Nucleotide sequence and transcriptional analysis of the DNA polymerase gene of Bombyx mori nuclear polyhedrosis virus

Abstract

A gene encoding a putative DNA polymerase (pol) of Bombyx mori nuclear polyhedrosis virus (BmSNPV) was cloned and sequenced. The gene included an open reading frame (ORF) encoding a polypeptide of 988 amino acids with a predicted molecular mass of 114.65 kDa. The deduced amino acid sequence of the BmSNPV pol ORF showed an overall identity of 96 and 45% to those of the Autographa californica NPV (AcMNPV) pol ORF and the Lymantria dispar NPV pol ORF, respectively, and contained sequences conserved in a variety of eukaryotic and viral replicative DNA polymerases. The BmSNPV pol lacked a canonical TATAA element but contained a G+C-rich sequence in the transcriptional initiation region. Analyses by Northern blot hybridization, RNase protection assay, primer extension, and 3' and 5' RACE (rapid amplification of cDNA ends) showed that at least seven different transcripts of approximately 3.1 kb that shared a common 3' end were expressed from the BmSNPV pol. The expression of these transcripts from BmSNPV pol was regulated differentially during virus infection. Transcription of five of the seven species initiated in the close vicinity of and within the motif 5'-GCGTGCT-3'. One transcript placed its initiation site within the motif 5'-AGAGCGT-3' and the remaining one within the motif 5'-GGCGGTGG-3'. The motifs 5'-GCGTGCT-3' and 5'-AGAGCGT-3' have been identified in pol and other genes of AcMNPV as conserved sequences containing transcriptional initiation sites, whereas the motif 5'-GGCGGTGG-3', which is arranged as a direct repeat in BmSNPV pol but not in AcMNPV pol, has not been defined as the sequence responsible for transcriptional initiation sites. The BmSNPV pol transcripts were detectable at 2 hr postinfection (p.i.), peaked at 10 hr p.i., and declined to a low level by 18 hr p.i. The expression of BmSNPV pol was not inhibited but delayed dramatically by the protein synthesis inhibitor cycloheximide upon treatment of infected cells, whereas aphidicolin, an inhibitor of DNA polymerase, inhibited BmSNPV pol transcription. These results suggest a complicated and unique mechanism for the regulation of BmSNPV pol expression.