Identification and expression of two baculovirus gp37 genes.

The gp37 genes of the Mamestra brassicae and Lymantria dispar multicapsid nucleopolyhedroviruses (MbMNPV and LdMNPV) have been identified and characterized. Both genes were similar to other baculovirus gp37 genes and to entomopoxvirus fusolin genes. Phylogenetic analysis showed that baculovirus gp37 genes and entomopoxvirus fusolin genes form two distinct and well-separated clades. There was no evidence of recent gene transfer between the two groups. The gp37 genes also showed a distant similarity to bacterial cellulose- and chitin-binding protein genes, but the significance of this is unclear. MbMNPV and LdMNPV gp37 were both transcribed from consensus baculovirus late transcription start sites. MbMNPV gp37 was additionally transcribed from a putative early transcription start site. Tunicamycin treatment of MbMNPV-infected cells confirmed that MbMNPV GP37 is N-glycosylated. Confocal immunofluorescence microscopy revealed that the protein is located exclusively in the cytoplasm, probably in the endoplasmic reticulum.

Identification of baculovirus gene that promotes Autographa californica nuclear polyhedrosis virus replication in a nonpermissive insect cell line.

A gene that promotes Autographa californica M nuclear polyhedrosis virus (AcMNPV) replication in IPLB-Ld652Y cells, a cell line that is nonpermissive for AcMNPV, was identified in Lymantria dispar M nuclear polyhedrosis virus (LdMNPV). Cotransfection of AcMNPV DNA and a plasmid carrying the LdMNPV gene into IPLB-Ld652Y cells results in AcMNPV replication. The gene maps between 43.3 and 43.8 map units on the 162-kbp genome of LdMNPV. It comprises a 218-codon open reading frame and encodes a polypeptide with a predicted molecular mass of 25.7 kDa. The predicted polypeptide is glutamic acid and valine rich and negatively charged, with a pI of 4.61. No protein sequence motifs were identified, and no matches with known nucleotide or peptide sequences were found in the AcMNPV genome or database searches that suggest how this gene might function. A recombinant AcMNPV bearing the LdMNPV gene overcomes a block in protein synthesis observed in AcMNPV-infected IPLB-Ld652Y cells. Using Southern blotting techniques, we were unable to identify a homolog in Orgyia pseudotsugata M nuclear polyhedrosis virus, a baculovirus that is routinely propagated in IPLB-Ld652Y cells. This suggests that the LdMNPV host range is unique among the baculoviruses studied to date. We named this gene hrf-1 (for host range factor 1).

A baculovirus gene with a novel transcription pattern encodes a polypeptide with a zinc finger and a leucine zipper.

An Autographa californica nuclear polyhedrosis virus gene encoding a 30-kilodalton polypeptide with two different sequence motifs characteristic of DNA-binding proteins was identified immediately downstream of the major capsid protein gene (vp39). The gene, CG30, was characterized by sequencing, transcriptional mapping, in vitro translation of hybrid-selected RNA, and comparison of the derived polypeptide sequence with published data bases. The initial ATG of the 792-base-pair CG30 open reading frame is two nucleotides downstream of the vp39 terminal TAA codon. Early transcripts of CG30 initiate within the vp39 coding sequence. At late times, bicistronic transcripts initiate from the vp39 promoter, continue through CG30, and terminate at the same site as the early transcripts. In vitro translation of hybrid-selected early CG30 RNA yields a polypeptide of 30 kilodaltons. The predicted CG30 polypeptide sequence has characteristics of a eucaryotic transcriptional activator and is novel in having two potential DNA-binding domains. A stretch of acidic residues bridges a zinc finger at the amino terminus and a leucine zipper with a flanking basic region at the carboxyl terminus.

Identification, sequence, and transcriptional mapping of the major capsid protein gene of the baculovirus Autographa californica nuclear polyhedrosis virus.

The gene encoding the major capsid protein of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) was identified, sequenced, and transcriptionally mapped. The location of the gene was determined by immunological screening of an expression library of AcMNPV open reading frame-beta-galactosidase fusions with an antibody raised to virus structural proteins. The DNA sequence of the corresponding region, which mapped within 56.6 and 58.0 map units on the AcMNPV genome, revealed a 1,040-base-pair open reading frame capable of encoding a 39-kilodalton polypeptide. The identity of the polypeptide was determined by Western blot (immunoblot) analysis of purified empty capsids with an antibody raised to the capsid-beta-galactosidase fusion protein. The identity of the peptide encoded by the gene was confirmed by immunoprecipitation of an in vitro translation product with RNA selected by hybridization to DNA sequences from the coding region of the gene. Transcripts of the capsid gene were analyzed by Northern (RNA) blots and mapped by nuclease protection and primer extension analysis. The capsid gene is transcribed maximally at 12 and 24 h postinfection but not in the presence of cycloheximide, a protein synthesis inhibitor, or aphidicolin, a viral DNA synthesis inhibitor, and is therefore classified as a late gene. The gene is transcribed in a counterclockwise direction with respect to the circular map. There are three transcriptional start sites, all containing the AGTAAG consensus sequence found at the start site of all late AcMNPV genes.