its mechanisms.We transfected A549 cells with pcDNA 3_TSPOAP1-AS1 plasmid and TSPOAP1-AS1 siRNA respectively and then detected viral replication(mRNA,protein and infectious progeny virions)and cellular antiviral innate immune response(ISRE luciferase reporter assay,Ifnb1 and ISGs expression).The results showed that overexpression of TSPOAP1-AS1 remarkably promoted IAV replication.In contrast,suppression of endogenous TSPOAP1-AS1 by siRNA significantly inhibited IAV replication.In addition,overexpression of TSPOAP1-AS1 repressed IAV ‐induced ISRE promoter activation,Ifnb1 and ISGs mRNA transcription.Conversely,the presence of TSPOAP1 ‐ AS1 siRNA significantly promoted ISRE promoter activity,and the transcription of Ifnb1 and ISG.These results indicate that TSPOAP1-AS1 promotes IAV replication and represses antiviral innate immune response.Taken together,we identify a new function of TSPOAP1-AS1 that facilitate IAV replication by suppressing host antiviral innate immune responses.Our data provides evidence for the host lncRNA utilized by viruses to support its replication.