Two Cypovirus-encoded MiRNAs Co-regulate Bombyx Mori GTP-binding Nuclear Protein Ran And Facilitate Virus Replication

Bombyx mori cypovirus(BmCPV)is one of the major pathogens of silkworm.Its genome consists of ten double-stranded RNA segments.The pathogenesis of BmCPV invading silkworm is still unclear.Many studies have shown that miRNA encoded by virus plays an important role in the process of virus infection and immune escape.Therefore,the study of miRNA derived from BmCPV is helpful to clarify the interaction mechanism between BmCPV and host silkworm.In our previous work,silkworm strain 4008,which is sensitive to BmCPV,was orally inoculated with BmCPV polyhedra,the midgut was dissected 72 hours post inoculation for deep sequencing of small RNAs.Several candidate miRNAs probably encoded by BmCPV genome were screened,and the target genes were predicted by bioinformatics methods.In this study,two candidate miRNAs,BmCPV-miR-1encoded by the first segment of the BmCPV genome and BmCPV-miR-3 derived from the third segment of the BmCPV genome were selected to study their regulatory effects on their target gene and their effect on viral replication in the BmCPV infected larvae.The main results are as follows:1.Expression patterns of miRNAs and the target gene in midgut of BmCPV infected silkworm larvae.Two specific miRNAs(BmCPV-mir-1 and BmCPV-miR-3)were detected by stem-loop RT-PCR in the midgut infected by BmCPV,and the results of qRT-PCR showed that the expression of these two miRNAs increased with the advance of infection.Bombyx mori GTP binding nuclear protein Ran(BmRan)was predicted with the both softwares miRanda and targetscan to be a common target gene for the two miRNAs,and the binding sites were all located in 3’-untranslated region(3’-UTR)of the target gene mRNA sequence.The results of qRT-PCR showed that the expression of BmRan gene was down-regulated with the advance of infection.2.BmCPV-miR-1 and BmCPV-miR-3 down-regulate expression of target gene BmRan.Since both BmCPV-miR-1 and BmCPV-miR-3 bind to the 3’-UTR of the target gene,it is speculated that they could down-regulate the expression of the target gene,and the regulatory effect of the two miRNAs might be in a synergistic pattern.The lentiviral expression vectors of BmCPV-miR-1,BmCPV-miR-3 and BmRan were constructed respectively and co-transfected with the three packaging plasmids into 293 T cells.The cells were collected at 24 h and 48 h after transfection for total RNA extraction,and the expression level of BmRan gene was detected by qRT-PCR.The results showed that both BmCPV-miR-1 and BmCPV-miR-3 could down-regulate the expression of BmRan gene,and the negative regulation effect of the two miRNAs on the expression of target gene was cumulative.Furthermore,chemically synthesized miRNA mimics and NC(negative control)were respectively transfected into BmN cells,the expression of target gene BmRan was detected at 24 h,48 h and 72 h after transfection.The results showed that the two miRNA mimics could down-regulate the expression of the target gene,and the down-regulation of target gene was more obvious in the cells transfected with both the two miRNAs together,which further proved that BmCPV-miR-1 and BmCPV-miR-3 played a negative role in regulating the expression of BmRan gene,the inhibition effect of the two miRNAs was stronger than that of any single miRNA.In order to prove the regulatory effect of the two miRNAs on target gene in silkworm,the synthesized miRNA mimics,inhibitors and NC were injected into the body cavity of silkworm.The midgut was taken at different time points after injection,and the total RNA was extracted for reverse transcription.The expression of BmRan gene was detected by qRT-PCR.The results showed that both BmCPV-miR-1 and BmCPV-miR-3 could negatively regulate the expression of target gene BmRan either in normal control or in BmCPV-infected larvae,and the down-regulation of target gene expression was more obvious by the combined action of the two miRNAs.3.BmCPV-miR-1 and BmCPV-miR-3 promote the replication of virus.The synthesized miRNA mimics,inhibitors and NC were injected into the fifth instar larvae of silkworm infected by BmCPV.The replication level of the second,fifth and tenth segments of virus genome were detected by qRT-PCR at different time points after injection.The results showed that the replication levels of the three segments of virus genome gradually increased with the progress of infection,but compared with NC injection group,their replication level in mimics injection group increased faster,while those in inhibitor injection group was lower.Therefore,it is speculated that BmCPV-miR-1 and BmCPV-miR-3 may promote virus replication by suppressing the expression of target gene BmRan.In conclusion,BmCPV-miR-1 and BmCPV-miR-3 are putative miRNAs encoded by BmCPV,negatively regulate the expression of target genes BmRan thus promote viral replication.GTP-binding nuclear protein Ran(BmRan)is a transporter involved in nucleocytoplasmic transport of pre-miRNAs.While some host miRNAs play an improtant role in defending virus infection.Therefore,it could be speculated that BmCPV-miR-1 and BmCPV-miR-3 bind to the 3’-UTR region of BmRan mRNA,negatively regulate the translation of the BmRan protein,thus inhibit the transport of pre-miRNA from the nucleus to the cytoplasm in host cells,reduce the generation of host miRNAs,and consequently create a favorable intracellular environment for BmCPV genome replication and the virus multiplication.