Expression Activities Of Late Gene Promoters Of Plutella Xylostella Granulovirus In An Non-permissive Cell Line

Baculoviridae is a family of viruses with rod-shaped,enveloped virions containing single circular,supercoiled,double-stranded DNA genomes.Plutella xylostella granulovirus?PlxyGV?belongs to betabaculovirus genus.Advance in research of molecular biology on PlxyGV has been relatively slow,due to unavalibility of stable permissive cell lines for in vitro replication of the virus.In this study,activities of a group of PlxyGV putative late expressed genes in a non-permissive cell line were analyzed in order to find out causes restraining in vitro replication of PlxyGV.?1?Reporter plasmids of ten PlxyGV putative late genes?an,e18,e25,gp41,p6.9,vp39,pk1,gran,orf21 and orf50?,which contained the coding sequence of the luciferase gene under control of the promoters of the individual viral genes,were constructed and used in transient assays with Sf9 cells that were non-permissive PlxyGV.The levels of the luciferase a ccumulated in the transfected cells were measured to evaluate activities of the indicadual late gene promoters.We found that the PlxyGV late promoters were not activated in the cells transfected by the indvadual reporter plasmids alone or together with a PlxyGV bacmid,which contained the complete genome of PlxyGV except the granuline gene.In contrast,significant levels of luciferase activity were detected in the cells transfected with the individual reporter plasmids together with AcMNPV bacmid bMON14272.?2?For comparison in expression activities of the late gene promoters between PlxyGV and AcMNPV,reporter plasmids containing luciferase gene under control of the individual promoters of corresponding AcMNPV homologous late genes were also constructed and used in transient expression assays.It was found that luciferase activities detected in the cells transfected with the individual PlxyGV late gene reporter plasmids and bMON14272 were close to or even higher than those in the cells with the corresponding AcMNPV late gene reporter plasmids.?3?The individual late promoter-luc chemira of PlxyGV and AcMNPV were inserted into bMON14272 separately to make reporter viruses for real-time analysis of expression activities of PlxyGV late promoters in comparison with the ones of AcMNPV,in context of AcMNPV genome.We found that pxPgp41?or pxPgp41L?and acPgp41,pxPvp39?or pxPvp39L?and acPvp39,pxPe18 with acPe18?or acPe18L?,pxPorf50 with acPp10 four groups,although the corresponding number contained in the core promoter motifs may be equal,but they trends differences activity changes with time significantly.pxPan and acPan,pxPpk1 with acPpkl two groups,there are significant differences in the level of the corresponding promoter activity of expression in the recombinant virus in infected cells,but they are substantially the same trends over time.And acPp6.9 and pxPp6.9,acPe25 and pxPe25,acPpolh and pxPgran,pxPorf21 and acPp10,these four groups,expressing activity of the corresponding promoter in the recombinant virus in infected cells have the same changes with time.?4?PlxyGV reporter bacmids containing p6.9 promoter-luc or gran promoter-luc and recombinant AcMNPV bacmids with ie1,lef8,p35 or vp39 deleted,were constructed.The PlxyGV reporter bacmids were transfected,together with ithe ndividual AcMNPV recombinant bacmids,into Sf9 cells to test effects of deletion of the AcMNPV genes on reporter gene expression.It was shown that high levels of luciferase activity were detected in the cells tranfected with either one of the PlxyGV reporter bacmids together with the vp39-knockout AcMNPV recombinant.In contrast,the luciferase activities detected in the cells transfected with the individual PlxyGV reporter bacmids together with the ie1-knockout AcMNPV recombinant were only lightly higher than those in the cells with individual reporter bacmids alone.The luciferase activities detected in the cells with the PlxyGV p6.9 reporter bacmid and the lef8 knockout or p35-knockout AcMNPV recombinant were 0.98 and 34.49 percent of that in the cells with the reporter bacmid and the vp39-knockout AcMNPV recombinant respectively.Similarly,the levels of luciferase activity detected in the cells with the PlxyGV gran reporter plasmid and the lef8-knockout or p35-knockout AcMNPV recombinant were 0.29 and 28.49 percent of the level in the cells with the reporter plasmid and the vp39-knockout AcMNPV recombinant respectively.These results suggested that the reporter gene expression from the PlxyGV late promoters in transfected Sf9 cells mainly replied on AcMNPV-encoded RNA polymerase,and that the native PlxyGV factors required for late genes expression were not efficiently produced even in presence of AcMNPV genome.?5?To determine if PlxyGV DNA replication could be rescued AcMNPV genome in the cells containing both PlxyGV and AcMNPV genomes,three different PlxyGV bacmids were transfected into Sf9 or Hi5 cells alone or together with an AcMNPV bacmid,and the DNA generated were analyzed by PCR in combination with DpnI digestion.As a result,DNA of PlxyGV synthesized after transfection were detected in the cells with the PlxyGV bacmid vPxG and the vp39-knockout or lef8-knockout AcMNPV recombinant.After co-transfection,we extraction the genome.Through PCR analysis we found that,PlxyGV genome can be replicated both in Sf9 cells and in Hi5 cells;AcMNPV ie1?p35 and gp64 could enhance the replication of its genome.In the presence of vAc^lef8KO or vAc^vp39KO,PlxyGV genome replication was further strengthened.