Insertion of the SfMNPV polyhedrin gene into an AcMNPV polyhedrin deletion mutant during viral infection

Abstract

The Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin deletion mutant, d10A, was cotransfected with the recombinant plasmid SfMNPV.HindIII-L, which contains the Spodoptera frugiperda nuclear polyhedrosis virus (SfMNPV) polyhedrin gene. An occlusion-positive hybrid virus was obtained which contained a DNA insertion at approximately 13 map units on the AcMNPV.d10A genome. A fine structure restriction map of cloned viral DNA fragments from this region revealed that most of the 1.7-kb AcMNPV.PstI-O fragment (13.4-14.7 map units) had been replaced with a 3.7-kb fragment, containing the SfMNPV polyhedrin gene with flanking sequences and the entire pUC8 plasmid. Subclones of this region were sequenced and the exact site of insertion was determined. Flanking the insert were 43 and 60 bp of unknown origin at the 5' and 3' ends, respectively. Transcription was examined in the region of the insertion in both wild-type AcMNPV and the AcMNPV/SfMNPV (Ac/Sf) hybrid. In the AcMNPV, a nested set of seven overlapping transcripts ranging from 2.2 to 5.3 kb was found, each with coterminal 3' ends. Only the 2.2-kb transcript was found to be expressed early and throughout infection. The SfMNPV insertion interrupted this transcriptional unit and produced a more complex pattern of transcription. Alterations included a nested set of three overlapping transcripts with coterminal 5' ends, including the SfMNPV polyhedrin mRNA, transcripts originating in AcMNPV and terminating in either SfMNPV or pUC8 sequences, and other minor transcripts. The SfMNPV polyhedrin gene was sequenced and the locations of the 5' and 3' ends of polyhedrin mRNA were mapped. An analysis of SfMNPV polyhedrin protein expression showed that the SfMNPV polyhedrin gene in the hybrid virus was expressed at approximately one-fourth the level of the wild-type AcMNPV polyhedrin gene. Expression of a beta-galactosidase gene under the control of the SfMNPV polyhedrin promoter in the AcMNPV.d10A mutant was also investigated.