MicroRNA-2361 Inhibits The Replication Of Bovine Infectious Rhinotracheitis Virus By Directly Targeting The EGR1 Gene

Infectious Bovine Rhinotracheitis virus(IBRV),also known as Bovine herpesvirus 1(BHV-1),is the pathogen of Infectious Bovine Rhinotracheitis,the disease often leads to respiratory infections,conjunctivitis,encephalitis,milk production decline in clinical symptoms,acute IBR respiratory infection can be secondary to bovine pathogenic bacterial pneumonia,causing large economic losses to livestock producers.Therefore,the molecular mechanism of virus replication,especially the research focus such as miRNA,needs to be further studied in order to find drug targets and provide clues and ideas for anti-IBRV new drug development..miRNAs are short noncoding single-stranded RNAs.And miRNAs can be produced to regulate gene expression by host or virus via base pairing interactions.They down-regulate gene expression by binding to the 3'-untranslated region(3'UTR)of target mRNA.There is growing evidence that miRNAs play a central role in viral replication.host cell miRNA can be significantly expressed during viral infection.Host miRNAs can cause the virus to alter the intracellular environment to evade the antiviral immune response,or enable the host cell to trigger an antiviral defense.In the herpesvirus,miRNA-649 can promote HSV-1 replication by targeting MALT1.However,the interaction between IBRV and host miRNAs has not been reported.In this study,samples of IBRV infected with MDBK at 12 h,24h,and 48 h were deeply sequenced.Compared with the control group,105 miRNAs were up-regulated at 12 h after infection,152 miRNAs were down-regulated,and 146 miRNAs were up-regulated at 24 h after infection,41 miRNA was down-regulated,242 miRNAs were up-regulated and 78 miRNAs down-regulated 48 h after infection.Subsequently,the differentially expressed differentially expressed genes were analyzed,and the miRNAs with consistent expression at three time points were selected for verification.The real-time PCR confirmed that miR-2361 was significantly down-regulated at different time points after IBRV infection.In order to detect the role of miR-2361 in IBRV infection,miR-2361 mimic was transfected into MDBK cells and tested for virus titer.It was found that miR-2361 can inhibit IBRV replication.To more efficiently screen for candidate target genes for miR-2361,we performed transcriptome sequencing by analyzing up-regulated genes after IBRV-infected MDBK cells,and combined with bioinformatics tools to predict the results,early growth response factor 1(EGR1)is Candidate target for miR-2361.Subsequently,the target association between miR-2361 and the 3'UTR of EGR1 was further validated using the dual luciferase reporter gene detection technique.Furthermore,overexpression of miR-2361 resulted in decreased levels of EGR1 mRNA and protein,indicating that the EGR1 gene is a target molecule for miR-2361.Furthermore,after overexpression of EGR1,IBRV replication can be promoted by detecting viral titers,while silencing EGR1 has the opposite effect.Further mechanistic studies indicate that EGR1 stimulates the IBRV UL46 promoter activity,whereas overexpression of miR-2361 inhibits the expression of EGR1 and UL46 genes,thereby inhibiting IBRV replication.In this study,miRNA and EGR1 genes,which may be involved in IBRV replication,were discovered by deep miRNA sequencing and transcriptome sequencing.The relationship between the two was found to be the first entry point.It was found that miR-2361 and EGR1 inhibited and promoted IBRV replication,respectively.miR-2361 provides a target molecule for antiviral drug development by targeting the EGR1 gene to inhibit the molecular mechanism of IBRV replication.