Gene expression from the delayed early promoters of Augographa california nuclear polyhedrosis virus (AcNPV) is regulated by the viral protein, IE1. The activity of IE1 is mediated through binding to viral enhancer elements. Recent experiments indicate that the function of IE1 may be regulated by phosphorylation. To better define the structure and function of this transactivator, the sites of phosphorylation will be determined by phosphopeptide analysis and amino acid sequencing. To determine the role of phosphorylation in the function of IE1, substitution mutants with exchanges in the phosphorylation sites will be constructed. The effect of these mutations on transactivation, DNA binding, and nuclear targeting of IE1 will be analyzed. Analysis of nuclear targeting involves the development of a microinjection protocol for expression and immunofluorescence analysis of IE1 in insect cells. In addition to a role in transcription, IE1 may be required for viral DNA replication. Preliminary data indicate that the viral enhancers also function as origins of replication in infected cells. To confirm this observation, an in vitro system for baculovirus DNA replication will be developed, using an SV40 DNA replication assay as a model system. Two periods of international collaboration are requested. An initial visit of 7 months to determine the phosphorylation sites and initiate studies on DNA replication. Then, a later visit of three months will be utilized to analyze the phosphorylation states and nuclear targeting of the mutant proteins. This research should provide insights into the mechanisms which regulate viral development and gene expression as well as increase our knowledge of the functions of cis- and trans-activating factors.
