The baculovirus transactivator IE1 binds to viral enhancer elements in the absence of insect cell factors

The transregulatory IE1 protein of Autograph californica nuclear polyhedrosis virus binds to the viral enhancer element hr5. To test whether IE1 binds independent of host cell factors, IE1 was translated in rabbit reticulocyte extracts nd tested for DNA binding activity by an electrophoretic mobilit...

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Bibliographic Details
Published in:Journal of Virology 1995-07, Vol.69 (7), p.4548-4551
Main Authors: Choi, J. (Baylor College of Medicine, Houston, TX.), Guarino, L.A
Format: Article
Language:English
Subjects:
ADN
Animals
AUTOGRAPHA CALIFORNICA
baculovirus
DNA-Binding Proteins - metabolism
Enhancer Elements, Genetic
EXPERIMENTACION IN VITRO
EXPERIMENTATION IN VITRO
GENOMAS
GENOME
Immediate-Early Proteins - metabolism
nuclear polyhedrosis virus
Nucleopolyhedrovirus - genetics
Phosphorylation
Protein Biosynthesis
PROTEINAS
PROTEINE
Rabbits
SECUENCIA NUCLEICA
SEQUENCE NUCLEIQUE
Spodoptera
Trans-Activators - metabolism
VIRUS POLIEDROSIS NUCLEAR
VIRUS POLYEDROSE NUCLEAIRE
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Description
Summary:The transregulatory IE1 protein of Autograph californica nuclear polyhedrosis virus binds to the viral enhancer element hr5. To test whether IE1 binds independent of host cell factors, IE1 was translated in rabbit reticulocyte extracts nd 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 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 relations 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 of analysis 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 of a non-specific DNA. These results suggest that binding of IE1 to DNA causes a conformational change in the protein that alters ability of IE1 to protein kinases
ISSN:0022-538X
1098-5514
DOI:10.1128/JVI.69.7.4548-4551.1995