The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter

Abstract

Control of adeno-associated virus (AAV) transcription from the three AAV promoters (p5, p19, and p40) requires the adenovirus E1a protein and the AAV nonstructural (Rep) proteins. The Rep proteins have been shown to repress the AAV p5 promoter yet facilitate activation of the p19 and p40 promoters during a productive infection. To elucidate the mechanism of promoter regulation by the AAV Rep proteins, the cellular factors involved in mediating Rep activation of the p19 promoter were characterized. A series of protein-DNA binding experiments using extracts derived from uninfected HeLa cells was performed to identify cellular factors that bind to the p19 promoter. Electrophoretic mobility shift assays, DNase I protection analyses, and UV cross-linking experiments demonstrated specific interactions with the cellular factor SP1 (or an SP1-like protein) at positions ؊50 and ؊130 relative to the start of p19 transcription. Additionally, an unknown cellular protein (cellular AAV activating protein [cAAP]) with an approximate molecular mass of 34 kDa was found to interact with a CArG-like element at position ؊140. Mutational analysis of the p19 promoter suggested that the SP1 site at ؊50 and the cAAP site at ؊140 were necessary to mediate Rep activation of p19. Antibody precipitation experiments demonstrated that Rep-SP1 protein complexes can exist in vivo. Although Rep was demonstrated to interact with p19 DNA directly, the affinity of Rep binding was much lower than that seen for the Rep binding elements within the terminal repeat and the p5 promoter. Furthermore, the interaction of purified Rep68 with the p19 promoter in vitro was negligible unless purified SP1 was also added to the reaction. Thus, the ability of Rep to transactivate the p19 promoter is likely to involve SP1-Rep protein contacts that facilitate Rep interaction with p19 DNA.

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

“…Plasmid pIM45 contains AAV nucleotides (nt) 145 to 4373 in a pBS M13ϩ vector (34). Mutants in the p19 promoter have been described elsewhere (42). Mutations in the p40 promoter region were constructed in the background of pIM45 by using oligonucleotide-directed mutagenesis as described previously (27).…”

“…The primers were 5Ј-end labeled with 50 Ci of [␥-32 P]ATP (3,000 Ci/mmol) and polynucleotide kinase (10 U; New England BioLabs). PCR conditions have been described previously (42). The PCRs were cycled 27 times through 45 s at 94°C, 45 s at 59°C, and 30 s at 72°C.…”

“…The reaction conditions for DNase I footprinting experiments were identical to those for the EMSA except that the reaction volume was increased to 50 l and 1 g of poly(dI-dC) was used as a nonspecific competitor as described previously (42).…”

“…Transactivation of the p19 and p40 promoters is dependent on an RBE in either the p5 promoter or the TR (41). Furthermore, we have demonstrated that the cellular factor Sp1 and a cellular AAV-activating protein (cAAP) are required to mediate Rep activation of the p19 promoter (42). Both of these proteins bind to sites just upstream of the p19 message start.…”

“…Both of these proteins bind to sites just upstream of the p19 message start. Sp1 appears to mediate Rep activity at the p19 promoter by facilitating Rep interaction with the p19 promoter through direct protein contacts between these proteins (42). Similar contacts may occur between cAAP and Rep, but these have not been directly demonstrated.…”