The gene encoding gp64, the envelope glycoprotein of the budded virus (BV) of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV), was mapped to the HindIII-E fragment of of the viral genome and expression of the gp64 gene was examined at various times postinfection. To locate the gp64 gene, a cross-reacting monoclonal antibody (AcV5) (A. W. Hohmann and P. Faulkner, 1983, Virology, 125, 432-444) directed against the gp64 protein of the Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) was used to screen a lambda gt11 expression library of OpMNPV and insert DNAs from immunopositive recombinants were used for Southern hybridization mapping. The gp64 gene was sequenced and transcription of the gp64 gene was examined by Northern blot, S1 nuclease, and primer extension analysis. Two sets of gp64 transcripts were detected during infection: a single early transcript which initiated at -43 nt and four late transcripts which initiated at -152, -167, -174, and -175 nt relative to the start of the gp64 open reading frame. Comparison of the gp64 early transcription initiation site with several other early baculovirus genes revealed a four-nucleotide consensus sequence (CAGT) which is conserved at the early transcription initiation sites of the IE-1 and 39K genes. The four late gp64 transcripts initiated at two of the four upstream ATAAG motifs. All gp64 mRNAs appear to be coterminal at the 3' end. Analysis of the nucleotide sequence of the gp64 gene revealed that the late gp64 mRNAs are bicistronic, consisting of a three amino acid minicistron located 70 nt upstream of the 509 amino acid gp64 open reading frame. Early transcripts do not contain the minicistron. The 1527-nt gp64 open reading frame encodes a predicted protein of 509 amino acids with a molecular weight of 58 kDa. The predicted gp64 protein contains seven potential N-linked glycosylation sites and hydrophobic N- and C-termini characteristic of signal and membrane anchor sequences found on envelope glycoproteins. By western blot analyses and indirect immunofluorescence microscopy, we show that the gp64 protein is present at early times (6 hr) postinfection and accumulates in the infected cell, moving to the periphery at later times postinfection. Western blot comparisons of the temporal expression of the gp64 protein with the p39 capsid protein revealed that these two virion structural protein genes differ significantly in the timing of their initial expression. The upstream regulatory regions, open reading frames, and predicted proteins from the OpMNPV and AcMNPV gp64 genes were compared.(ABSTRACT TRUNCATED AT 400 WORDS)