Molecular dissection of cis-acting regulatory elements from 5'-proximal regions of a vaccinia virus late gene cluster

Abstract

Promoter elements responsible for directing the transcription of six tightly clustered vaccinia virus (VV) late genes (open reading frames [ORFs] Dll, D12, D13, Al, A2, and A3) from the HindIII D/A region of the viral genome were identified within the upstream sequences proximal to each individual locus. These regions were identified as promoters by excising them from the VV genome, abutting them to the bacterial chloramphenicol acetyl transferase gene, and demonstrating their ability to drive expression of the reporter gene in transient-expression assays in an orientation-specific manner. To delineate the 5' boundary of the upstream elements, two of the VV late gene (Al and D13) promoter:CAT constructs were subjected to deletion mutagenesis procedures. A series of 5' deletions of the ORF Al promoter from-114 to-24 showed no reduction in promoter activity, whereas additional deletion of the sequences from-24 to +2 resulted in the complete loss of activity. Deletion of the ORF Al fragment from-114 to-104 resulted in a 24% increase in activity, suggesting the presence of a negative regulatory region. In marked contrast to previous 5' deletion analyses which have identified VV late promoters as 20to 30-base-pair cap-proximal sequences, 5' deletions to define the upstream boundary of the ORF D13 promoter identified two positive regulatory regions, the first between-235 and-170 and the second between-123 and-106. Background levels of chloramphenicol acetyltransferase expression were obtained with deletions past-88. Significantly, this places the ORF D13 regulatory regions within the upstream coding sequences of the ORF Al. A high-stringency computer search for homologies between VV late promoters that have been thus far characterized was carried out. Several potential consensus sequences were found just upstream from RNA start sites of temporally related promoter elements. Three major conclusions are drawn from these experiments. (i) The presence of promoters preceding each late ORF supports the hypothesis that each is expressed as an individual transcriptional unit. (ii) Promoter elements can be located within the coding portion of the upstream gene. (iii) Sequence homologies between temporally related promoter elements support the notion of kinetic subclasses of late genes.

Detailed phenotypic characterization of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase

“…Specific termination of transcription does not occur late during infection, so late transcripts are also heterogeneous at their 3' ends (22). Although the 5'-flanking regions of late genes are clearly essential in conferring late transcription specificity to these genes (2,6,17,25,45), the relationship of these sequences to poly(A) heads or to a typical promoter sequence is not yet understood.…”

Vaccinia virus vectors: new strategies for producing recombinant vaccines.

“…Viral transcriptional apparatus. VV transcribes its genome by using unique viral enzymes, viral transcription signals, and ancillary transcription factors (103,104,183). From an operational standpoint, this means that, if one abuts a foreign gene directly to a VV promoter element, one can be reasonably sure that the foreign sequences will be actively transcribed during the course of the viral infection.…”

Fine structure mapping of five temperature-sensitive mutants in the 22- and 147-kilodalton subunits of vaccinia virus DNA-dependent RNA polymerase

“…When DNA replication begins, expression of most early genes ceases and late gene expression is initiated. The early and late viral genes that have been characterized to date possess specific cis-acting sequences at their 5' ends which are essential for transcription by the viral RNA polymerase (4,7,10,18,32,33). Transcription of early genes terminates in response to a specific DNA sequence, therefore the early transcripts are of a distinct size (24, 34).…”