BINDING AND REPRESSION OF THE LATENCY-ASSOCIATED PROMOTER OF HERPES-SIMPLEX VIRUS BY THE IMMEDIATE-EARLY 175K PROTEIN

We demonstrate that the immediate early 175K protein (IE175K) of herpe s simplex virus type 1 binds to the cap site of the latency-associated promoter (LAP) in an unusual manner. The complex formed on the LAP ca p site was significantly larger than that formed on the 1E175K cap sit e and the requirements for binding were qualitatively distinct with re spect to both the primary sequence determinants at the site, and the r egions of 1E175K protein required for binding compared to those for th e IE175K cap site. Although purified IE175K was sufficient for this la rger complex formed on the LAP cap site, the DNA-binding domain was un able to bind efficiently. This contrasted strikingly with the IE175K c ap site where, using precisely analogous probes, the DNA-binding domai n exhibited a strong interaction. Surprisingly, from dissociation kine tics we show that binding of the intact protein to the LAP cap site is considerably more stable than the binding of IE175K to its own cap si te (half-lives of the complexes 15 min and < 1 min respectively), and this was reflected in more efficient repression of LAP-driven expressi on than IE175K promoter-driven expression by IE175K. Moreover, primary sequence requirements for IE175K binding to the LAP cap site region d iffered from previously identified IE175K recognition sequences in tha t in addition to a partially conserved consensus sequence, neighbourin g bases were necessary for binding. Although the LAP cap site exhibits a pseudopalindromic arrangement of core consensus sites, we show that this is not the basis for the higher order, more stable binding to th is region. Together these results indicate that IE175K forms an unusua l complex at the LAP cap site, broadening the range of previously defi ned sequences recognized by IE175K.