Sequence and in vivo transcription of Lacanobia oleracea granulovirus egt

We have determined the nucleotide sequence and located the major in vivo transcript termini of the Lacanobia oleracea granulovirus (LoGV) egt gene. The open reading frame encodes a 460-amino acid polypeptide having extensive sequence similarity to ten nucleopolyhedrovirus (NPV) ecdysteroid UDP-glucosyltransferase (EGT) proteins; the degree of similarity is particularly high within several previously identified EGT 'domains', and eight invariant amino acid residues are conserved. A phylogenetic tree, constructed by the neighbour joining method, showed LoGV EGT to be the most highly diverged of the eleven baculovirus sequences compared. Database searching revealed that part of a published DNA sequence from Cryptophlebia leucotreta granulovirus appears to encode the N-terminal region of EGT, and the relative genomic locations of the egt and granulin genes in that virus were compared with their positions in LoGV. In infected L. oleracea larvae, egt is transcribed predominantly as a 1.6 kb mRNA. Primer extension analysis suggested that the major egt 5' transcription terminus is located within a baculovirus late gene promoter motif (GTAAG), in contrast to the early gene promoter contexts determined by others for three NPV egt mRNA 5' ends. An early transcriptional start site is also used in LoGV egt expression, but at a much lower level. The 3' terminus of egt mRNA was identified by sequencing DNA fragments generated by rapid amplification of cDNA ends, and is located 58-62 nucleotides beyond the translation stop codon.