Location and nucleotide sequence of the 25K protein missing from baculovirus few polyhedra (FP) mutants

Wild-type and few polyhedra (FP) mutants of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) were studied to identify and sequence the gene encoding the 25-kDa (25K) protein normally present in AcMNPV-infected Spodoptera frugiperda cells but which is often missing from FP mutant-infected cells. Our previous study had mapped two overlapping late transcripts to the insertion site of host cell DNA within the HindIII-I fragment (33.8 to 37.7 map units) of wild-type AcMNPV. An FP mutant, AcFP875-2, had a 1.6-kbp insertion of S. frugiperda DNA near the 5' end of these transcripts which by S1 analysis were shown to initiate within the host cell sequence. Primer extension analysis revealed that the transcription start for this gene in wild-type virus occurred within a conserved 12-base sequence found near the transcription start sites of several baculovirus late and hyper-expressed genes. A similar 12-base sequence was found at the transcription start site within this 1.6-kbp pair host cell DNA sequence in AcFP875-2. mRNAs from wild-type virus-infected cells were hybridization-selected using a 542-bp SalI subfragment of the 3.2-kbp EcoRI-HindIII fragment (35.0 to 37.7 map units). These mRNAs directed the synthesis of a 25K protein which in size was identical to the 25K protein in wild-type virus-infected cells and the translation product of a 1.15-kb cRNA transcribed from a RsaI fragment (36.4 to 37.4 map units). Comparison of gel band patterns following partial proteolysis of the translation product of the 1.15 cRNA and the 25K protein from wild-type virus-infected cells revealed that the two proteins were closely related if not identical. Nucleotide sequence analysis within this EcoRI-HindIII fragment revealed an open reading frame which encodes a 25K protein. Insertion of the Escherichia coli lacZ gene encoding the beta-galactosidase enzyme into the transcribed portion of this EcoRI-HindIII fragment yielded a recombinant virus which lacked a 25K protein and exhibited an altered (FP) plaque phenotype.