In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals

Abstract

A vaccinia virus gene that is expressed throughout the reproductive cycle was found to have two sets of RNA start sites approximately 55 nucleotides apart. The site nearest to the coding segment is used early in infection and the one further upstream is used after DNA replication. A series of 5' to 3' deletions were made in the promoter region, and the truncated DNA segments were then ligated to the coding portion of the procaryotic chloramphenicol acetyltransferase gene to measure expression. The effects of these mutations on chloramphenicol acetyltransferase synthesis were determined in a vaccinia virus helper-dependent transient expression system and by forming infectious vaccinia virus recombinants that contain the chimeric genes. Deletions extending up to 31 nucleotides before the late RNA start site had no effect on either early or late expression. Removal of an additional 15 nucleotides produced a dramatic decrease in late expression but had no effect on early expression. The latter was not diminished until the deletion was extended from 31 to 24 nucleotides before the early RNA start site. These results were confirmed by transcriptional analyses. We concluded that this vaccinia virus gene has two promoters and that the regulatory signals for each are located within 31 nucleotides of their sites of transcription.

Discontinuous transcription or RNA processing of vaccinia virus late messengers results in a 5′ poly(A) leader

“…The virus does in fact have its own transcription and replication machinery (Moss, 1976). Furthermore, recent studies concerning the functional analysis of viral promoters confirm the earlier observation that they possess unique features that are not found in their eukaryotic counterparts (Cochran et al, 1985b;Rose1 and Moss, 1985;Hanggi et al, 1986).…”

“…The site of transcription initiation of late vaccinia mRNA has been determined thus far by Sl mapping experiments using genomic DNA as a probe (Cochran et al, 1985b;Rose1 and Moss, 1985;Bertholet et al, 1985;Hanggi et al, 1986). The reason for this is that primer elongation experiments are complicated by a high degree of complementarity in late vaccinia RNA transcripts, both DNA strands are transcribed, genes can be overlapping, and there is readthrough of the RNA polymerase (Plucienniczak et al, 1985;Smith et al, 1984).…”

“…The regulatory signals controlling the transcription and the time of gene activation reside in very short stretches of approximately 20 to 30 bp. This has been shown by translocation of promoter fragments by means of homologous recombination (Cochran et al, 1985b;Rose1 and Moss, 1985;Hanggi et al, 1986).…”

Vaccinia virus produces late mRNAs by discontinuous synthesis

“…Several studies have demonstrated that the sequences involved in gene regulation in vaccinia virus reside in the 5'flanking region of early and late genes (Weir and Moss, 1984;Bertholet et al, 1985;Cochran et al, 1985). Recent analyses (Bertholet et al, 1986;Hanggi et al, 1966) have revealed unexpected features of the putative promoter region of a strongly expressed late gene that encodes a major structural polypeptide of molecular weight 11,000 (11K gene).…”

Poxviral promoters for improving the immunogenicity of MVA delivered vaccines

“…It was originally described as two promoters, because it has two different transcriptional start sites, 55nt apart. 41 The native version of p7.5 promoter is found in two copies driving the expression of C23L and B29R ORFs in the ITR (Inverted Terminal Repeats) of poxvirus genomes. Other early and late promoters have also been characterised such as the promoter of the TK gene, 42 the F7L promoter, 43 or the H5R promoter.…”