Function Of BmNPV-encoded CircRNA And Effect Of Down-regulatinge M~6A-related Gene Expression On Viral Proliferation

With the development of high-throughput technology,more and more circRNAs have been discovered and identified,and have been proved to have functions of binding to miRNAs and proteins,regulating gene transcription,and translating proteins.Besides,circRNAs are found to be involvoed in the process of cell proliferation and differentiation,cancer occurrence.It is of great significance m⁶A modification?as one of the most common methylation modification methods,has been found to be involved in the regulation of viral proliferation and replication in recent years.Silkworm bloody pus caused by Bombyx mori Nucleopolyhetiroviru.s?BmNPV?brings huge losses to the sericulture industry every year.There is no report about the formation of circRNA by BmNPV transcripts and the regulation of proliferation and replication of BmNPV by m⁶A modification.In this study,the expression of circRNAs in the midgut and normal midgut after BmNPV infection was compared by high-throughput sequencing.A total of 353 circRNAs were found to be significantly different in the midgut of BmNPV infection compared with the normal midgut.241 circRNA expression was up-regulated and 112 circRNA expression was down-regulated.Analysis of GO and KEGG showed that silkworm-derived circRNA may participate in the regulation of apoptosis,endocytosis and ubiquitination through interaction with miRNA.Further,high-throughput sequencing data of midgut circRNA of BmNPV-infected silkworm was compared with BmNPV genome,and BmNPV-derived circRNA was found.Based on the divergent primer PCR and Sanger sequencing,three BmNPV-derived circRNAs were identified,named circ₀03,circ₀04,circ₀10.The results of q-PCR showed that after 12 h of BmNPV infection,circ₀03 began to accumulate and gradually accumulated.The parental gene lef-6 began to express after 6 h of infection.It was found that circ₀03 could inhibit BmNPV proliferation in a dose-dependent manner and promote BmN from silkworm culture cells from G1 phase.Enter the S phase.Circ₀04 began to be produced 12 hours after BmNPV infection,and the expression of its parental gene odv-e66 was also detected at the same time.Up-regulation of circ₀04 expression level could activate the expression of Dicer2,an innate immune related gene in silkworm.Circ₀10 was produced 6 hours after BmNPV infection,and its parental gene odv-e18 expression was also detected simultaneously.Cellular immunofluorescence results showed that all three circRNAs could co-localize with NF90/NF110 and Rig-I.The circRNA-pull down results show that circ₀10 may function by interacting with proteins.These results indicate that BmNPV can express functional circRNAs to play a role in regulating viral infection processes and in virus-host interactions.The effect of m⁶A modification on BmNPV proliferation remains unclear.The expression levels of METTL3,METTL14 and YTHDF3 genes related to m⁶A were decreased by RNAi,and the expression of Bm59 gene of BmNPV was down-regulated,and the results of western blot showed that down-regulation of METTL3 resulted in an increase in odv-e25 protein level and YTHDF3 expression.Down-regulation caused down-regulation of odv-e25 protein,suggesting that cell methylation levels affect viral proliferation.