| Influenza A viruses(IAV)primarily targets respiratory epithelial cells and produces clinical outcomes ranging from mild upper respiratory infection to severe pneumonia. Micro RNAs(mi RNAs), a family of small noncoding RNAs controlling translation and transcription of many genes, play important roles in the regulation of various biological processes, including the interaction between virus and host. Previous studies show that the virus infection alters expression of cellular mi RNAs, which in turn can modify the intracellular microenvironment to help virus infection, replication and proliferation. Our preliminary findings from mi RNA expression profile indicate that IAV infection induced the expression of host mi R-9. This study was designed to explore the role of mi R-9 in IAV infection.First, we fully detected the expression of mi R-9 in A549 cells with different IAV subtypes infection for the indicated time. The results confirmed IAV infection significantly induced the expression of mi R-9, and its expression peak appeared earlier than drastic expression of IAV matrix protein(M)gene and nucleoprotein(NP)gene. Next, the study association between mi R-9 and IAV subtypes infection indicated that the overexpression of mi R-9 significantly enhanced the viral structural protein gene expression on RNA and protein levels, and infectious progeny virus, while knockdown of mi R-9 significantly inhibited IAV replication. To investigate the mechanism of mi R-9 on IAV replication, we predicted and obtained 4452 potential target genes for mi R-9 by Targetscan. We used bioinformatic methods, such as GO annotation analysis, to resolve these target genes, and chose MCPIP1 as a potential target gene of mi R-9 to verify in the following experiments.Previous studies have demonstrated that MCPIP1 not only negatively regulated inflammatory signaling through its deubiquitinase and RNAse activity, but also exhibited broad-spectrum antiviral effects through viral RNA binding and degradation. In function aspects, we secondly completely verified the inhibition of IAV replication by MCPIP1. The results showed that MCPIP1 overexpression abrogated viral structural protein genes expression on RNA and protein levels and infectious progeny virus while inhibiting the expression of MCPIP1 significantly promoted the proliferation of IAV. Moreover, previous studies have also demonstrated that MCPIP1 specifically recognized stem-loop structure and regulated of gene expressionin in post-transcriptional level. We hereby used half-life of RNA experiment to examined the effect of MCPIP1 on IAV m RNA stability. The results showed that overexpression MCPIP1 shorten the IAV M and NP gene m RNA half-life, and inhibited IAV RNA expression. Meanwhile, we predicted the m RNA secondary structure of IAV PR8 strains. The results showed that many stem-loop structures were presented in the predicted secondary structure of each IAV gene suggesting that MCPIP1 could potentially bind and degrade IAV m RNA, then inhibit viral gene expression.To demonstrate the role of mi R-9/MCPIP1 axis in IAV replication, we finally carried out the following experiments. By luciferase reporter gene assay, Real-time PCR and western blot, we confirmed that mi R-9 directly target and negatively regulate MCPIP1 expression. After mi R-9 and MCPIP1 cotransfection we detected levels of viral structural proteins expression and infectious progeny virus, and the results showed that mi R-9 through direct inhibition, at least in part by MCPIP1 way to promote replication of IAV.Taken together, IAV hijacks host mi R-9 to regulate intracellular microenvironment and effectively suppresses host antiviral response by mi R-9/MCPIP1 interaction in order to complete virus infection and replication. |
