The Function of DNA Binding in the Regulation of HSV-1 Gene Expression by ICP4

Abstract

Herpes simplex virus is an important model in the study of temporally regulated gene expression in eukaryotic cells. Three classes of genes - immediate early, early, and late - are sequentially expressed during the course of lytic infection. One immediate early gene product, ICP4, is required for transactivation of most early and late genes; it is also implicated in repression of immediate early gene expression. ICP4's mechanism(s) of action is/are not yet understood; although ICP4 binds to specific sequences of DNA, whether this is necessary for transregulation by ICP4 is not clear. To gain a better understanding of how the ability of ICP4 to bind DNA relates to its transregulatory activities, I introduced ICP4 binding sites into a simple model promoter within the viral genome. Two sets of construct were made in which an ICP4 binding site (or mutant site) was placed either downstream or upstream of a TATA box, reproducing the spacing found in (i) the native 𝘐𝘊𝘗4 promoter and (ii) the native 𝘐𝘊𝘗0 promoter (respectively). The promoter of HSV-1 𝘜𝘓24𝘣 (a nonessential gene in tissue culture) was replaced with these model promoters and levels of transcripts accumulating from these constructs during lytic infection assayed by primer extension. I found that an ICP4 site placed either upstream or downstream of a TATA box shifted kinetics of expression from E/leaky L to true L. Neither the strength of the TATA box nor the helical orientation of the ICP4 binding site with respect to the TATA box affected this result.