Identification and in vivo characterization of the Epiphyas postvittana nucleopolyhedrovirus ecdysteroid UDP-glucosyltransferase

The genome of Epiphyas postvittana Nucleopolyhedrovirus (EppoMNPV) contains an ecdysteroid UDP-glucosyltransferase (egt) gene. The egt gene was comple tely sequenced and surrounding open reading frames identified. EppoMNPV egt is 1479 nucleotides in length encoding a protein with a predicted molecula r mass of 55 kDa. Analysis of upstream sequence revealed dual TATA boxes an d two CGT upstream activating region motifs. Mapping of the 5' terminus of the egt transcript identified a major transcript produced from an adenine r esidue 29 nucleotides downstream from the distal TATA box. No transcript wa s detected from a late promoter motif (GTAAG). Characterization of egt tran scripts showed that poly-adenylation occurs at the 3' terminus. EppoMNPV eg t transcripts were first detected in infected Epiphyas postvittana larvae b y Northern hybridization at 9 h post-infection (hpi) and EGT enzyme activit y was detected at 9 hpi in haemolymph from infected larvae. EppoMNPV EGT ca n conjugate the sugars from both UDP-glucose and UDP-galactose to ecdysone in vitro. Localization assays performed using radiolabelled ecdysone demons trated that the conjugation of glucose or galactose, from the respective UD P-sugar, led to the prevention of the uptake of ecdysone by SF-21 cells. We propose that EGT functions to prevent the uptake of ecdysone or 20-hydroxy ecdysone by metabolite and target cells respectively.