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.

The Adenovirus Genome Contributes to the Structural Stability of the Virion

“…The late regions (L1–L5) are transcribed from an alternatively spliced transcript. Recently, it was shown that the regions encoding the L4-22K and L4-33K proteins are initially expressed at low levels from a novel promoter located within the L4 region [ 24 ], and these proteins act to fully activate the MLP [ 25 ]. There are also four small products produced at intermediate/late times of infection, including the structural protein IX (pIX), and the IVa2 protein that helps package viral DNA into immature virions [ 26 ].…”

Novel splicing and open reading frames revealed by long-read direct RNA sequencing of adenovirus transcripts

“…At 24 hpi, however, viral gene expression shifted to be dominated by late gene expression, as well as early transcripts derived from the E1B locus ( Figure 3B ). At late times post infection we also saw the E3-Fiber transcript, 19K/IX, and E4-DBP transcripts increase dramatically, potentially implicating these messages as novel late transcripts, with expression as abundant as the recently described late UXP transcript [47,48]. Furthermore, while all permutations of the X, Y, and Z leaders preceding Fiber were previously detected by short-read sequencing these could not be phased to full-length transcript isoforms [53,70].…”

“…Due to the spurious nature of low-level AdV splicing events [53], we set abundance thresholds for the highly abundant viral late transcripts of 500 reads for short-read junctions, and at least ten events detected in the long-read sequencing when collapsed by FLAIR [69]. Using this method, we readily detected other recently discovered viral isoforms, such as multiple splice sites preceding the pVII ORF [53], the so-called X, Y, and Z leaders embedded in E3 and preceding L5-Fiber [53,70], and the newly described UXP [47,48]. Using full-length RNAs, we were able to detect novel splice sites producing canonical ORFs that only differ in UTRs for L4-100K, L4-33K, L4-pVIII, and E4orf6/7.…”

“…The MLTU encodes for primarily structural capsid components or proteins involved with packaging of new virions, and their expression ultimately leads to the death of the host cell. Recently, a novel late gene, UXP, was discovered on the reverse strand of the genome [47,48]. The UXP promoter is located between E4 and E2 on the reverse strand of the genome, and splices downstream to the exons within the E2A region to continue translation of an ORF in an alternate reading frame to that of DBP.…”

Comparison of the Life Cycles of Genetically Distant Species C and Species D Human Adenoviruses Ad6 and Ad26 in Human Cells

“…The U exon protein (UXP) is expressed from the complementary l-strand of the viral genome from an exon (U) located within E3, with two subsequent exons located in the 100K and DBP coding regions (Table 2) (43). Activation of the U promoter element located in fiber has been shown to occur after, and to be dependent on, Ad genome replication (44). In concordance with these observations, transcripts from U are found at later time points, consistent with late gene transcripts.…”