The baculovirus transactivator IE1 binds to viral enhancer elements in the absence of insect cell factors.
The transregulatory IE1 protein of Autographa californica nuclear polyhedrosis virus binds to the viral enhancer element hr5. To test whether IE1 binds independently of host cell factors, IE1 was translated in rabbit reticulocyte extracts and tested for DNA binding activity by an electrophoretic mobility shift assay. Complexes with the hr5 probe were detected with translation reaction mixtures primed with ie1 RNA but not with control translation reaction mixtures. However, the DNA-protein complexes formed with IE1 translated in vitro migrated more slowly than complexes formed with IE1 that was transiently expressed in insect cells. Phosphatase treatment of the translation reactions resulted in an increase in the mobility of the DNA-protein complexes, suggesting that hyperphosphorylation was responsible for the altered migration. To further verify that IE1 was capable of binding DNA in the absence of host cell factors, an N-terminal truncation of IE1 was synthesized in vitro, and shown to interact with hr5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of IE1 translated in vitro revealed that the mobility of the protein was heterogeneous. This pattern was altered by translation in the presence of an oligonucleotide corresponding to the IE1 specific binding site but was not affected by translation in the presence of a nonspecific DNA. These results suggest that binding of IE1 to DNA causes a conformational change in the protein that alters the accessibility of IE1 to protein kinases.
Choi
J
J
Department of Biology, Texas A&M University, College Station 77843-2128, USA.
Guarino
L A
LA
D000818:Animals; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D017874:Immediate-Early Proteins / Q000378:metabolism*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011817:Rabbits; D018411:Spodoptera; D015534:Trans-Activators / Q000378:metabolism*
C069183:Ac-IE-1 protein, Autographa californica nucleopolyhedrovirus; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D015534:Trans-Activators