title: Characterization of an early gene accelerating expression of late genes of the baculovirus Autographa californica nuclear polyhedrosis virus.
abstract: The region of the Autographa californica nuclear polyhedrosis virus (AcMNPV) encompassing the EcoRI T fragment (29.0 to 30.1 map units) was characterized by DNA sequencing, transcriptional mapping, and site-directed mutagenesis. The largest transcript from this region, an early 1.7-kilobase (kb) poly(A)+ RNA, encompassed three tandem, nonoverlapping open reading frames (ORFs). The largest of these ORFs, ETL, was proximal to the 5' end of the transcript and had the capacity to encode a 28-kilodalton (kDa) polypeptide. A recombinant virus, vETL beta gal, containing the Escherichia coli beta-galactosidase (beta gal) gene fused to the N-terminal two-thirds of the ETL ORF, produced blue plaques in the presence of a chromogenic indicator of beta gal and wild-type levels of polyhedra in cell culture. This recombinant was also infectious in insect larvae by oral administration of occluded virus. Comparison of vETL beta gal and wild-type viral proteins pulse-labeled at various times postinfection (p.i.) revealed (i) absence of a virus-induced 28-kDa polypeptide, (ii) early expression of a large (approximately 130-kDa) polypeptide which may be the ETL-beta gal fusion protein, (iii) a delay in expression of early 35 and 40-kDa polypeptides, and (iv) a 4- to 6-h delay in the expression of late proteins in vETL beta gal-infected cells. Cycloheximide did not inhibit synthesis of the 1.7-kb RNA but did inhibit its shutoff, which occurs at 12 h p.i. in the absence of inhibitors. Thus, the ETL gene product is apparently an early 28-kDa protein which is necessary, directly or indirectly, for timely expression of many other AcMNPV genes. The promoter-leader regions of the 1.7-kDa transcript showed significant sequence similarities to the leader of the AcMNPV IE-1 gene. The middle ORF within the 1.7-kb transcript, ETM, would encode a hydrophobic polypeptide of 113 amino acid residues. ETS, a small ORF within and proximal to the 3' end of the 1.7-kb transcript, was also transcribed as a set of smaller (approximately 0.5-kb) RNAs initiated heterogeneously in the region between ETL and ETS and persisting throughout infection.
authors.affiliation: Department of Genetics, University of Georgia, Athens 30602.
authors.forename: A M
authors.initials: AM
authors.lastname: Crawford
authors.forename: L K
authors.initials: LK
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003513:Cycloheximide / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects; D005814:Genes, Viral; D007304:Insect Viruses / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012333:RNA, Messenger / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D014764:Viral Proteins; D003513:Cycloheximide