A mechanism for negative gene regulation in Autographa californica multinucleocapsid nuclear polyhedrosis virus.
The Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) ie-1 gene product (IE-1) is thought to play a central role in stimulating early viral transcription. IE-1 has been demonstrated to activate several early viral gene promoters and to negatively regulate the promoters of two other AcMNPV regulatory genes, ie-0 and ie-2. Our results indicate that IE-1 negatively regulates the expression of certain genes by binding directly, or as part of a complex, to promoter regions containing a specific IE-1-binding motif (5'-ACBYGTAA-3') near their mRNA start sites. The IE-1 binding motif was also found within the palindromic sequences of AcMNPV homologous repeat (hr) regions that have been shown to bind IE-1. The role of this IE-1 binding motif in the regulation of the ie-2 and pe-38 promoters was examined by introducing mutations in these promoters in which the central 6 bp were replaced with BglII sites. GUS reporter constructs containing ie-2 and pe-38 promoter fragments with and without these specific mutations were cotransfected into Sf9 cells with various amounts of an ie-1-containing plasmid (pIe-1). Comparisons of GUS expression produced by the mutant and wild-type constructs demonstrated that the IE-1 binding motif mediated a significant decrease in expression from the ie-2 and pe-38 promoters in response to increasing pIe-1 concentrations. Electrophoretic mobility shift assays with pIe-1-transfected cell extracts and supershift assays with IE-1-specific antiserum demonstrated that IE-1 binds to promoter fragments containing the IE-1 binding motif but does not bind to promoter fragments lacking this motif.
Leisy
D J
DJ
Department of Microbiology, Oregon State University, Corvallis 97331-7301, USA. leisyd@bcc.orst.edu
Rasmussen
C
C
Owusu
E O
EO
Rohrmann
G F
GF
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D017874:Immediate-Early Proteins / Q000235:genetics* / Q000378:metabolism; D009036:Moths / Q000821:virology; D009154:Mutation; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011401:Promoter Regions, Genetic; D018411:Spodoptera / Q000166:cytology; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014764:Viral Proteins
C069183:Ac-IE-1 protein, Autographa californica nucleopolyhedrovirus; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D015534:Trans-Activators; D014764:Viral Proteins; C072773:IE2 protein, polyhedrosis virus