The Mechanism Of Teleost MicroRNA In Regulating The Proliferation Of SHVV And CyHV-3

Virus infection has instigated great financial ruin to the aquaculture industry in China and the pathogenic mechanism of most aquatic animal viruses is still unclear.As a class of small non-coding RNAs,micro RNA show a vital regulatory role in the process of virus-infected hosts.Prior research confirmed that Snakehead vesiculovirus(SHVV)and Cyprinid herpesvirus 3(CyHV-3)infection could cause differential expression of miRNA in the host,and overexpression of differential miRNA can effectively regulate virus proliferation.Therefore,this study intends to study the regulatory mechanism of miRNA in virus-infected hosts,which included:(1)To investgate the regulatory mechanism of miR-214 inhibiting SHVV proliferation by targeting viral or host genes.The infection of SHVV could reduce the expression of miR-214 in striped snakehead(SSN-1)cells in a time-and dose-dependent manner.Notably,transfecting with miR-214 mimic significantly inhibits,whereas miR-214 inhibitor promotes,SHVV replication,suggesting that miR-214 acts as a negative regulator of SHVV replication.A dual-luciferase reporter assay revealed that N and P of SHVV were the target genes of miR-214.Our study further demonstrated that miR-214 suppressed the expression of N and P in SSN-1 cells co-transfected with miR-214 mimic/inhibitor and plasmid respectively expressing N or P,and overexpression either N or P could restore miR-214-mediated inhibition of SHVV replication.Furthermore,our results showed that the P protein could inhibit SHVV-induced IFN-2 production,and overexpression of miR-214 restored the P-mediated inhibition of IFN-2 production.Taken together,these results suggest that miR-214 is downregulated during SHVV infection,and in turn,the downregulated miR-214 promoted viral N and P expression and resulted in less IFN-2 production,thus facilitating SHVV replication.Transcriptomic sequencing of miR-214 overexpressed SSN-1 cells were used to investigate the host target genes of miR-214.The target gene of miR-214 was identified as AMP-activated protein kinase(AMPK)from the downregulated genes.Remarkably,si RNA knockdown of AMPK inhibited SHVV replication while promoting IFN-2production,similar to overexpression of miR-214.Furthermore,we discovered that knocking down of cellular miR-214 restored si AMPK-mediated inhibition of SHVV replication,demonstrating that miR-214 suppressed SHVV replication through targeting AMPK.In addition to our prior finding,our study also found that miR-214 was downregulated in the presence of SHVV.(2)To investgate the regulatory mechanism of miR-155/miR-722 regulates the proliferation of Koi herpesvirus 3(Cyprinid herpesvirus 3,CyHV-3)by targeting host and viral genes.We performed miRNA high-throughput sequencing on CyHV-3 infected CCB cells,and the results showed that CyHV-3 infection caused the up-regulation of miR-155/miR-722.Transfection of miR-155/miR-722 mimics can inhibit the proliferation of CyHV-3,indicating that miR-155/miR-722 is a negative regulator of CyHV-3proliferation.Sequence analysis revealed that activated protein kinase(AMPK)is a potential target gene of miR-155.The dual luciferase reporter system confirmed that AMPK is the target gene of miR-155.Overexpression of miR-155 can inhibit the expression of AMPK and meanwhile promoting the up-regulation of IFN activated by viral infection;overexpression of AMPK can inhibit the expression of IFN.Therefore,we propose a model: CyHV-3 infection up-regulates the expression of miR-155,and up-regulated miR-155 can down-regulate the expression of the target gene AMPK,thereby promoting the expression of host IFN,and ultimately inhibiting the proliferation of CyHV-3.The ORF89 gene of CyHV-3 is a potential target gene of miR-722,and the dual luciferase reporter system also confirmed this conjecture.CCB cells were transfected with miR-722 mimic and ORF89 fluorescent eukaryotic expression plasmids.The result indicated that miR-722 significantly inhibited the expression of ORF89 protein by detecting the amount of fluorescence and the expression level of viral proteins.Overexpression of ORF89 markedly reduced the expression of IFN and interferonstimulate genes(ISGs).Mechanically,ORF89 interacted with and degraded IRF3,and inhibited the entry of IRF3 into the nucleus by suppressing the dimerization of IRF3.Moreover,ORF89-mediated suppression of IFN expression could be restored by adding miR-722.The research results provide a solid theoretical basis for understanding the pathogenic mechanism of SHVV/CyHV-3 and the development of new antiSHVV/CyHV-3 drugs.Meanwhile,it also provides innovative concepts and a reliable theoretical basis for the research of micro RNA in regulating aquatic viruses.