Characteristics Of Circular Noncoding RNA That Modulate Influenza Virus Replication And Its Mechanism Basis

Influenza A virus is an enveloped,single-stranded,negative-sense RNA virus,which belongs to the orthomyxovirus family.The worldwide prevalence of influenza A virus causes major public health security problems and poses severe threat to the survival and development of human.The interaction between cellular factors,including host proteins and non-coding RNAs(nc RNAs)and influenza virus plays roles in the replication and pathogenicity of influenza virus.Thus,identification of these host factors and their underlying mechanisms can provide important insights for the development of strategies to inhibit viral infection.Previous studies have shown that a number of host proteins interact with different influenza virus proteins to promote or inhibit viral replication at different stages of the virus life cycle.Circular RNAs(circ RNAs),a new member of the long non-coding RNA families,have been identified in a variety of organisms including plants,animals,and humans.Circ RNAs are generated from pre-m RNA through the process of back-splicing,and are categorized into exonic circ RNAs,exon-intron circ RNAs,intronic circ RNAs,intergenic circ RNAs,and antisense circ RNAs according to their sequences.Circ RNAs play an important role in cellular regulation by some mechanisms,including interacting with mi RNA or protein and translation to produce peptides,which are involved in various biological processes,such as gene transcriptional regulation,RNA splicing and protein transport.In comparison to cellular proteins,the effects of nc RNAs on influenza virus replication are not well understood.To investigate whether circ RNAs regulate influenza virus replication,we detected 11,620 distinct circ RNAs(> 200 nt)in A549 cells by deep-sequencing and bioinformatic analysis,and 411 of them were differentially expressed in influenza virus-infected A549 cells.We then selected six circ RNAs that were abundant in A549 cells and highly differentially expressed after viral infection,and verified that four circ RNAs were upregulated and two circ RNAs were downregulated by q RT-PCR after A549 cells were infected with H9N2 virus.Sanger sequencing results further verified their circular properties and RT-PCR results showed that the expression levels of these six circ RNAs were viral dose-and infection time-related.By using gene silencing and overexpression experiment,we further found a novel intronic circ RNA could significantly antagonized influenza virus replication in A549 cells and we named it AIVR(Antagonizes influenza virus replication).To investigate the mechanism of how AIVR inhibit the influenza virus replication,we first found that AIVR predominantly localized in the cytoplasm by fluorescence in situ hybridization analysis and subcellular fractionation experiment.By circ RNA precipitation and dual luciferase reporter assay,we further indicated that AIVR worked as a micro RNA(mi RNA)sponge.We found that hsa-mi R-330-3p,one of the mi RNAs absorbed by AIVR can facilitate influenza virus replication in A549 cells.In addition,we found hsa-mi R-330-3p bound the m RNA of CREBBP,which is an important component of the nucleoprotein complex IFN-β enhanceosome and degraded the m RNA of CREBBP and inhibited its expression.We deeply found that AIVR could absorb hsa-mi R-330-3p and then promoted the expression of CREBBP.Furthermore,AIVR-overexpression significantly increased the levels of INF-βin the influenza virus-infected A549 cells.In summary,the newly identified intronic circ RNA-AIVR is upregulated upon influenza virus infection and is an important part of cellular innate IFN-β antiviral response.Our study provides new insights into the roles of circ RNAs in the cellular innate antiviral response.