Construction and analysis of an Autographa californica nuclear polyhedrosis virus mutant lacking the polyhedral envelope

Abstract

An Autographa californica nuclear polyhedrosis virus (AcMNPV)-specific protein of 34 kDa (pp34) is shown to be involved in the morphogenesis of the polyhedral envelope of baculoviruses. The region of the AcMNPV genome encompassing EcoRI-H and -S (map positions 82.6–85.8) contains five open reading frames (ORFs) forming one transcriptional unit. The bacterial β-galactosidase ( lacZ) gene was inserted in phase with the N-terminal 12 amino acids of ORF3, thereby intervening this gene. A recombinant (AcMNPV/p34DZ5) was selected by the expression of lacZ (blue plaques). Protein analysis by SDS-PAGE and immunoblotting indicated that Spodoptera frugiperda cells infected with the recombinant lacked pp34. Electron microscopy of recombinant-infected cells showed that the electron-dense “spacers”, normally present in wild-type AcMNPV-infected cells, were also absent. These results indicated that pp34 is dispensable for nonoccluded virus replication and is involved in the morphogenesis of the polyhedral envelope. The recombinants were infectious to fourth instar larvae of Spodoptera exigua. Recombinant polyhedra were more sensitive to weak alkali. This supports the hypothesis that the absence of the polyhedral envelope and the efficient release of occluded virions are responsible for the increased virulence of polyhedral envelope negative mutants.