Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

Abstract

We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up-and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit 0-globin sequences placed in the dispensable El region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.The adenovirus major late promoter (MLP) has been studied extensively as a model for eucaryotic promoters. It contains a classic TATA box, necessary for correct and quantitative transcription in vitro (11,22,49), to which binds a transcription factor termed IID (41). As with many other eucaryotic promoters, a second sequence upstream from the cap site also affects the efficiency of transcription. This sequence element, centered at -58, is necessary for efficient transcription in vitro and in vivo (20,23,31) and is capable of binding a second transcription-promoting HeLa cell protein (7,32,41) termed USF.In addition to these well-characterized upstream sequence elements, the MLP contains striking direct repeats of nonalternating GC pairs flanking the TATA box (see Table 1). It has been suggested, on theoretical grounds, that these repeats and similar sequences in cellular promoters may form a structure which acts as a "trap" for...

Factors responsible for the higher transcriptional activity of extracts of adenovirus-infected cells fractionate with the TATA box transcription factor.

“…To be certain that these assays measured the transcriptional activity of MLTF, we tested the activities of the reconstituted transcription reac-VOL. 8,1988 r R; 'IT,t(N r, i" 2), pMLP -66 to +33 (lanes 3 and 4), and pMLP -51 to +33 (lanes 5 and 6). The Si-protected, specifically initiated RNAs were 820 nucleotides for the pMLP -66 to +192 template and 661 nucleotides for the pMLP -66 to +33 and pMLP -51 to +33 templates.…”

Transcription from the adenovirus major late promoter uses redundant activating elements.

“…The viability of a particular mutant MLP was tested by the methods described previously (Reach et al, 1990). After inactivation of the restriction enzyme, the fragment was co-transfected (Graham and Van der Eb, 1973), on A549 human lung carcinoma cells (Giard et al, 1991), with a right terminal adenovirus genomic fragment extending from the XhoI site at bp 8244 from the phenotypically wild type virus LLX1 (Brunet et al, 1987). After inactivation of the restriction enzyme, the fragment was co-transfected (Graham and Van der Eb, 1973), on A549 human lung carcinoma cells (Giard et al, 1991), with a right terminal adenovirus genomic fragment extending from the XhoI site at bp 8244 from the phenotypically wild type virus LLX1 (Brunet et al, 1987).…”

“…DNA preparations of the mutation-containing plasmid were restricted with EcoRI, which cleaves at the left terminus. After inactivation of the restriction enzyme, the fragment was co-transfected (Graham and Van der Eb, 1973), on A549 human lung carcinoma cells (Giard et al, 1991), with a right terminal adenovirus genomic fragment extending from the XhoI site at bp 8244 from the phenotypically wild type virus LLX1 (Brunet et al, 1987). Recombination in the overlapping sequence between bp 8244 and bp 9523 will yield a full-length genome, and, if the mutation does not confer a lethal phenotype, will give rise to plaques on the transfection plates.…”

The adenovirus major late transcription factor USF is a member of the helix-loop-helix group of regulatory proteins and binds to DNA as a dimer.

“…Sequences within the adenovirus major late (AdML) promoter that are required for its efficient expression in vitro and in vivo have been identified by extensive mutational analysis in several laboratories (Hu and Manley 1981;Hen et al 1982;Concino et al 1984;Miyamoto et al 1984;Yu and Manley 1984;Sawadogo and Roeder 1985;Logan and Shenk 1986;Brunet et al 1987;Lee et al 1988;Smale and Baltimore 1989). Altogether, these studies have identified three promoter elements; the upstream activating element (UAE) centered at position -63 to -52, the TATA box located at position -31 to -25, and the initiator at position -6 to + 4.…”