Biogenesis Mechanism Of BmCPV MicroRNAs And Function Of BmCPV-miR-1

MicroRNAs（miRNAs）are small non-coding RNAs that function as novel gene expression regulators at the post-transcriptional level.Not with standing that the biogenesis and function of miRNAs are well-understood in eukaryotes,little is known about RNA virus-encoded miRNAs.Bombyx mori cypovirus（BmCPV）is a double-stranded RNA virus with a genome of 10 discrete RNA segments that causes cytoplasmic polyhedrosis disease in silkworm larvae.To date,the interaction between BmCPV and silkworm remains largely unclear.Herein,we decided to investigate the molecular mechanism of the infection between BmCPV and silkworm through studying the function of miRNAs encoded by BmCPV on the host antiviral defense.1.BmCPV-encoded small RNA sequencingIn this study,small RNA library was constructed firstly,and then small RNA sequencing was performed on the midguts of BmCPV-infected silkworm.Totally,34.7M raw reads and 33.6M clean reads were yielded.18-23 nt long sequences represented 72.76%of the raw reads.Through analyzing the data of small RNA sequencing,12 miRNAs precursors and 253 probable miRNAs were obtained.The target prediction was performed on the miRNAs with high sequencing abundance.The result showed that one miRNA can target many genes of silkworm,and also,one host gene might be regulated by several viral miRNAs.Some miRNAs might participate in the interaction between virus and host through regulating the genes of the immune-associated pathways.From these pathways,we found an immune-associated host gene,Bombyx mori inhibitor of nuclear factor kappa-B kinase subunit beta（Bm IKKβ）,is a target gene of BmCPV-miR-1 derived from BmCPV genome RNA segment 1.BmCPV-miR-1 can bind to 3’UTR of Bm IKKβm RNA.BmCPV-miR-1and Bm IKKβwere set as our research objects to explore their interactions and the impacts on BmCPV multiplication.2.BmCPV can encode miRNAsPossibility of BmCPV encoding miRNAs was investigated taking BmCPV-miR-1 as an example.Stem-loop PCR was extensively used to detect the expression of miRNAs.Herein,Stem-loop PCR was performed to detect the expression of BmCPV-miR-1.The result showed that BmCPV-miR-1 could be detected in the BmCPV-infected silkworm but not in the normal silkworm.And the expression of BmCPV-miR-1 increased significantly with the progression of the viral infection.These results demonstrate that BmCPV-miR-1 is encoded by BmCPV genome RNA rather than the degraded fragments of the viral genome.In addition,we constructed the overexpression vector p IZT-mir-1 to study the biogenesis of BmCPV-miR-1 at the cell level.After the transfection of Bm N cells with p IZT-mir-1,BmCPV-miR-1 could be detected in the Bm N cells but not in the cells transfected with p IZT.And q PCR result showed that the expression of BmCPV-miR-2increased gradually at 24～72 HPT,which indicated that BmCPV-miR-1 could be generated from its precursor BmCPV-mir-1.The above results demonstrate that BmCPV can encode miRNAs.3.Biogenesis mechanism of BmCPV-encoded miRNAsBiogenesis mechanism of BmCPV-encoded miRNAs was investigated using RNA interference（RNAi）in vivo and in vitro.Bm N cells were transfected with siRNAs to silence the genes related to the generation of miRNAs,including Dicer-1（Dcr1）,Dicer-2（Dcr2）,Argonauate-1（AGO1）and Argonauate-2（AGO2）.Then expression of BmCPV-miR-1 in the Bm N cells with corresponding gene silenced and transfected with p IZT-mir-1 vector were detected using q PCR.As a result,the expression of BmCPV-miR-1 was suppressed in Bm N cells where AGO1,AGO2 and Dcr1 were silenced,whereas BmCPV-miR-1 expression was elevated when the Dcr2 was knocked down.These results suggested that AGO1,AGO2,Dcr1 and Dcr2 proteins might be related to the biogenesis of viral miRNAsMoreover,the genes（Dcr1,Dcr2,AGO1 and AGO2）in the BmCPV-infected silkworm were silenced through injecting siRNAs.The expression of BmCPV-miR-1 in the BmCPV-infected silkworm were detected using q PCR.As a result,si-AGO1 and si-AGO2could not silence the expression of AGO1and AGO2 in the BmCPV-infected silkworm.When Dcr1 was silenced in the BmCPV-infected silkworm larvae,the expression of BmCPV-miR-1 was significantly suppressed.In contrast,BmCPV-miR-1 was significantly elevated when the Dcr2 protein expression was knocked down.The above results suggest that Dcr1 protein play an important role in the biogenesis of BmCPV miRNA,while Dcr2 is inversely correlated with the biognesis.Furthermore,viral replication was detected in the BmCPV-infected silkworm larvae where the Dcr1 or Dcr2 protein expression was knocked down.The result showed that viral