Transcriptional enhancer activity of hr5 requires dual-palindrome half sites that mediate binding of a dimeric form of the baculovirus transregulator IE1.
The hr5 enhancer element stimulates early viral transcription and may function as an origin of DNA replication for Autographa californica nuclear polyhedrosis virus (AcMNPV). The smallest functional unit of hr5 is a 28-bp repeat consisting of an imperfect palindrome (28-mer). To identify essential sequences and examine the molecular basis of hr5 activity, the effects of site-directed mutations on transcriptional enhancement by the 28-mer and binding of the AcMNPV transregulator IE1 were investigated. In transfection assays and infections with AcMNPV recombinants, activation of a basal viral promoter required sequences within both halves of the 28-mer. Basal promoter activation also required a critical spacing between these half sites. Mobility shift assays indicated that hr5 probes containing a single 28-mer were bound by in vitro-synthesized IE1. Competition assays using DNA fragments that contained mutated 28-mers demonstrated that both half sites were required for optimal binding of IE1. Similar assays using mutated 28-mer DNAs and nuclear extracts indicated that the relative affinity with which AcMNPV infection-specific proteins bound to the 28-mer was similar to that of in vitro-synthesized IE1. By using a combination of DNA binding and antibody supershift assays, it was demonstrated that IE1 binds to the 28-mer as a dimer. Collectively, these findings support a model in which symmetrical IE1 binding and simultaneous interaction with each half site are required for IE1-mediated transcriptional enhancement by hr5. Thus, sequence-specific binding may be one of the mechanisms by which IE1 directly or indirectly transregulates baculovirus gene expression.
Rodems
S M
SM
Institute for Molecular Virology, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin-Madison 53706, USA.
Friesen
P D
PD
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral / Q000378:metabolism; D004268:DNA-Binding Proteins; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral; D017874:Immediate-Early Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D017924:Nucleopolyhedroviruses / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D018411:Spodoptera; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D015533:Transcriptional Activation
C069183:Ac-IE-1 protein, Autographa californica nucleopolyhedrovirus; D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D015534:Trans-Activators