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 (α genes) is induced by the a gene trans-inducing factor (αTIF), a virion structural protein. The cis-acting site in the 5′ untranscribed domain of a genes was previously reported to be the sequence 5′-GYATGN-TAATGARATTCYTTGNGGG noncoding (where Y is a pyrimidine, R is a purine, N is any base), which binds a host protein designated αH1 (also termed the octamer binding protein, OTF-1, Oct-1, etc.) and which, together with this and possibly other proteins, forms complexes with αTIF. To determine the role of the various components of this cis-acting site and of other sequences shared by the a genes, we constructed 17 mutants spanning 110 nucleotides of the promoter domain of the α27 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 α genes were expressed and (ii) the capacity of the mutated sequence to form complexes containing αTIF and αH1 proteins. We report that (i) transversions in the "TAAT" sequence abolished both complex formation and induction of the chimeric α gene, (ii) mutations in the octamer binding site sequence upstream from TAAT or of the downstream GARAT abolished αTIF complex formation and also reduced α mRNA accumulation, (iii) mutations in a "CAAT" box also reduced expression of mRNA without affecting the formation of DNA-protein complexes containing αTIF, and (iv) mutations in sequences immediately downstream from TAATGARAT and in a pair of GA-rich elements reduced a 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 αH1 octamer binding site ATGNTAAT and the GARAT sequence play a significant role in the induction of a genes. For optimal gene expression, however, additional elements down-stream from the GARAT sequence and in other regions of the a promoter must be present.