multiplication was suppressed 20～50-fold in the BmCPV-infected silkworm larvae where the Dcr1 was knocked down,while was enhanced 0.8×10⁴～1.4×10⁵-fold in the BmCPV-infected silkworm larvae when Dcr2 was silenced.The above results demonstrate that Dcr2 protein plays a vital role in the host antiviral defense,and the suppression of Dcr2enhance the viral replication,which leads to the high expression of BmCPV-miR-1.4.Regulation of BmCPV-miR-1 on the target gene Bm IKKβTo study the regulation of BmCPV-miR-1 on Bm IKKβ,we firstly detected the expression of Bm IKKβin the Bm N cells transfected with p IZT-mir-1 and that in the silkworm infected with BmCPV.The results indicated that the expression of Bm IKKβdecreased gradually to more than 50%in the Bm N cells transfected with p IZT-mir-1 over time during 24-72 HPT,which suggested that BmCPV-miR-1 could repress the expression of Bm IKKβin Bm N cells.In the BmCPV-infected silkworm,the expression of Bm IKKβwas increased up to～18 folds at 24 hours post injection（HPI）,whereas was repressed significantly in the period 48～96 HPI.As reported,BmCPV infection can activate host Imd pathway,which may enhance the expression of Bm IKKβto induce the generation of IKK complex.Therefore,the expression of Bm IKKβwas increased significantly at the initial period of viral infection.With the progression of viral infection,more and more BmCPV-miR-1 was generated to repress the translation of the target gene Bm IKKβand lead to the degradation of Bm IKKβm RNA.The above results indicate that BmCPV-miR-1 can regulate negatively the expression of Bm IKKβ.Moreover,the dual-luciferase assay was performed to corroborate the regulation of BmCPV-miR-1 on the expression of Bm IKKβat the protein level indirectly.The plasmids pmir GLO-IKKb-wt containing BmCPV-miR-1 binding site and pmir GLO-IKKb-mut with a mutated BmCPV-miR-1 binding site were respectively co-transfected into 293T cell with BmCPV-miR-1 mimics,then luciferase activity was detected.The results showed that firefly luciferase activity in the cells co-transfected with pmir GLO-IKKb-wt and BmCPV-miR-1mimics decreased about 40%,while that in the cells co-transfected with pmir GLO-IKKb-mut and BmCPV-miR-1 mimics,or pmir GLO-IKKb-wt and NC,was approximately 100%.The result indicates that BmCPV-miR-1 functions as a negative regulator via binding to the 3’UTR of target m RNA.5.BmCPV-miR-1 enhances BmCPV replicationBmCPV-miR-1 agomirs and the inhibitor BmCPV-miR-1 antagomirs were chemically synthesized and injected into the 5^thinstar silkworm larvae infected with BmCPV.The expression of Bm IKKβand viral multiplication were detected by q PCR.The result showed that the expression of Bm IKKβin the group injected with BmCPV-miR-1 agomirs was significantly suppressed at 48～96 HPI,which indicated that higher expression of BmCPV-miR-1 could enhance the suppression effect of BmCPV-miR-1 on the target Bm IKKβexpression.In contrast,expression of Bm IKKβin the BmCPV-miR-1 antagomirs group was significantly elevated at 24～96 HPI,which suggested that the miR-1 antagomirs mitigated the effects of BmCPV-miR-1 on the expression of Bm IKKβto a certain extent.These results further confirmed that BmCPV-miR-1 regulates negatively the expression of Bm IKKβ.The viral proliferation was estimated by analyzing the transcription of three segments of the BmCPV RNA genome,S2,S5 and S10.The results showed that the transcript level of viral genomic segment S2,S5 and S10 was increased in all groups injected with NC agomirs,miR-1 agomirs or miR-1 antagomirs,respectively,which suggested that BmCPV infected and replicated in the silkworm.However,the BmCPV proliferation rate was significantly enhanced in the miR-1 agomirs group at 48～96 HPI,which implied that higher expression of BmCPV-miR-1 could enhance the viral replication.In contrast,the BmCPV proliferation in the miR-1 antagomirs group was significantly suppressed,which indicated that suppression of BmCPV-miR-1 could repress the viral replication.The above results demonstrate that BmCPV-miR-1 can promote the replication of BmCPV.In summary,this study demonstrates that BmCPV-miR-1 is one of the miRNAs encoded by BmCPV,its biogenesis is dependent on the protein Dicer-1.In addition,BmCPV-miR-1 can represses the expression of the target gene Bm IKKβby binding to its m RNA 3’UTR,which fine-tunes the host NF-κB signaling pathway and consequently enhances viral replication.Our results are helpful to understand the molecular mechanism of BmCPV infecting silkworm and provide new evidence supporting the hypothesis that RNA viruses can generate miRNAs to modulate antiviral host defense.