Identification of a Previously Unrecognized Promoter That Drives Expression of the UXP Transcription Unit in the Human Adenovirus Type 5 Genome

Abstract

We previously identified an adenovirus (Ad) protein named U exon protein (UXP) encoded by a leftward-strand (l-strand) transcription unit. Here we identify and characterize the UXP promoter. Primer extension and RNase protection assays mapped the transcription initiation site at 32 nucleotides upstream of the UXP gene initiation codon. A series of viral mutants with mutations at two putative inverted CCAAT (I-CCAAT) boxes and two E2F sites were generated. With mutants lacking the proximal I-CCAAT box, the UXP mRNA level decreased significantly to 30% of the Ad type 5 (Ad5) mRNA level as measured by quantitative reverse transcription-PCR. Decreased UXP was also observed by immunoblotting and immunofluorescence. UXP mRNA and protein levels were similar to those of Ad5 for mutants lacking the distal I-CCAAT box or both putative E2F sites. Ad DNA levels were similar in mutant- and wild-type Ad5-infected cells during the late stage of infection, strongly suggesting that the decreased UXP mRNA and protein from mutants lacking the proximal I-CCAAT box was due to decreased promoter activity. Electrophoretic mobility shift assays (EMSA) indicated that a cellular factor binds specifically to the proximal I-CCAAT box of the UXP promoter. An in vitro luciferase reporter assay demonstrated that basal promoter activity lies between bp -158 and +30 of the transcription initiation site. No E1A-mediated promoter transactivation was observed in 293 cells compared with A549 cells. Thus, we propose that there is a previously unidentified Ad5 promoter that drives expression of the UXP transcription unit. This promoter is embedded within the gene for fiber, and it contains a proximal I-CCAAT box critical for UXP mRNA transcription.