Molecular cloning and analysis of transcription initiation in the Anagrapha falcifera multiple nucleocapsid...

Using Autographa californica multiple nucleocapsid polyhedrosis virus (AcMNPV) polyhedrin to probe the Southern blots of Anagrapha falcifera multiple nucleocapsid polyedrosis virus (AfMNPV), we identified the location of the AfMNPV polyhedrin gene within the 7.2 kb EcoRI fragment. The 7.2 kb EcoRI fragment of AfMNPV was cloned and the nucleotide sequences of the polyhedrin coding region and its flanking regions were determined. Nucleotide sequence analysis indicated the presence of an open reading frame (ORF) of 735 nucleotides (nt) which could encode 245 amino acids with a predicted molecular mass of 29 kDa. The nucleotide sequences within the coding region of the AfMNPV polyhedrin shared 80% similarity with the polyhedrin gene from AcMNPV but were most closely related to Bombyx mori NPV with 92% sequence identity. The size of the AfMNPV polyhedrin mRNA, determined by the Northern blot analysis, was estimated to be 1200 nt. The consensus promoter sequence (ATAAG) for the baculovirus very late gene was also observed. Two degenerate poly(A) tailing signals were found immediately downstream of the translational stop codon. The transcription initiation site, mapped by primer extension analysis, was found to be at T located 24 nt upstream from the A of the translation initiation codon. This site is located 26 nt downstream from the second A of the consensus TAAG, the transcription initiation site of most other NPVs.