Functional analysis of a 603 nucleotide open reading frame upstream of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus

Abstract

The sequence of the 2000 nucleotides immediately upstream of the polyhedrin gene of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus has been determined. Comparative analysis of the data identified a 603 nucleotide open reading frame (ORF) separated from the polyhedrin gene coding sequences by 156 nucleotides and in the opposite strand of DNA. Northern hybridization analysis of polyadenylated RNA from infected cells highlighted a 3.7 kb species produced maximally at 12 h post-infection, but not in the presence of cycloheximide. Preliminary nuclease S1 analysis of the 5' end of this RNA suggested that it initiated at a position very close to that of the polyhedrin mRNA start site. Deletion of a portion of the ORF 603 from viruses containing the normal polyhedrin gene and the lacZ gene in lieu of polyhedrin did not affect replication in cell culture or the production of beta-galactosidase protein. A virus which lacked the ORF 603 gene but produced polyhedrin had similar infectivity in Trichoplusia ni larvae compared to the wild-type virus. The chloramphenicol acetyltransferase (CAT) gene was also inserted in lieu of the ORF 603 in a virus containing the lacZ gene instead of the polyhedrin (Ac.CAT.lacZ). Analysis of CAT expression revealed that a maximum level was reached at 16 h p.i. and that transcription was initiated in Ac.CAT.lacZ at the same site as for the normal gene.