Repression of the 0 Gene by ICP 4 during a Productive Herpes Simplex Virus Infection

During a productive infection by herpes simplex virus type 1 (HSV-1), ICP4, the major regulatory protein encoded by the 4 gene, binds to its transcription initiation site and represses the accumulation of 4 RNA. Evidence suggests that the degree of repression by ICP4 is a function of the absolute distance of an ICP4 binding site 3 from a TATA box. However, repression of HSV-1 gene expression by ICP4 through binding sites located 5 of TATA boxes, as in the case of the 0 gene, has not been adequately addressed. To this end, recombinant 0 promoters with various arrays of ICP4 binding sites flanking the 0 TATA box were constructed and recombined into the HSV-1 genome. Our results demonstrate the following. (i) Destruction of the endogenous 0 ICP4 binding site, located 5 of the TATA box, results in derepression of 0 protein and RNA accumulation in infected Vero cells. (ii) The degree of 0 derepression is equivalent to that reported for the 4 gene following destruction of the ICP4 binding site at the 4 mRNA cap site in HSV-1. (iii) Introduction of an ICP4 binding site at the 0 mRNA cap site represses the accumulation of 0 RNA greater than threefold relative to the wild type. (iv) Changes in the abundance of 0 protein and RNA in infected cells do not affect replication or growth of HSV-1 in tissue culture. Our findings are consistent with the conclusion that 0 transcription is repressed by ICP4. These results demonstrate that repression by ICP4 can occur through binding sites located 5 of virus gene TATA boxes in HSV-1. Thus, models addressing repression of HSV-1 gene expression by ICP4 should incorporate the role of binding sites located 5 , as well as 3 , of virus gene TATA boxes.