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) region of human cytomegalovirus by alternative splicing. The IE gene products regulate subsequent viral and cellular gene expression. We previously reported that cotransfection of a genomic clone of the major IE region stimulated transient expression of chloramphenicol acetyltransferase driven by the dihydrofolate reductase (DHFR) promoter and that an intact E2F site was required for the trans activation (M. Wade, T. F. Kowalik, M. Mudryj, E.-S. Huang, and J. C. Azizkhan, Mol. Cell. Biol. 12:4364-4374, 1992). With the availability of cDNA clones for the individual major IE proteins, we sought to determine which of these proteins exerted this effect and whether the IE protein(s) interacted with E2F. In this study, we use cotransfection to demonstrate that the 55- and 86-kDa major IE proteins from the IE2 region can each moderately trans activate the DHFR promoter and that the 72-kDa IE1 protein stimulates DHFR transcription to a much higher level. Furthermore, trans activation 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 also demonstrate by in vitro pull-down assays that the 72-kDa IE1 protein can specifically interact with the DNA binding domain of E2F1 (amino acids 88 to 191) in the presence of nuclear extract. Moreover, antibodies to either E2F1 or IE72 will immunoprecipitate both E2F and IE72 from cells that stably express IE72, and antibody to E2F1 will immunoprecipitate IE72 from normal human fibroblast cells infected with human cytomegalovirus.
D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000096:biosynthesis; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D017781:Genes, Immediate-Early; D005814:Genes, Viral; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000378:metabolism*; D008970:Molecular Weight; D016296:Mutagenesis; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D056465:Retinoblastoma-Binding Protein 1; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis*; D050680:Transcription Factor DP1; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins
D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D011993:Recombinant Fusion Proteins; D056465:Retinoblastoma-Binding Protein 1; D050680:Transcription Factor DP1; D014157:Transcription Factors; D014764:Viral Proteins; D013762:Tetrahydrofolate Dehydrogenase; D015500:Chloramphenicol O-Acetyltransferase