A 25-kDa Protein Is Associated with the Envelopes of Occluded Baculovirus Virions

Abstract

Antiserum produced against preoccluded virions of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was used to screen an OpMNPV λgt11 expression library. One of the immunoreactive clones contained an insert that hybridized to a portion of the OpMNPV HindIII-P fragment. A 2-kb region of HindIII-P was sequenced and found to contain three open reading frames, each preceded by a late promoter element. The insert from the λgt11 clone was derived from the third open reading frame, which encodes a predicted protein of 25 kDa. The λgt11 insert was cloned into a pMALcR1 bacterial expression vector and the fusion protein was expressed, isolated, and used for antibody production. This antiserum detected a doublet of approximately 25 kDa on Western blots of a time course of OpMNPV-infected Lymantria dispar cells. The protein was first detected at low concentration by 18 hr p.i.; by 36 hr p.i., the protein concentration had increased significantly and remained at this level for the duration of the time course. Similar results were seen on Western blots of Autographa californica MNPV (AcMNPV)-infected Spodoptera frugiperda cells. No evidence of O- or N-linked glycosylation of the OpMNPV p25 was found. Immunoelectron microscopy showed that p25 was present in the nuclei of OpMNPV-infected cells and localized to the envelopes surrounding polyhedron-derived virions.