Two Leaky-Late HSV-1 Promoters Differ Significantly in Structural Architecture

Abstract

The HSV-1 VP5 and VP16 transcripts are expressed with leaky-late (γ1) kinetics and reach maximal levels after viral DNA replication. While the minimal VP5 promoter includes only an Sp1 site at −48, a TATA box at −30, and an initiator (Inr) element at the cap site, here we show that elements upstream of −48 can functionally compensate for the mutational loss of the critical Sp1 site at −48. To determine whether this is a general feature of leaky-late promoters, we have carried out a detailed analysis of the VP16 promoter in the context of the viral genome at the gC locus. Sequence analysis suggests a great deal of similarity between the two. Despite this, however, mutational analysis revealed that the 5′ boundary of the VP16 promoter extends to ca. −90. This region includes an Sp1 binding site at −46, CAAT box homology at −77, and “E box” (CACGTG) at −85. Mutational and deletional analyses demonstrate that the proximal Sp1 site plays little or no role in promoter strength; despite this it can be shown to bind Sp1 protein using DNA mobility shift assays. Like the VP5 promoter, the VP16 promoter also requires an initiator element at the cap site. The VP16 Inr element differs in sequence from that of the VP5 promoter, and its deletion or mutation has a significantly smaller effect on promoter strength. The difference between these two Inr elements was confirmed by our finding that the VP16 initiator element binds to the 65-kDa YY1 transcription factor, and the VP5 Inr element competes poorly for the binding between the VP16 element and infected cell proteins in comparative bandshift assays. While the VP16 Inr sequence is identical to that of several murine TATA-less promoters, the VP16 Inr requires a TATA box for measurable activity.