Function Of BmCPV CircRNAs And Effect Of Overexpression Of BmCPV Single Gene On Virus Proliferation

With the development of high-throughput sequencing and bioinformatics,more and more circRNAs have been discovered.It has been proved that some circRNAs can bind to miRNAs,interact with RNA binding protein,participate in gene transcription,splicing regulation and so on.It also plays an important role in cell proliferation,differentiation,survival,aging,many other life processes and the occurrence of many diseases.Bombyx mori cypovirus?BmCPV?is a typical species of CPV1.It can infect the midgut cells of silkworm and cause the silkworm to develop the disease of cytoplasmic polyhedrosis.And leading to the poor harvest of sericulture.Viruses are the major pathogens of human,animal and plant diseases,however there are no reports on the formation of circRNAs by viral transcription products.In this study,we identified 295 BmCPV-derived circRNAs from the midgut tissues of BmCPV-infected silkworms through high-throughput sequencing.The length of these circRNAs ranging from 100 nt to 3800 nt,mainly concentrated at 200-1000 nt.The circRNAs are distributed in all 10 fragments of the BmCPV genome,but are mainly distributed on the positive strand of the genome.After statistical analysis of splicing signals on both sides of BmCPV circRNAs junction site,it was found that the formation of viral circRNAs do not follow the typical GT-AG splicing rules,and also varies in the positive and negative strands of the genome.GO and KEEG analysis showed that BmCPV circRNAs may regulate the bombesin receptor signaling pathway,fructose2,6-bisphosphate metabolic process,MAPK signaling pathway,Jak-STAT signaling pathway and so on.By sequencing of the PCR products and Northern blot,we further confirmed the existence of BmCPV circRNAs in the midgut of silkworm infected with BmCPV.In addition,we also found that in the transformed cell line which overexpressed the single BmCPV gene and BmCPV virus particles were detected theBmCPV circRNAs.These new findings suggest that BmCPV forms functional circRNAs via reverse splicing,which provides a new perspective for understanding the transcriptional regulation of virus genes and the interaction between virus and host.In order to better explain the function and mechanism of the virus producing circRNAs,we have carried out further research on BmCPV circ₀48.qPCR revealed that circ₀48 began to formation after BmCPV infected BmN cells 12 hours and accumulated gradually.The results of cellular immunofluorescence showed that circ₀48 was localized in the cytoplasm of BmN.The results of transfection showed that up-regulation of circ₀48 level inhibited the proliferation of BmCPV and up-regulated the expression of serine / threonine protein phosphatase,which is a congenital immune-related gene of Bombyx mori.In addition,by RNA pull down and MS identification,it was found that the Elongation factor 1-alpha,ATP synthase subunit beta might interact with the circ₀48.The informatics analysis showed that circ₀48had IRES sequence and maybe encode 6 kinds of polypeptides ORF-V,ORF-1,ORF-2,ORF-3,ORF-4 and ORF-5.The antigenic peptides of the above peptides were further synthesized and mixed antibodies were prepared.The results of cellular immunofluorescence assay showed that circ₀48 may have the potential of coding peptides.It was also found that the antigenic peptides derived from ORF-3 and ORF-5could inhibit the proliferation of BmCPV virus.Antigenic peptides derived from ORF-5can induce cell aggregation and present pathological changes.These results preliminarily indicate that RNA of circ₀48 can play function by interacting with proteins and encoding small peptides.The genomic fragment of the S1-S10 dsRNAs of BmCPV is monocistron,and the role of each genomic fragment in viral replication remains unclear.We studied the effects of vp3,vp7,polh and its corresponding non starting codon gene vp3-mut,vp7-mut,and polh-mut,also S2 and S5 fragments on the proliferation and replication of BmCPV.It was found that overexpression of vp7,vp7-mut could promote the proliferation of BmCPV,and overexpression of s5?nsp5?could inhibit the proliferation of BmCPV.However,we had not found effect on BmCPV virus proliferationafter after overexpression of vp3/vp3-mut,polh/ polh-mutwe.The results of qPCR detection showed that the expression of vp3/ vp3-mut and vp7/ vp7-mut had no significant effect on the cell immunity and the expression of apoptosis related genes.In addition,overexpression of S5?nsp5?also promoted the transformation of bmn cells from S phase to G2 phase.