Identification And Functional Study Of A Putative MiRNA BmCPV-miR-10 Derived From Bombyx Mori Cypovirus

Bombyx mori cypovirus(BmCPV),a typical RNA virus,is one of the important pathogens of silkworm.The mechanism of its interaction with the silkworm immune defense system during infection is still not yet clear.Researches have demonstrated that virus-encoded micro RNAs play crucial roles in regulating host-pathogen interaction.At present,few reports are available on miRNAs encoded by insect viruses,especially RNA viruses and on their functions.Therefore,identification and functional analysis of miRNAs encoded by BmCPV genome could reveal its function in the process of pathogen-host interaction,display a new mechanism of miRNA-mediated regulation on RNA virus replication and also enrich the virus-encoded miRNA family.In our previous deep sequencing of small RNAs in the midgut of silkworm larvae infected with BmCPV,several putative BmCPV-encoded miRNAs were identified in the virus infected midgut.Their target genes were predicted respectively against the silkworm and the BmCPV genomes.GO and KEGG pathway analysis revealed that the predicted target genes are involved in apoptosis,cell cycles,RNA degradation,PPAR signal pathway,splicing and repair of nucleic acid,etc.In the present study,a candidate miRNA,BmCPV-miR-10,encoded by the tenth segment of BmCPV genome was evaluated for its regulation on target genes and the impact on virus replication.Major findings are as follows.1.Expression patterns of BmCPV-miR-10 and its target genes in midgut of BmCPV infected silkworm.The expression of BmCPV-miR-10 was detectable in BmCPV-infected silkworm midgut via stem-loop RT-PCR,indicating that it is BmCPV-encoded miRNA.Target gene prediction revealed that BmCSDE1(Bombyx mori cold shock domain containing protein E1)gene and BmR2D2(Bombyx mori R2D2 protein)gene are candidate target genes for BmCPV-miR-10 with the binding site located in 3’UTR region of BmCSDE1 and CDS region of BmR2D2 mRNAs.As the time advances after BmCPV infection,the expression of BmCPV-miR-10 was gradually increased in the midgut of silkworm larvae,while the expression of both the two target genes gradually reduced.2.BmCPV-miR-10 down-regulates the expression of target genes BmCSDE1 and BmR2D2.With its binding sites on the two target genes,it was speculated that BmCPV-miR-10 could negatively regulate the expression of the target genes.In order to verify the regulation effect of BmCPV-miR-10 on BmCSDE1 and BmR2D2,lentiviral expression vectors for expressing BmCPV-miR-10 and target genes were constructed respectively and transfectedinto HEK293 T cells with packaging plasmids,and meanwhile,the synthesized BmCPV-miR-10 mimics were transfected into Bm N cells.The qRT-PCR results showed that BmCPV-miR-10 could down-regulate the expression of both BmCSDE1 and BmR2D2.By the injection of BmCPV-miR-10 mimics and inhibitors into the silkworm larvae infected with BmCPV and the following qRT-PCR detection of the target gene expression,it was demonstrated that the expression level of both the two target genes was lower in the mimics-injected silkworm larvae while higher in the inhibitor-injected ones than that in the NC-injected group.These indicated that increasing the level of BmCPV-miR-10 could down-regulate the expression of BmCSDE1 and BmR2D2,while inhibition of BmCPV-miR-10 could increase expression of the target genes,implying that BmCPV-miR-10 could also down-regulate the expression of the two target genes in vivo in the silkworm.3.BmCPV-miR-10 could promote BmCPV replication.The changes in expression level of the second and tenth segments of BmCPV genome were detected by qRT-PCR after injecting BmCPV-miR-10 mimics,inhibitors and NC to BmCPV-infected silkworm larvae.The results showed that the expression of both the second fragment RNA-S2 and the tenth fragment RNA-S10 of BmCPV genome increased gradually in BmCPV infected silkworm midgut as the infection advanced,however,the expression level of the two genomic fragments increased more rapidly in the mimics-injected silkworm larvae,and significantly lower in the inhibitors-injected ones than that in the NC-injected group,indicating that BmCPV-miR-10 could promote viral replication through down-regulating the expression of the target genes.In conclusion,BmCPV-miR-10 is a putative miRNA encoded by BmCPV,negatively regulate the expression of target genes BmCSDE1 and BmR2D2 thus promote viral replication.Apoptosis and RNAi are important mechanism for host to defense viral infection.CSDE1 can up-regulate the expression of pro-apoptotic factors thus accelerate apoptosis.R2D2 is an important component in RNA-indcued silencing complex(RISC)for RNAi or miRNA pathways,directly affecting the functions of host RNAi or miRNAs.Therefore,it can be spculated that,in the process of BmCPV infection to silkworm,BmCPV-miR-10 could inhibit the apoptosis and RNAi/miRNA pathways of the infected cells through down-regulating the expression of the two target genes,thus creating an optimal inernal environment for virus replication.