TAF-LIKE FUNCTIONS OF HUMAN CYTOMEGALOVIRUS IMMEDIATE-EARLY PROTEINS

The human cytomegalovirus (HCMV) major immediate-early (IE) proteins I EP86 (IE2(579aa)) and IEP72 (IE1(491aa)) can transcriptionally activat e a variety of simple promoters containing a TATA element and one upst ream transcription factor binding site, In our previous studies, trans criptional activation was shown to correlate with IEP86 binding to bot h the TATA-box binding protein (TBP) and the transcription factor boun d upstream, IEP72 often synergistically affects the activation by IEP8 6, although it has not previously been shown to directly interact in v itro with IEP86, TBP, or transcription factors (e.g., Spl and Tef-1) b ound by IEP86. We report biochemical and genetic evidence suggesting t hat the major IE proteins may perform a function similar to that of th e TBP-associated factors (TAFs) which make up TFIID. Consistent with t his 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, dT AF(II)110, and hTAF,130, Regions on major IE proteins which mediate bi nding have been defined. In addition, our data indicate that both IEP7 2 and IEP86 can bind simultaneously to hTAF(II)130, suggesting that th is TAF may provide bridging interactions between the two proteins for transcriptional activation and synergy, In agreement, a transcriptiona l activation mutant of IEP72 is unable to participate in bridging. Con firmation that these in vitro interactions were relevant was provided by data showing that both IEP72 and IEP86 copurify with TFIID and coim munoprecipitate with purified TFIID derived from infected cell nuclei, To further support a TAF-like function of the IE proteins, we have fo und 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 essen tial for rescue and that IEP72 augments its effect, and that mutations which affect TAF interactions are debilitated in rescue. Our data, sh owing that the IE proteins can bind with TFIID and rescue a ts transcr iptional defect in TAF(II)250, support the model that the IE proteins perform a TAF-like function as components of TFIID.