Identification Of AcMNPV Micro RNA MR40 And Preliminary Investigation Of Its Target Genes

Baculoviruses are insect-specific and insect-pathogenic viruses that encompass a group of arthropod-specific DNA viruses with a circular covalently closed genome of 80-180 kb. The Baculoviridae are divided into four genera. The viruses of Lepidoptera are divided into Alpha-and Betabaculoviruses encompassing the NPVs and GVs, respectively, and those infecting Hymenoptera and Diptera are named Gammaand Deltabaculoviruses, respectively. Autographa californica multiple nuclear polyhedrosis virus(AcMNPV) is a representative of baculovirus, which contains a genome of 134 kbp and about 151 open reading frames(ORFs). It is also the most intensively studied member of Baculoviridae family. Therefore, investigation of microRNAs encoded by AcMNPV is significant for the understanding of gene regulation mechanism of baculovirus and the host-pathogen interactions.As noncoding RNAs between 19-24 nucleotides(nt) in size that regulate gene expression by binding to the complementary sequence of the target mRNAs, microRNAs(microRNAs) have emerged as key players in the regulation of different biological processes in eukaryotes. An increasing number of studies suggest that viruses can also encode microRNAs to regulate their own gene expression or manipulate their host expression. In our previous study, we predicted six AcMNPVencoded microRNAs, and one of them was verified that can regulate the expression of Ac-ODV-E25.Here, we demonstrate another microRNA encoded by AcMNPV, named MR40. Firstly, Northern blot as well as Real Time PCR were carried out to analyzed the expression of MR40 in AcMNPV-infected sf-9 cells at different time points after infection and mock-infected cells. The results show that MR40 is a microRNA encoded by AcMNPV and its expression reaches an approximate maximum at 6 h p.i. Then the MiRanda program was used for predicted viral target genes of MR40. Five target candidate genes were generated, among which lef-6 had the higest score. In addition, dual luciferase reporter assay was performed to verify the influence of MR40 on the expression of five target candidate genes.We found that MR40 was able to downregulate the expression of lef-6, lef-11, Ac-orf49 and Ac-orf63. Finally, our results show that MR40 over-expression could downregulate BV infectivity but had no impact on the polyhedra.