IDENTIFICATION OF A PROMOTER-SPECIFIC TRANSACTIVATION DOMAIN IN THE HERPES-SIMPLEX VIRUS REGULATORY PROTEIN ICP4

ICP4 is expressed during the immediate-early phase of infection by her pes simplex virus (HSV) and activates transcription of viral genes dur ing subsequent phases of productive infection. Several members of the alpha-herpesvirus family encode regulatory proteins that have extensiv e homology with ICP4 and exhibit a transactivation domain (TAD) at the N terminus. The portions of ICP4 required for nuclear localization, D NA binding, and dimerization have been defined, but a domain that is s pecifically required for transactivation has not been identified. We h ave defined a promoter-specific ICP4 TAD by analysis of the activity o f GAL4-ICP4 fusion proteins cotransfected into HeLa cells with a lucif erase reporter gene linked to a promoter with five GAL4 binding sites. The transactivation activity of GAL4-ICP4 hybrids is located entirely within the first 139 residues of ICP4 and is significantly less poten t than the activity of GAL4-TAD hybrids derived from ICP4 homologs. IC P4 residues 97 to 109 are a critical component of this N-terminal TAD. Transient transfection assays performed with nonfusion forms of ICP4 and luciferase genes linked to the HSV glycoprotein D (go) or thymidin e kinase (tk) promoter revealed that ICP4 residues 97 to 109 are requi red for induction of the go promoter but are not required for inductio n of the tk promoter. Comparative experiments with ICP4 homologs revea led that the pseudorabies virus TAD is a potent activator of the go pr omoter and a weak activator of the fk promoter. Complementation assays revealed that loss of ICP4 residues 97 to 109 reduced the yield of vi rus from infected cells nearly 500-fold compared to wild-type ICP4. We conclude that ICP4 residues 97 to 109 are a core component of a promo ter-specific transactivation domain that is required for efficient rep lication of herpes simplex virus.