title: TAF-like functions of human cytomegalovirus immediate-early proteins.
abstract: The human cytomegalovirus (HCMV) major immediate-early (IE) proteins IEP86 (IE2(579aa)) and IEP72 (IE1(491aa)) can transcriptionally activate a variety of simple promoters containing a TATA element and one upstream transcription factor binding site. In our previous studies, transcriptional activation was shown to correlate with IEP86 binding to both the TATA-box binding protein (TBP) and the transcription factor bound upstream. IEP72 often synergistically affects the activation by IEP86, although it has not previously been shown to directly interact in vitro with IEP86, TBP, or transcription factors (e.g., Sp1 and Tef-1) bound by IEP86. We report biochemical and genetic evidence suggesting that the major IE proteins may perform a function similar to that of the TBP-associated factors (TAFs) which make up TFIID. Consistent with this model, we found that the major IE proteins interact with a number of TAFs. In vitro, IEP86 bound with drosophila TAF(II)110 (dTAF(II)110) and human TAF(II)130 (hTAF(II)130), while IEP72 bound dTAF(II)40, dTAF(II)110, and hTAF(II)130. Regions on major IE proteins which mediate binding have been defined. In addition, our data indicate that both IEP72 and IEP86 can bind simultaneously to hTAF(II)130, suggesting that this TAF may provide bridging interactions between the two proteins for transcriptional activation and synergy. In agreement, a transcriptional activation mutant of IEP72 is unable to participate in bridging. Confirmation that these in vitro interactions were relevant was provided by data showing that both IEP72 and IEP86 copurify with TFIID and coimmunoprecipitate with purified TFIID derived from infected cell nuclei. To further support a TAF-like function of the IE proteins, we have found that the IE proteins expressed from the intact major IE gene, and to a lesser extent IEP86 alone, can rescue the temperature-sensitive (ts) transcriptional defect in TAF(II)250 in the BHK-21 cell line ts13. Analyses of mutations in the major IE region show that IEP86 is essential for rescue and that IEP72 augments its effect, and that mutations which affect TAF interactions are debilitated in rescue. Our data, showing that the IE proteins can bind with TFIID and rescue a ts transcriptional defect in TAF(II)250, support the model that the IE proteins perform a TAF-like function as components of TFIID.
authors.affiliation: Department of Microbiology and Graduate Group of Molecular Biology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6142, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Lukac
authors.forename: N Y
authors.initials: NY
authors.lastname: Harel
authors.forename: N
authors.initials: N
authors.lastname: Tanese
authors.forename: J C
authors.initials: JC
authors.lastname: Alwine
mesh_terms: D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D006224:Cricetinae; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila; D005091:Exons; D016679:Genome, Viral; D006801:Humans; D017874:Immediate-Early Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012867:Skin; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000378:metabolism*; D019473:Transcription Factors, TFII / Q000302:isolation & purification / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D011993:Recombinant Fusion Proteins; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D019473:Transcription Factors, TFII