Inducible gene expression from vaccinia virus vectors.
A system for inducible gene expression by vaccinia virus (VV) vectors utilizing the Escherichia coli lac I repressor/operator system is described. A VV recombinant that expresses the lac I repressor protein from the constitutively active 7.5K promoter was constructed. Gel retardation experiments showed that a protein present in extracts of cells infected with this virus, but not wild-type virus, bound to the lac operator and that this binding was inhibited by IPTG. A series of VV recombinants were constructed that contained a 21-bp synthetic operator sequence(s) at different positions between the VV late 4b promoter and the firefly luciferase gene. Cells were co-infected with one of these viruses and the VV recombinant expressing the lac I repressor protein in the presence or absence of IPTG, and the level of luciferase activity was determined. Single operators positioned 19, 11, or 6 bp downstream of the promoter resulted in the 30, 50, or 97% inhibition of luciferase activity, respectively, while two operators increased the inhibition to greater than 99.9%. Addition of 1.25 mM IPTG at any time after infection restored 90% of enzyme activity from viruses containing a single operator, but reversal was only 50% when two operators were present. Both elements of the lac I inducible system were functional and stable in the genome of single recombinant virus. S1 nuclease protection of virus mRNA confirmed that luciferase expression was controlled at the transcriptional level and that IPTG did not affect transcription of endogenous VV genes. The utility of this inducible expression system for functional analyses of endogenous VV genes is demonstrated by the controlled expression of a gene encoding a 14-kDa protein and the correlation of 14-kDa expression with a biological property of the virus, namely plaque size phenotype. Plasmid vectors that are generally applicable to the inducible expression of genes by VV recombinants are described.
Department of Pathology, University of Cambridge, United Kingdom.
J F
JF
Rodriguez
G L
GL
Smith
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D005822:Genetic Vectors; D007763:Lac Operon; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism; D014158:Transcription, Genetic; D014616:Vaccinia virus / Q000235:genetics*; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification
D015345:Oligonucleotide Probes; D012097:Repressor Proteins; D014764:Viral Proteins