The characterization of the Autographa californica nuclear polyhedrosis virus IE2 protein

Abstract

The Autographa californica nuclear polyhedrosis virus (AcNPV) expresses several immediate-early genes in infected cells. One of these, ie2 (formerly called IEN), was mapped by its ability to augment IE1 - mediated transactivation of the delayed early 39k gene. IE2 also stimulated the expression of reporter genes linked to the ie1 promoter. To better understand the function of the ie2 gene product in infected cells, polyclonal antiserum specific for IE2 was prepared after overexpressing the protein in bacteria. Immunoblot analysis of cell extracts prepared at various times during virus infection indicated that IE2 protein was expressed from 0 to 12 hpi, but was undetectable during the later times of infection. To demonstrate directly that the increase in the experssion of IE1 cat was due to increased mRNA, primer extension analyses were performed. Quantitative primer extension assays revealed that cells cotransfected with pIE1 and pIE2 contained higher levels of IE1 mRNA than cells transfected with pIE1 alone. To directly demonstrate that IE2 is a transcritional regulator, in vitro transcription assays were performed. THe nuclear extracts containing IE2 were more active than the control extracts in transcription of a C-free cassette linked to the ie1 promoter. Imuunodepletion of IE2 from the transfected extracts reduced the transcriptional activity to that of the control extracts. Nuclear extracts prepared from IE2-transfected cells also more actively transcribed the adenovirus major late promoter. Taken together, these results demonstrate that IE2 is a transcriptional regulator. Computer analysis of IE2 amino acid sequences showed that IE2 contains several sequence motifs that are common to transcriptional activators. Sequential deletion of amino-terminal sequences of IE2 gradually decreased the activity of the protein. Carboxy-terminal truncations of IE2 resulted in a more dramatic loss of transactivation activities. Analysis of IE2 internal-deletion mutants demonstrated that the acidic region between amino acids 198 and 206 possessed significant transactivation potential for all target promoters tested. In addition, the squelching phenomena exhibited by wild type IE2 and IE2 deletion mutants containing the acidic amino acid-rich region indirectly demonstrated that this region may be involved in interactions with factors of the basic transcription machinery.