Plutella xylostella granulovirus late gene promoter activity in the context of the Autographa californica multiple nucleopolyhedrovirus genome

Abstract

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 (PlxyGV) were cloned into a transient expression vector and the alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome, and compared with homologous late gene promoters of AcMNPV in Sf9 cells. In transient expression assays, all PlxyGV late promoters were activated in cells transfected with the individual reporter plasmids together with an AcMNPV bacmid. In infected cells, reporter gene expression levels with the promoters of PlxyGV e18 and AcMNPV vp39 and gp41 were significantly higher than those of the corresponding AcMNPV or PlxyGV promoters, which had fewer late promoter motifs. Observed expression levels were lower for the PlxyGV p6.9, pk1, gran, p10a, and p10b promoters than for the corresponding AcMNPV 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 AcMNPV 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 PlxyGV gran, p10c, and pk1. The results of this study demonstrated that PlxyGV late gene promoters could be effectively activated by the RNA polymerase from AcMNPV, implying that late gene expression systems are regulated by similar mechanisms in alphabaculoviruses and betabaculoviruses.

IE1 of Autographa californica Multiple Nucleopolyhedrovirus Activates Low Levels of Late Gene Expression in the Absence of Virus RNA Polymerase

“…2A ) were used to transfect Sf9 cells seeded in a 96-well plate (1 μg/well), respectively. vAc Pvp39.luc ( 34 ) were used as a wild-type control. Luciferase activity generated in each well was measured 48 hpt.…”

“…3A ). To identify the factor(s) associated with the activation of the reporter gene expression under the control of Pvp39, the individual cosmid clones were screened by transient assay in which the individual cosmid clones were transfected into Sf9 cells together with a reporter pPacvp39.luc that contained a copy of luc under the control of Pvp39 ( 34 ). As shown in Fig.…”

“…The late expression reporter plasmids pPacvp39.luc, pPacpolh.luc, pPacp6.9.luc, pPace25.luc, pPace18.luc, pPacgp41.luc, and pPgp16.gfp were constructed as described previously ( 34 , 44 ). These plasmids contain a copy of the luciferase gene ( luc ) or enhanced green fluorescence protein gene ( gfp ) under the control of the promoters of the designated AcMNPV late genes.…”

Autographa californica multiple nucleopolyhedrovirus ac75 is required for egress of nucleocapsids from the nucleus and formation of de novo intranuclear membrane microvesicles

“…Plasmids pP acvp39 -luc, pP acgp41 -luc, pP acpk1 -luc, pP acan -luc, pP acp6.9 -luc, pP ace18 -luc and pP acpolh -luc, which separately contain the reporter gene ( luc ) linking with individual promoters of AcMNPV vp39 , gp41 , p6 . 9 , pk1 , an , e18 , and polh [ 15 ], were transformed into E . coli DH10B containing vAc ac75ko to generate reporter bacmids vAc ac75ko-Pvp39-luc , vAc ac75ko-Pgp41-luc , vAc ac75ko-Pp6.9-luc , vAc ac75ko-Ppk1-luc , vAc ac75ko-Pan-luc , vAc ac75ko-Pe18-luc , and vAc ac75ko-Ppolh-luc , and transformed into E .…”