Viral Replication Mechanism Of HvAV-3h Regulated By Their Early Genes

Early genes of double-strand DNA(ds DNA)virus are genes transcribed prior to the viral DNA replication,and they are involved in the activation of viral gene transcriptional cascade in the viral initial infectious stages.Early genes play important roles in virus replication and infection.Ascoviruses are insect specific ds DNA virus with great potential for biological control,and they are safe to non-target organisms.Besides,ascovirus exhibited outstanding ability of rapid and efficient DNA replication in the previous studies.The pathogenesis of ascovirus is significantly different from that of other ds DNA insect viruses(such as baculovirus),and the mechanism of ascoviral DNA replication has not been clarified,furthermore,the viral replication is closely related to the transcription and expression of early genes,thus,this study was performed to explore the molecular features and functions of early genes of Heliothis virescens ascovirus 3h(HvAV-3h),so as to lay a foundation for revealing the efficient DNA replication mechanism of ascovirus.The main results of this study are as follows:1.HvAV-3h early genes and sequence analysis: DNA polymerase inhibitor was used to inhibit the replication of HvAV-3h on Spodoptera exigua fat body cell line(Se FB).A total of25 early genes were screened.All early genes were transcribed at 3 h after HvAV-3h infection.Analyzed the sequence of early gene encoded protein,its relative molecular weight was between 11 and 145 k Da,the theoretical isoelectric point ranged from 5.0 to 9.5,and the secondary structure was mainly composed of strand,helix,and coil.Sequence homology analysis showed that early gene has a high degree of similarity with the virus strain of Heliothis virescens ascovirus.2.Transcription pattern of HvAV-3h early genes: Using 5’ RACE and 3’ RACE technology to obtain the non-coding region sequence of early genes.The results showed that early genes contain multiple transcription start sites and multiple transcription termination sites.It has a sequence of poly A at the start region of 5’ end.The A + T content of 5’untranslated region is higher than the G + C content.5’ untranslated region contains the early initiation elements TATA box and CAGT,and the late initiation element TAAG.And a highly conserved motif of AAAATTGATT at 40～90 nt upstream of the start codon.However,none of the early genes had a poly(A)structure at the 3’ end.The secondary structure of 3’untranslated region showed a complex stem-loop structure.In addition,using insect expression vector p IZ/V5-His to construct a eukaryotic expression vector control by early genes(contains 5’ untranslated region and translated region),transfected cells in vitro and found that early genes can be transcribed independently of genomic DNA.3.Spatio-temporal distribution and protein expression phase of HvAV-3h early genes:Using the prokaryotic expression vector p ET-28a(+)to construct a prokaryotic expression vector to induce,express,purify and prepare polyclonal antibodies for the early gene encoded protein.The polyclonal antibodies of 20 early genes encoding proteins were prepared.Detected the protein expression phases by Western Blot and the results showed that ORF19,ORF23,ORF47,ORF52,ORF115,ORF126,ORF129,ORF152 and ORF179 were expressed at 24～96 hpi(hours post infection).ORF174 expressed at 0～96 hpi.Detected the distribution of early genes on virions and found that the transcripts of orf8,orf20,orf23,orf33,orf47,orf52,orf115,orf129,orf132,orf164,orf172,orf173,orf174,orf179 and orf185 exist on HvAV-3h virions.ORF19,ORF23,ORF47,ORF52,ORF115 and ORF152 proteins exist on virus particles and which means these proteins are structural proteins.In addition,the expression of ORF33,ORF36,ORF81,ORF124,ORF132,ORF153,ORF164,ORF172,ORF173 and ORF185 was not detected.4.HvAV-3h early genes regulate viral replication at the in vitro level: Synthesize ds RNA of early genes and perform RNAi interference on Se FB infected HvAV-3h.The results show that the early genes have positive and negative regulatory effects on virus replication at the in vivo level.The silencing of orf19,orf23,orf52,orf115,orf129 and orf179 lead to the replication of HvAV-3h inhibited significantly.orf126 and orf152 silenced increased the virus genomic DNA which means promote the replication of HvAV-3h.orf174 silenced increased virus replication significantly at 6 and 12 hpi,and decreased significantly at 48 hpi.5.HvAV-3h early genes regulate viral replication at the in vivo level: The prepared antiserum of the early gene encoded protein was used to block with HvAV-3h.Then using the blocked virus to infected S.exigua.The results show that the early genes also have positive and negative regulatory effects on virus replication at the in vitro level.The virus replication began to change significantly at 48 hpi.ORF52,ORF126 and ORF152 antisera can significantly inhibit the replication of HvAV-3h in S.exigua;ORF19,ORF47,ORF129,ORF174 and ORF179 antisera can significantly promote the replication of HvAV-3h in S.exigua.ORF23 and ORF115 antisera have no effect on HvAV-3h replication.Analysis the survival curve of infected S.exigua found that after HvAV-3h blocked with ORF47,ORF126 and ORF152 antisera,the survival rate of larvae significantly decreased and the survival time was shortened.