摘要
Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus(Plxy GV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus(Ac MNPV) genome, and compared with homologous late gene promoters of Ac MNPV in Sf9 cells. In transient expression assays, all Plxy GV late promoters were activated in cells transfected with the individual reporter plasmids together with an Ac MNPV bacmid. In infected cells, reporter gene expression levels with the promoters of Plxy GV e18 and Ac MNPV vp39 and gp41 were significantly higher than those of the corresponding Ac MNPV or Plxy GV promoters,which had fewer late promoter motifs. Observed expression levels were lower for the Plxy GV p6.9,pk1, gran, p10 a, and p10 b promoters than for the corresponding Ac MNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the Ac MNPV polh, p10,and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of Plxy GV gran, p10 c, and pk1. The results of this study demonstrated that Plxy GV late gene promoters could be effectively activated by the RNA polymerase from Ac MNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.
Within Baculoviridae, little is known about the molecular mechanisms of replication in betabaculoviruses, despite extensive studies in alphabaculoviruses. In this study, the promoters of nine late genes of the betabaculovirus Plutella xylostella granulovirus（Plxy GV） were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus（Ac MNPV） genome, and compared with homologous late gene promoters of Ac MNPV in Sf9 cells. In transient expression assays, all Plxy GV late promoters were activated in cells transfected with the individual reporter plasmids together with an Ac MNPV bacmid. In infected cells, reporter gene expression levels with the promoters of Plxy GV e18 and Ac MNPV vp39 and gp41 were significantly higher than those of the corresponding Ac MNPV or Plxy GV promoters,which had fewer late promoter motifs. Observed expression levels were lower for the Plxy GV p6.9,pk1, gran, p10 a, and p10 b promoters than for the corresponding Ac MNPV promoters, despite equal numbers of late promoter motifs, indicating that species-specific elements contained in some late promoters were favored by the native viral RNA polymerases for optimal transcription. The 8-nt sequence TAAATAAG encompassing the ATAAG motif was conserved in the Ac MNPV polh, p10,and pk1 promoters. The 5-nt sequence CAATT located 4 or 5 nt upstream of the T/ATAAG motif was conserved in the promoters of Plxy GV gran, p10 c, and pk1. The results of this study demonstrated that Plxy GV late gene promoters could be effectively activated by the RNA polymerase from Ac MNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.
基金
supported by the fund of Hubei Key Laboratory of Genetic Regulation and Integrative Biology
