Characterization of equine herpesvirus 1 harboring mutant forms of the immediate-early protein

Equine herpesvirus type 1 (EHV-1) possesses a sole diploid immediate early gene (IE) that encodes the major regulatory protein of 1,487 amino acids capable of modulating gene expression from both early and late promoters and also of trans-repressing its own promoter. Generation of an IE deletion virus showed that the IE gene is essential for viral growth in cell culture and is required for both early and late gene expression. The IE protein is comprised of discrete functional domains that mediate a multitude of activities. Sequence alignment of the EHV-1 IE protein and Alphaherpesvirus homologs defined five colinear regions that harbor these functional domains. A potent transcriptional activation domain (TAD) lies within amino acids 3 to 89, while regions 2 and 3 harbor the DNA binding domain (DBD; aa 422--597). Additionally, a serine-rich tract (SRT) motif (aa 181--220) lies within region 2 of the IE protein. The nuclear localization signal (NLS; aa 963--970) lies within region 3, and region 5 may contain a transcriptional enhancement function. This laboratory generated a library of constructs (linker-insertion, deletion, and nonsense mutants) that encode forms of the IE protein harboring mutations within all five regions of the IE protein. Mutations within the IE ORF were assessed for their effects on IE function in productive infection and for the ability to complement an IE null virus generated by lacZ insertion. Seventeen IE mutant viruses were generated by a specially designed recombination system. Characterization of these mutant viruses demonstrated that the TAD, SRT, DBD, and NLS of the IE protein are required for viral replication in tissue culture. Additionally, it was demonstrated that the carboxyl-terminus of the IE protein is important for maximal IE transactivation activation activity. Studies to elucidate the mechanism by which the IE protein mediates transactivation revealed that the IE protein interacts with transcription factor TFIIB but not TFIID, indicating that its mechanism differs from that of the ICP4 homolog. Characterization of the 17 EHV-1 mutants revealed that sequences totaling 43% of the IE protein are not essential for viral replication in cell culture.