Characterization of the acidic domain of the IE1 regulatory protein from Orgyia Pseudotsugata multicapsid nuclear polyhedrosis virus

• Main Author: Forsythe, Ian James
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/6480

This study presents a detailed analysis of the acidic amino-terminal region of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) transactivator IE1. The N-terminal region of IE1 is rich in acidic amino acids and has been hypothesized to be an acidic activation domain (AAD). Removal of the N-terminal 126 amino acids containing the acidic domain of IE1 resulted in complete loss of transactivation activity, indicating that this region is essential for transactivation. The OpMNPV acidic domain was replaced with the archetype AAD of herpes virus VP16 and the acidic rich region of Autographa californica multicapsid nuclear polyhedrosis virus (AcMNPV) IE1. These chimeric constructs were fully active in transient assays. This study therefore represents the first direct evidence that the acidic-rich N-terminal region of OpMNPV IE1 functions as an AAD. The chimeric OpMNPV IEls containing the VP16 and AcMNPV IE1 AADs consistently transactivated a reporter gene to higher levels than the OpMNPV IE1 AAD, suggesting that VP16 and AcMNPV IE1 are more efficient AADs than the OpMNPV IE1 AAD. Transactivation by all the chimeric constructs is enhanced synergistically when cotransfected with IE2 into Lymantria dispar (Ld652Y) and Spodoptera frugiperda (Sf9) cells. To define the critical regions of the AAD of OpMNPV IE1 a fine deletion analysis was performed. Both N - to C-terminal and C- to N-terminal deletions of the OpMNPV AAD were constructed to define functional domains within the OpMNPV IE1 AAD. At least two potential activation core domains were identified at amino acids 28-43 and amino acids 113- 124. Similar to the core activation domains of VP16 and GAL4, both OpMNPV IE1 subdomains contained predominately acidic and bulky hydrophobic amino acids.