ABSTRACT
We have identified the sequence elements that are required for adeno-associated virus type 2 p40 promoter activity. Mutation of specific promoter elements showed that two Sp1 sites at approximately -50 (Sp1-50) and -70 (GGT-70) bp upstream of the start of the p40 messages were necessary for maximal promoter activity. As expected, the TATA site at -30 was also essential. In vitro DNA binding experiments confirmed that the Sp1-50 and GGT-70 sites were bound by Sp1 or Sp1-like proteins. Two other transcription elements, the ATF-80 and AP1-40 sites, may play a role in p40 activity. Mutation of these elements resulted in a modest decrease in p40 transcription, but DNA binding experiments did not clearly demonstrate binding of transcription factors to these sites. In contrast, a major late transcription factor site at -110 was shown to bind the transcription factor, but mutation of this site had no effect on p40 activity. In a previous report, we have shown that transactivation of the p40 promoter by the viral Rep proteins required an upstream Rep binding element (in the terminal repeat or the p5 promoter), an unidentified p19 promoter element, and a p40 promoter element (D. J. Pereira and N. Muzyczka, J. Virol. 71:1747-1756, 1997). Here we demonstrate that the CArG-140 element in the p19 promoter and the Sp1-50 element in the p40 promoter are the specific p19 and p40 elements required for Rep induction of p40. As in the case of the p19 promoter, Sp1 facilitates interaction of Rep with the p40 promoter by interaction of the two proteins. Furthermore, electron microscopy experiments demonstrated that when Rep is bound to an upstream Rep binding element, it can interact with a proximal Sp1 site by protein contacts and create a loop in the intervening DNA. This finding suggests a common mechanism whereby the Rep binding element in the TR or the p5 promoter induces p19 and p40 activity by interaction with their respective Sp1 sites.
