One hundred base pairs of 5' flanking sequence of a vaccinia virus late gene are sufficient to temporally regulate late transcription.

Abstract

A vaccinia virus late gene coding for a major structural polypeptide of 11 kDa was sequenced. Although the 5' flanking gene region is very A+T rich, it shows little homology either to the corresponding region of vaccinia early genes or to consensus sequences characteristic of most eukaryotic genes. Three DNA fragments (100, 200, and 500 base pairs, respectively), derived from the flanking region and including the late gene mRNA start site, were inserted into the coding sequence of the vaccinia virus thymidine kinase (TK) early gene by homologous in vivo recombination. Recombinants were selected on the basis of their TK-phenotype. Cells were infected with the recombinant viruses and RNA was isolated at 1-hr intervals. Transcripts initiating either from the TK early promoter, or from the late gene promoter at its authentic position, or from the translocated late gene promoters within the early gene were detected by nuclease S1 mapping. Early after infection, only transcripts from the TK early promoter were detected. Later in infection, however, transcripts were also initiated from the translocated late promoters. This RNA appeared at the same time and in similar quantities as the RNA from the late promoter at its authentic position. No quantitative differences in promoter efficiency between the 100-, 200-, and 500-base-pair insertions were observed. We conclude that all necessary signals for correct regulation of late-gene expression reside within only 100 base pairs of 5' flanking sequence.Vaccinia virus, a member of the poxvirus family, contains a large double-stranded DNA genome of 180 kilobase pairs. Expression of this large amount of genetic information is temporally well-regulated. Early genes are transcribed shortly after penetration of the virus particles into the host cell. After DNA replication, late genes encoding predominantly structural polypeptides are expressed. The molecular basis for this temporal regulation is not understood.In contrast to other animal DNA viruses, which replicate in the nucleus of the infected cells and which use the host cell RNA polymerase to transcribe their genes, vaccinia replicates in the cytoplasm and utilizes its own transcription system. A multisubunit RNA polymerase (1, 2) and enzymes involved in modification of RNA (3, 4) have been isolated from purified virus particles. The mRNAs made by these enzymes are not spliced (5, 6) but have cap structures (7) and poly(A) tails (8), which are characteristic features of eukaryotic mRNAs. As might be expected, recent evidence suggests that vaccinia virus has evolved its own regulatory signals for gene expression, First, vaccinia genes are transcribed in cell-free extracts prepared from infected cells but not in extracts from non-infected cells (9), which suggests that the host-cell transcriptional machinery does not recognize vaccinia promoter elements. Second, the 5' flanking regions of four early genes that have been sequenced (10-13) lack the regulatory elements characteristic of the corresponding regions of most cel...

Cloning and Characterization of the Gene Encoding the Major Protein of the A-type Inclusion Body of Cowpox Virus

“…However, the ATI promoter is more A/T-rich than the already well known VV late promoter sequences, perhaps explaining why ATI is so strongly expressed. Indeed, a gene coding for the 11K VV protein and carrying an A/T-rich promoter has been reported to be expressed strongly in VV-infected cells (Bertholet et al, 1985). Furthermore, expression of a gene coding for the 28K protein was greatly reduced following a point mutation from A to G in a run of eight As between -15 and -7 upstream of the structural gene (Weir & Moss, 1987).…”

“…Structural and functional analyses of viral polypeptides synthesized in infected cells have revealed at least two late gene subsets that can be defined temporally (Pennington, 1974;Ichihashi et aL, 1971). The fine structures of several early and late VV genes have been determined (Bertholet et al, 1985(Bertholet et al, , 1986Cochran et al, 1985;Plucienniczak et al, 1985 ;H~inggi et al, 1986;Rosel et al, 1986;Weir & Moss, 1987;Weinrich & Hruby, 1986;Niles et al, 1986;Earl et al, 1986), and comparison of nucleotide sequences upstream from these genes has revealed that VV promoters lack both eukaryotic and prokaryotic consensus sequences. Instead they have characteristic signals (Venkatesan et al, 1981(Venkatesan et al, , 1982Weir & Moss, 1983) recognized by VV RNA polymerase but not by the cellular RNA polymerase II (Puckett & Moss, 1983).…”

Production and characterization of recombinant mouse neurotrophin‐3

“…1. First, the coding sequence for mouse NT-3 was amplified from cloned genomic DNA and inserted into the recombination plasmid 1 Ik-ATA-18 [33], downstream of the cloned promoter that normally regulates the expression of an abundant 3 I-kDa protein component of the wild-type vaccinia virion. In plasmid 11 k-ATA-NT-3, the chimeric promoter/NT-3 cassette is flanked by DNA sequences derived from the vaccinia virus thymidine kinase (tk) gene.…”

Dengue 2 Virus Envelope Protein Expressed by a Recombinant Vaccinia Virus Fails to Protect Monkeys against Dengue

“…The VV shuttle plasmid pKB3 contains part of the VV TK gene interrupted by a unique EcoRI restriction enzyme site adjacent to the P11 late promoter of the 11K VV protein (Sarov & Joklik, 1972;Bertholet et al, 1985;Esposito et al, 1988). The cDNA of clone p30-VD2 was subcloned into this EcoRI site (Fig.…”