| Influenza A virus is an RNA virus with a single strand,a negative strand,segments,and with enveloped.It is a member of the Orthomyxoviridae family.The influenza A virus has become one of the primary objectives of prevention and control for public health safety and is also a major focus of virology study since it is simple to mutate,challenging to prevent and manage,and responsible for many historical large-scale influenza epidemics.With the continuous research,the infection mechanism of influenza virus has been studied more at the protein level,but whether the virus can generate circular RNA(circRNA)and its biological function has not been reported.As a result,this study was carried out to investigate the circRNA of the influenza A virus,and the findings were as follows:1.Based on the bioinformatics predictions,we tested potential viral circRNAs and discovered that the negative-stranded RNA expressing the H1N1 nucleoprotein(NP)gene generate circNP37 via divergent primer amplification.The circular structure of this circRNA was further confirmed by rolling circle amplification,ribonuclease R(RNase R)digestion assay,full circle sequence amplification,and non-trans backsplice product verification assay.2.To assess the biological role of circNP37 in virus infection,circNP37 deletion virus was rescued,and infection tests were performed to determine the influence of circNP37 on influenza virus replication.The results demonstrated that circNP37 deletion reduced viral replication,transcription,and progeny virus titers,and that circNP37 overexpression plasmid restored viral replication after returning to circNP37.Furthermore,circNP37 oligonucleotide knockdown revealed a decrease in viral replication following circNP37 reduce.It is clear that circNP37 promotes viral replication.3.To elucidate the mechanism by which circNP37 exerts its biological effects,we first discovered that circNP37 was primarily localized in the cytoplasm during viral infection using nucleoplasmic segregation experiments,and hypothesized that circNP37 might function by interacting with micro RNAs(miRNAs);then we predicted that 7 miRNAs interacting with circNP37,and it was discovered that four of the miRNAs exhibited elevated expression in the absence of circNP37 viral infection,and that circNP37 could pull down two of the four miRNAs in the circRNA pull-down assay.Finally,a dual luciferase assay revealed that hsa-miR-361-5p in two different miRNAs had a direct targeting effect on circNP37;finally,the effect of hsa-miR-361-5p on circNP37 was studied under viral infection and non-infection conditions,and circNP37 was found to be inhibited by hsa-miR-361-5p;and further validation of the critical binding site for the interaction of circNP37 with miR-361-5p by p LC5 GFP fluorescence changes.4.To further investigate the mechanism of the effect of hsa-miR-361-5p on viral replication,we firstly predicted the possible viral m RNAs targeted by hsa-miR-361-5p by bioinformatics and verified by miRNA pull-down and m RNA pull-down,and found that hsa-miR-361-5p targeted PB2-m RNA.The effect on PB2 expression was investigated by knocking down and overexpressing hsa-miR-361-5p under viral infection and transfection of PB2 expression plasmid alone,and it was discovered that hsa-miR-361-5p could reduce PB2 protein expression.5.To confirm the relationship between circNP37/hsa-miR-361-5p/PB2-m RNA,hsa-miR-361-5p was up-and down-regulated under wild-type(WT)virus infection and non-infection conditions to examine PB2 transcription and translation,and the presence of hsa-miR-361-5p inhibited circNP37’s promoter effect on PB2 expression.The data imply that circNP37 enhances PB2-m RNA translation during virus infection by sponging hsa-miR-361-5p,thereby increasing virus replication.In summary,this work identified the presence of influenza virus circRNA for the first time and proved its role and mechanism in the viral infection process. |
