The nucleotide sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was completed and analyzed. It is composed of 131,990 bases with a G + C content of 55% and contains 152 putative genes of 150 nucleotides or greater. Major differences in gene content and arrangement between OpMNPV and the Autographa californica MNPV were found. These include the presence in OpMNPV of three complete iap gene homologs, two conotoxin gene homologs, two protein tyrosine phosphatase homologs, and genes encoding homologs of dUTPase and the large and small subunits of ribonucleotide reductase. Seven major intergenic repeated regions were identified. Five of these are homologous regions that are related to similar regions from other baculoviruses.

To investigate the regulation of pl0 and polyhedron envelope protein (PEP) gene expression and their role in polyhedron development, Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis viruses lacking these genes were constructed. Recombinant viruses were produced, in which the pl0 gene, the PEP gene or both genes were disrupted with the fl-glucuronidase (GUS) or fl-galactosidase (lacZ) genes. GUS activity under the control of the PEP protein promoter was observed later in infection and its maximal expression was less than 10% the level for pl0 promoter-GUS constructs. Tissues from O. pseudotsugata larvae infected with these recombinants were examined by electron microscopy. Cells from insects infected with the pl0 viruses lacked pl0-associated fibrillar structures, but fragments of polyhedron envelope-like structures were observed on the surface of some polyhedra. Immunogold labelling of cells infected with the pl0-GUS + virus with an antibody directed against PEP showed that the PEP was concentrated at the surface of polyhedra. Although polyhedra produced by pl0 and PEP gene deletion mutants demonstrated what appeared to be a polyhedron envelope by transmission electron microscopy, scanning electron microscopy showed that they had irregular, pitted surfaces that were different from wild-type polyhedra. These data suggested that both pl0 and PEP are important for the proper formation of the periphery of polyhedra.

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Two types of envelope fusion proteins have been identified in lepidopteran baculoviruses. GP64 is found in Autographa californica multinucleocapsid nucleopolyhedrovirus, Orgyia pseudotsugata multinucleocapsid nucleopolyhedrovirus (OpMNPV), and other relatively closely related viruses, while Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV), which lacks GP64, utilizes LD130 as its envelope fusion protein. Homologs of ld130 have since been found not only in all the sequenced gp64-minus virus genomes, but also in the genomes of gp64-containing viruses. In addition, they are evolutionarily related to the envelope proteins of certain insect retroviruses. In this report, the characterization of a LD130 homolog (OP21) from OpMNPV, which also contains gp64, is described. Western blot analysis of extracts of OpMNPV-infected Lymantria dispar cells, using antibodies generated against OP21, identified an infected cell-specific doublet of 85 and 89 kDa. These bands were first observed at about 6 h p.i. and were present at all later time points. Such analyses also demonstrated that OP21 was associated with budded virions. Tunicamycin treatment of OpMNPV-infected cells indicated that OP21 is N-glycosylated. Studies employing NP-40 to remove the envelope from budded virions indicated that the majority of OP21 remained associated with the nucleocapsid fraction, whereas all GP64 was removed. Confocal immunofluorescence microscopy showed that OP21 and GP64 have a similar pattern of distribution on the membrane of cells infected with OpMNPV. Immunoelectron microscopy of budded virions also showed similar patterns of localization for both OP21 and GP64.

Polyclonal antiserum produced against preoccluded virions from the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) was used to screen an OpMNPV lambda gt11 expression library. The insert from one of the immunoreactive phage isolates hybridized to OpMNPV orf86 (p91), a 2460-bp (819 amino acids) open reading frame that encodes a predicted protein of 91 kDa. Antibodies generated against a maltose binding protein-P91 fusion detected a band of approximately 91 kDa on Western blots of extracts of OpMNPV-infected Lymantria dispar cells. This band was first observed at 18 hr p.i. and was present at all later time points. Similar results using this antiserum were seen with a time course of Autographa californica-infected Spodoptera frugiperda cells. Localization of P91 by confocal immunofluorescence microscopy showed that the protein was concentrated near the nuclear membrane and at late times p.i. was most concentrated near polyhedra. Immunoelectron microscopy indicated that P91 was present in both the capsid and envelope surrounding the capsid of occlusion-derived virions. Fractionation studies employing NP-40 and Western blot analysis indicated that P91 was associated with the capsid structure.

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An open reading frame homologous to AcMNPV ORF9 (ORF1629) was characterized in the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV). Sequence analysis indicated that the OpMNPV homolog (called ORF2) encoded a protein predicted to contain 545 amino acids with a molecular weight of 61 kDa. The first 80 amino acids did not have a counterpart in the AcMNPV homolog. The remainder of the ORF was poorly conserved with 29% amino acid identity overall with the AcMNPV ORF. However, the amino terminal 150 amino acids of AcMNPV ORF9 demonstrated about 45% amino acid sequence identity with OpMNPV ORF2 and conserved runs of proline residues were present in internal regions of both molecules. Transcriptional mapping indicated the ORF2 transcripts were initiated at a late promoter sequence, ATAAG, beginning about 24 hr p.i. These transcripts terminated near the 3' end of the ORF2 reading frame. Antibodies were produced against a fusion protein derived from the bacterial gene encoding the maltose binding protein and most of the ORF2 sequence. These antibodies reacted with a protein of 69 kDa on Western blots and the protein was found to be associated with virions isolated from both polyhedra and budded virus. The OpMNPV ORF2 antiserum also reacted with the AcMNPV ORF9 gene product. Immunoelectron microscopic analyses indicated that ORF2 was associated with the ends of the capsids which contain the basal structure. This end appears to be oriented away from both the virogenic stroma and membranes involved in intranuclear envelopment. In addition, as virions bud from the nucleus into the cytoplasm, this end also appears to be oriented away from the nuclear membrane.

The envelope fusion protein from a baculovirus pathogenic for Lymantria dispar was characterized. N-terminal sequence analysis determined that it was cleaved downstream of predicted signal peptide and furin cleavage motifs. Mutation of the furin motif resulted in a protein that was not cleaved and did not mediate fusion. Mutagenesis of three charged amino acids in a conserved sequence with the features of a fusion peptide resulted in significant reduction of the ability of the constructs to mediate fusion. None of the mutations inhibited transport of the proteins to the cell surface. In addition, the mutations of the predicted fusion peptide region yielded no inhibition of cleavage. No difference in cleavage was detected between constructs expressed in Spodoptera frugiperda or L. dispar cells.