Abstract
The IE1 protein of Autographa californica nuclear polyhedrosis virus (Ac/WNPV) is a major transregulatory protein for viral gene expression. IE1 transactivates delayed-early promoters through binding to viral enhancer elements. To study the binding properties of IE1, electrophoretic mobility shift assays were conducted using protein produced in bacteria or rabbit reticulocyte extracts. The resuits reveaied that iEi expressed in bacieria was not abie to bind to hr5. IE1 synthesized in vitro formed retarded DNA-protein complexes, suggesting that IE1 was capable of binding to hr5 without insect cell proteins. Translation of IE1 in the presence of hr5 increased the DNA binding activity of the protein. The mobility of the protein was faster when it was synthesized in the presence of its specific DNA binding site than in the presence of a non-specific DNA. Phosphatase treatment of in vitro translated IE1 resulted in an increase in the mobility of the protein, indicating that phosphorylation was responsible for the altered pattern. To establish the temporal expression profile of IE1 during infection, a polyclonal antiserum was raised against E. coli-expressed IE1. Immunoblot analysis of infected cell extracts revealed that four electrophoretically distinct forms of IE1 were synthesized by 36 h postinfection and accumulated through 72 h postinfection. Metabolic labeling with carrier-free phosphate showed that IE1 is a phosphoprotein, confirming that differential phosphorylation accounts for the different electrophoretic species. Electrophoretic mobility shift assays indicated that the DNA binding activity of IE1 did not correlate with the total amount of protein and was inhibited by phosphatase treatment, indicating that a specific phosphorylation state of IE1 is required for enhancer binding. To better understand the transregulatory properties of IE1, the temperaturesensitive mutant (Ribiero et al., J. Virol. 68; 1075-1084), ?sB821, was investigated. The teB821-IE1 showed wild-type levels of DNA-binding activity whether the proteins were expressed at the permissive or non-permissive temperature, however the binding activity of the mutant protein was sensitive to a brief heat shock at 33°C. Furthermore, temperature shift-up experiments showed that IE1 was required for the high level expression of polyhedrin.
Choi, Jene (1994). Studies of the role of phosphorylation in the function of IE1, a viral transregulatory protein of the Autographa californica nuclear polyhedrosis virus. Texas A&M University. Texas A&M University. Libraries. Available electronically from
https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -1554199.