Characterization Of The RNA Transcription Profile Of Bombyx Mori Bidensovirus

Bombyx mori bidensovirus(BmBDV)is the unique member of genus Bidensovirus of the Bidnaviridae family,and which is the only ssDNA bipartite virus encoding DNA plolymerase by itself as yet.The replication mechanism of BmBDV is unclear,which may replicate in a protein-initiated,that is similar to adenovirus.BmBDV has a non-enveloped,spherical,icosahedral structure,which exclusively infects the columnar cells of the larvae midgut epithelium,causing chronic densonucleosis disease.BmBDV has an ambisense genome that contains two linear ssDNA molecules(VD1 and VD2).VD1 contains four open reading frames(ORFs;ORF1 to ORF4),which encode nonstructural protein 2(NS2),nonstructural protein 1(NS1),a major structural protein(VP),and DNA polymerase(PolB),respectively.VD2 contains two ORFs,encoding nonstructural protein 3(NS3)and minor capsid structural protein(P133).At present,the complete gene transcription strategy of BmBDV is still unknown,which greatly hinders the study of virus infection and replication mechanism.A major impediment to Bidensovirus studies is the lack of permissive insect cells that support virus replication in vitro.Therefore,the viral RNA can be obtained from the larvae midgut post infection.We explored the characterization of the RNA transcription profile of BmBDV by RACE,RT-qPCR and luciferase assays.The transcription of viral genes in different phases was analyzed by RT-PCR,and determined 72 h post infection was the best time to study viral transcription.Then,the RACE experiment was conducted.The size and number of each gene transcripts of the virus were determined by RACE experiments,and the cDNA sequence of the virus was reconstructed.which indicated that VD1 produced four transcripts and VD2 produced two transcripts.Each transcript corresponding to an ORF,alternative splicing of any mRNA transcripts was not observed.For VD1,NS1/2 genes produced two independent transcripts under control of two overlapping promoters P5/5.5,and shared one canonical AAUAAA polyadenylation signal.The VP transcript was driven by P21,which overlapped with the 3' end of the NS transcript by 89 nts.PolB was driven by P97,which overlapped with the 3' end of the VP transcript by 4 nts.For VD2,NS3 and P133 transcripts were driven by P10 and P89,respectively.NS3 and P133 transcripts have no overlap.The RT-qPCR was used to analyze the number of transcripts of NS1/2 genes.Two pairs of primers(F1/R and F2/R)were designed to distinguish transcription differences.Specific primers of the NS2 transcript(F2/R)were used to amplify NS2-specific transcripts.Specific primers of NS transcripts(F1/R)amplified not only NS1 transcripts but also NS2 transcripts.The results of RT-qPCR showed that the copies of the product amplified by primer F2/R was significantly higher(that was more than 50%)than that amplified by primer F1/R.Therefore,it was further proved that the NS1/2 gene produced two independent transcripts.The functions of two overlapping promoters P5/5.5 of NS1/2 genes were studied by dual-luciferase reporter system.We made constructs in which the intact upstream promoter elements and downstream promoter element were cloned into the luciferase reporter plasmid(PGL3-Basic)and the functional elements will be mutated.The constructs were transfected into insect cells and promoter activity was determined using the Dual-Luciferase Reporter Assay System.P5/5.5 either is weak promoter,but its activity is enhanced in the presence of DPE.The NS1 transcripts are under the control of P5/5.5.NS2 ATG may be in a region that is negatively regulated for the P5/5.5 promoters,which has the effect of reducing promoters activities.The co-expression of NS1/2 genes was detected by western.The sequence of NS1/2 genes was cloned into pIB/V5-His vector to construct the co-expression plasmid of NS1/2 genes.The N terminus of NS2 fused HA epitope encoding sequence were amplified by PCR and the NS1 TAG was mutated to TAC,which cloned into pIB/V5-His vector.NS1 and NS2 proteins have been identified by western.It indicated that NS1/2 genes can be co-expressed in Hi5 cells.