INTERACTION OF THE 72-KILODALTON HUMAN CYTOMEGALOVIRUS IE1 GENE-PRODUCT WITH E2F1 COINCIDES WITH E2F-DEPENDENT ACTIVATION OF DIHYDROFOLATE-REDUCTASE TRANSCRIPTION

Three polypeptides are produced from the major immediate early (IE) re gion of human cytomegalovirus by alternative splicing. The IE gene pro ducts regulate subsequent viral and cellular gene expression. We previ ously reported that cotransfection of a genomic clone of the major IE region stimulated transient expression of chloramphenicol acetyltransf erase driven by the dihydrofolate reductase (DHFR) promoter and that a n intact E2F site was required for the trans activation (M. Wade, T. F . Kowalik, M. Mudryj, E.-S. Huang, and J. C. Azizkhan, Mel. Cell. Biol . 12:4364-4374, 1992). With the availability of cDNA clones for the in dividual major IE proteins, we sought to determine which of these prot eins exerted this effect and whether the IE protein(s) interacted with E2F. In this study, we use cotransfection to demonstrate that the jj and 86-kDa major IE proteins from the IE2 region can each moderately t rans activate the DHFR promoter and that the 72-kDa IE1 protein stimul ates DHFR transcription to a much higher level. Furthermore, trans act ivation through the 72-kDa IE1 protein is in part E2F dependent, while activation by the 55- and 86-kDa IE proteins is E2F independent. We a lso demonstrate by in vitro pull-down assays that the 72-kDa IE1 prote in can specifically interact with the DNA binding domain of E2F1 (amin o acids 88 to 191) in the presence of nuclear extract. Moreover, antib odies to either E2F1 or IE72 will immunoprecipitate both E2F and IE72 from cells that stably express IE72, and antibody to E2F1 will immunop recipitate IE72 from normal human fibroblast cells infected ,vith huma n cytomegalovirus.