The adeno-associated virus type 2 p40 promoter requires a proximal Sp1 interaction and a p19 CArG-like element to facilitate Rep transactivation

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.

The transcription strategy of bovine adeno-associated virus (B-AAV) combines features of both adeno-associated virus type 2 (AAV2) and type 5 (AAV5)

“…Based on our recent observations Ye and Pintel, 2007;Ye et al, 2006), and together with previously established AAV2 transcription activation models (Pereira and Muzyczka, 1997), we summarize in Fig. 5 an overview of the capsid gene transcription strategies employed by these three different AAVs.…”

“…1E). Therefore, although genetically closer to AAV5, the transcription pattern of B-AAV was more similar to that of AAV2, whose large Rep proteins bind a RBE and recruits transcription factors for capsid promoter activation (Pereira and Muzyczka, 1997). Based on our previous comparison study between AAV2 and AAV5 (10), this could have been due to either differences in the activation ability of the two Rep proteins (Ye et al, 2006), and/or the nature of the cis sequences around the capsid promoters themselves (Ye and Pintel, 2007).…”

“…Basal AAV2 P40 transcription activity is low in both E1A-and E1B-expressing 293 cells and HeLa cells, and this is at least partially due to the lack of AP1 and CRE-binding sites adjacent to P40, which are present in AAV5 and B-AAV P41 (Ye and Pintel, 2007). Activation relies on the AAV2 large Rep protein in the presence of helper viral functions, and Pereira et al (Pereira and Muzyczka, 1997) have proposed that this activation is mediated by a loop structure formed via the interaction between AAV2 Rep, transcription factor SP1, and their respective binding sites in the viral ITR/P5 and P40 regions (Fig. 5, top panel).…”

“…Filled ovals represent Rep binding elements (residing in ITRs at the end of each genome, and an extra one in the P5 region for AAV2). Shaded oval in the top panel represents SP1 transcription factor previously shown to interact with the AAV2 Rep protein (Pereira and Muzyczka, 1997). Adenovirus genes or unidentified cellular genes induced by adenoviral infection involved in Rep activation are shown in a cluster of empty ovals.…”

“…The upstream AP1 and CRE transcription factor binding sites are in rectangles and labeled with "+". The top panel is derived from previous study by Pereira et al (Pereira and Muzyczka, 1997). The middle panel is based on our previous observations (Ye and Pintel, 2007), and the bottom panel is based on this study.…”