Identification and characterization of the IE-1 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus.
The IE-1 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) was mapped between 95.7 and 97.1 map units on the viral genome. Sequence analysis of the OpMNPV IE-1 gene (OpIE-1) identified an open reading frame that coded for a predicted protein of 560 amino acids with a molecular weight of 64,775. Transcriptional analysis of OpMNPV-infected Lymantria dispar (LD652Y) cells identified two RNAs homologous to the OpIE-1 open reading frame that were 1.7 and 1.9 kb in size. The 1.7-kb transcript could be detected by 0 hr postinfection (hr p.i.) and the steady-state levels increased up to 48 hr p.i. The 1.9-kb message appears to be spliced and has peak expression from 4 to 6 hr p.i. but can still be detected at late times p.i. Comparison of the OpIE-1 and Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) IE-1-predicted proteins revealed that the N-terminal region had very low sequence identity (21%) but had maintained an acidic profile, whereas the C-terminal region showed 55% amino acid identity. Transient assay analysis showed that OpIE-1 was able to trans-activate the AcMNPV delayed early reporter gene construct p39CAT in both LD652Y cells and Spodoptera frugiperda (Sf9) cells. The expression of p39CAT trans-activated by OpIE-1 was also found to be enhanced by the AcMNPV hr enhancer sequences. The OpIE-1 promoter was linked to the chloramphenicol acetyl transferase gene and deletion analysis was used to identify regions involved in the regulation of this gene. This analysis revealed that the OpIE-1 promoter contained regions that were responsive to a transcriptional activator that was specific to Sf9 cells. In addition it was shown that OpIE-1 could trans-activate its own promoter and that for maximal expression this required sequences between -420 and -330 relative to the transcriptional start site. These data suggest that OpIE-1 is autoregulated during normal viral infection of insect cells.
Theilmann
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Agriculture Canada Research Station, Vancouver, British Columbia.
Stewart
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D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D005814:Genes, Viral; D007313:Insecta; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016366:Open Reading Frames; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D015719:Single-Strand Specific DNA and RNA Endonucleases