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

Abstract

The genome of Epiphyas postvittana Nucleopolyhedrovirus (EppoMNPV) contains an ecdysteroid UDP-glucosyltransferase (egt) gene. The egt gene was completely sequenced and surrounding open reading frames identified. EppoMNPV egt is 1479 nucleotides in length encoding a protein with a predicted molecular mass of 55 kDa. Analysis of upstream sequence revealed dual TATA boxes and two CGT upstream activating region motifs. Mapping of the 5' terminus of the egt transcript identified a major transcript produced from an adenine residue 29 nucleotides downstream from the distal TATA box. No transcript was detected from a late promoter motif (GTAAG). Characterization of egt transcripts showed that poly-adenylation occurs at the 3' terminus. EppoMNPV egt transcripts were first detected in infected Epiphyas postvittana larvae by Northern hybridization at 9 h post-infection (hpi) and EGT enzyme activity was detected at 9 hpi in haemolymph from infected larvae. EppoMNPV EGT can conjugate the sugars from both UDP-glucose and UDP-galactose to ecdysone in vitro. Localization assays performed using radiolabelled ecdysone demonstrated that the conjugation of glucose or galactose, from the respective UDP-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-hydroxyecdysone by metabolite and target cells respectively.