EXPRESSION OF THE IE1 TRANSACTIVATOR OF AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS DURING VIRAL-INFECTION

The immediate-early IE1 protein of Autographa californica nuclear poly hedrosis virus (AcMNPV) is an important regulator of viral gene transc ription. To provide a tool for further analysis of the expression and function of IE1, a polyclonal antiserum was raised against IE1 express ed in bacteria. Immunoblot analysis of infected cell lysates was used to monitor the accumulation of IE1 throughout the viral life cycle. Wh en extracts were prepared in the presence of phosphatase inhibitors, o nly one protein band was detected on SDS-polyacrylamide gels. However, in the absence of phosphatase inhibitors, at least four distinct elec trophoretic species were detected. Mobility shift assays were conducte d using an enhancer DNA probe and whole cell extracts prepared at diff erent times postinfection. Results indicated that the enhancer-binding activity of IE1 increased from 4 to 72 hr postinfection. DNA-protein complexes formed with infected cell extracts migrated more slowly than those formed with transfected cell extracts. This effect was more pro nounced with extracts prepared in the presence of phosphatase inhibito rs. Supershift experiments with IE1 antiserum confirmed that IE1 was a component of DNA-protein complexes in both transfected and infected c ell extracts. A titration experiment was done to determine the minimal amounts of IE1 required for activation of the 39k promoter in the pre sence and absence of a cis-linked enhancer element. These analyses ind icated that the intracellular levels of IE1 are not sufficient for enh ancer-independent activation of the 39k promoter during the early phas e of viral infection. Quantitative immunoblots revealed that the amoun t of IE1 in budded virus was less than 0.68 mole per mole of viral DNA , suggesting that IE1 is not a structural protein of AcNPV. (C) 1995 A cademic Press, Inc.