Mutational analysis of the promoter region of the alpha 27 gene of herpes simplex virus 1 within the context of the viral genome.
In herpes simplex virus 1-infected cells, the first set of genes to be expressed (alpha genes) is induced by the alpha gene trans-inducing factor (alpha TIF), a virion structural protein. The cis-acting site in the 5' untranscribed domain of alpha genes was previously reported to be the sequence 5'-GYATGNTAATGARATTCYTTGNGGG noncoding (where Y is a pyrimidine, R is a purine, N is any base), which binds a host protein designated alpha H1 (also termed the octamer binding protein, OTF-1, Oct-1, etc.) and which, together with this and possibly other proteins, forms complexes with alpha TIF. To determine the role of the various components of this cis-acting site and of other sequences shared by the alpha genes, we constructed 17 mutants spanning 110 nucleotides of the promoter domain of the alpha 27 gene and made a series of chimeric genes. Each chimeric gene embodying one set of these mutations was inserted into the viral genome and measurements were made of (i) accumulated mRNA under conditions in which only alpha genes were expressed and (ii) the capacity of the mutated sequence to form complexes containing alpha TIF and alpha H1 proteins. We report that (i) transversions in the "TAAT" sequence abolished both complex formation and induction of the chimeric alpha gene, (ii) mutations in the octamer binding site sequence upstream from TAAT or of the downstream GARAT abolished alpha TIF complex formation and also reduced alpha mRNA accumulation, (iii) mutations in a "CAAT" box also reduced expression of mRNA without affecting the formation of DNA-protein complexes containing alpha TIF, and (iv) mutations in sequences immediately downstream from TAATGARAT and in a pair of GA-rich elements reduced alpha mRNA expression whereas mutations between these elements had no effect on the accumulation of the mRNA. The results are consistent with the conclusion that both the alpha H1 octamer binding site ATGNTAAT and the GARAT sequence play a significant role in the induction of alpha genes. For optimal gene expression, however, additional elements downstream from the GARAT sequence and in other regions of the alpha promoter must be present.
Spector
D
D
Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, IL 60637.
Purves
F
F
Roizman
B
B
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D018139:Simplexvirus / Q000235:genetics*; D014709:Vero Cells; D015678:Viral Structural Proteins / Q000235:genetics*
D015678:Viral Structural Proteins