Identification And Functional Study Of BmCPV-miR-1 Encoded By Bombyx Mori Cypovirus

microRNAs(miRNAs)are 18–25 nucleotides(nts)small non-coding RNAs processed from the stem-loop precursor termed as the pre-miRNA.Furthermore,they regulate the expression of target genes at the posttranscriptional level and play important roles inregulating various cellular processes.In recent years,growing evidenceshave shown thatviruscan encode miRNAs,which regulated the host and its own gene expression to suit its life in the host.As a typical RNA virus,BmCPVwas found in previous studyto be able to encode functional miRNAs by our team,regulating the expression level of host cell genes.In this paper,silkworm and BmCPV were used as the experimental materials.Through the deep sequencing of small RNA,the miRNAs specifically expressed in the BmCPV-infected silkworm were screened out.One of the BmCPV-miR-1 was selected for further study,and the target gene was predicted by software.Firstly,the qRT-PCR was used to detect the changes of miRNA and target gene,and the lentiviral expression vectors containing miRNA precursor and target gene were constructed and transfected into HEK293 cells in order to verify the regulation of BmCPV-miR-1 on its target gene expression.Then the regulatory effect of BmCPV-miR-1 on its target gene were verified at the level of silkworm cells and in vivo respectively by using BmCPV-miR-1 mimics.Finally,the effect of BmCPV-miR-1 on the replication of the virus was verified by injecting mimics into the silkworm body.The main experimental results obtained are as follows:1.Changes of BmCPV-miR-1 and its target gene in silkworm midgut after infection with BmCPVThrough software analysis of small RNA deep sequencing dataand stem-loop RT-PCR identification,we got 8 miRNAs that may be encoded by the BmCPV genome.In this study,one of the miRNAs,BmCPV-miR-1,was selected for further study,and the result ofstem-loop qRT-PCRshowed that BmCPV-miR-1 was significantly up-regulated in the midgut of BmCPV-infected silkworms with infection time going.At the same time,miRanda and Targetscan were used to predicted a large number ofpotential target genes,and we chose the Bombyx moriinhibitor of apoptosis protein(BmIAP)gene,which could inhibit the apoptosis of silkworm cells.The result of qRT-PCR showed that the expression of BmIAP gene in the midgutsignificantly increased,and the trend is consistent with BmCPV-miR-1.2.BmCPV-miR-1 can up-regulate BmIAP gene expression(1)The lentiviral expression vector pLNHX-UTR-mCherry-IAP was constructed and transfected into HEK293 cells,then the transfection resultwas confirmedby observing red fluorescence of mCherry under the fluorescence microscope.However,the plasmid pLKO.3G-miR-1 was confirmed by detecting the green fluorescence.The cells were collected at 24 h and 48 h after transfection and Quantitative real-time PCR was used to detect the regulation of BmIAPgene by BmCPV-miR-1.The result showed that BmCPV-miR-1 can upregulate the expression of BmIAPgene,at the same time,the 5′UTR target site of BmIAP gene and BmCPV-miR-1 indeed can complementarily combine to form a stable structure by comparison and analysis.Therefore,we identified BmCPV-miR-1binds to the 5’UTR of BmIAP gene and positively regulates its expression level.(2)In order to further verify the regulatory effect of BmCPV-miR-1 on BmIAP gene in silkworm,BmCPV-miR-1 mimics and negative control NC sequences were synthesizedand transfected into BmN cellsrespectively.The cells were collected at 24 h,48hand 72 h after transfectionand the expression of BmIAP gene was detected by qRT-PCR.The result showed that the BmIAP gene was up-regulated at 24 h after transfection of BmCPV-miR-1 mimics,but the expression was not significantly increased within 48 h,unexpectedly,the expression level was 72 h rapidly increased,and the level of up-regulation was extremely significant.There was no significant difference in gene expression between the NC group and the control group.This result directly proves that BmCPV-miR-1 positively regulates the expression of BmIAP gene.(3)BmCPV-miR-1mimics were injected into the silkworm at the dose of 1 μg and 2 μg by using the body cavity injection technique.The fat bodies and midgut tissues of the silkworm were taken at different time points after injection to obtain total RNA reverse transcription.The results showed that BmIAP gene was slightly up-regulated in both fat body and midgut tissue when injected with low dose of mimics,whereas BmIAP gene was highly up-regulated in both tissues when injected with high dose of mimics,and the difference was extremely significant.Compared with theexperimental group,there was no significant difference in the expression level of BmIAP genein the groupinjected DEPC water and NC.It is further proved that BmIAP gene is one of the target gene of BmCPV-miR-1,and BmCPV-miR-1 can positively regulate the expression of the target gene.3.BmCPV-miR-1 can promote the replication of BmCPVBmCPV-miR-1 mimics,inhibitors and NC were injected into the body cavity of 5thinstar silkworm larvae infected with BmCPV.Silkworm midgut tissues were collected at different time points to extract total RNA reverse transcription.The second fragment(coding RNA-dependent RNA polymerase)and the tenth fragment(encoding polyhedrin)of viral genomic RNAwas detected by qRT-PCR.The results showed that both RNA fragments of the virus were at a low level of replication within 48 h,and all three groups showed a rise from 72 h.The largest increase was in the group injected with mimics,followed by the NC group,with the least increase in inhibitors,and this trend is even more pronouncedat 96 h.This is due to the increasing number of viruses in the midgut following the infection with BmCPV,but the mimics up-regulate the expression of the BmIAP gene,which in turn inhibits cell apoptosis and provides a better breeding environment for the virus,while inhibitors down-regulation of the BmIAP gene promotes apoptosis and is not conducive to virus reproduction.This demonstrated that BmCPV promotes its replication by encoding BmCPV-miR-1 to upregulate the BmIAP gene in the silkworm.The results of this study on BmCPV-miR-1 and its target gene BmIAP will provide new clues for the study of the immunological interaction mechanism between BmCPV and silkworm,and also provide new ideas for the research of other viral miRNAs.