Fig. 3: ICP4 binding and unfolding of G4 sequences embedded in the ICP4 promoter.

a Schematic representation of G4s present in the ICP4 promoter. Each G4 is indicated with the name of the oligo used in the study. The TAATGARAT region is shown as a blue box with the VP16 protein nearby; the TATA box is represented as a red box; the arrow indicates the position of the Transcriptional Start Site (TSS). The position of each of these features on the double helix with respect to the TSS is on scale. The G4s are shown according to the DNA strand in which they are embedded. b Western blot analysis of ICP4 cross-linking pull-down towards the indicated four G4s embedded in the ICP4 promoter. IE-CS is the reported ICP4 consensus sequence^{42, 60, 61}; SCR is a G-rich unfolded oligo; CTR is a control lane loaded with infected cell nuclear extracts not subjected to the pull-down procedure. MW: molecular weight (Marker VI, Applichem). The upper panel shows the unbound ICP4 fraction, the middle panel the washed out ICP4 fraction, the lower panel the ICP4 fraction bound to each oligo. The multiband appearance of ICP4 from HSV-1 infected cells in WB has been reported⁹¹ and reflects the presence of differently post-transcriptionally processed isoforms. The experiment was independently performed in duplicate. Each replicate was tested once in each experiment. c CD spectra of the indicated G4 sequences embedded in the ICP4 promoter folded in potassium phosphate buffer (20 mM PB, 2 mM KCl) and incubated in the absence/presence of ufICP4 (10x). ICP4 spectrum was subtracted from G4-ICP4 complex spectra. Two independent experiment encompassing one replicate per condition were performed. Representative spectra form one measurement are shown.