Parallel G-quadruplexes recruit the HSV-1 transcription factor ICP4 to promote viral transcription in herpes virus-infected human cells

Figures

• 

  Fig. 1. ICP4 binding and unfolding of G4-folded oligonucleotides. a CD spectra of the biotinylated oligonucleotides used in the cross-linking-pull-down assay, folded in potassium phosphate buffer (20 mM PB, 80 mM KCl). Spectra were acquired from two independent experiments with two replicates each; spectra from one measurement are shown. b Western blot analysis of ICP4 binding to the indicated G4s (parallel: un2L2, myc and VEGF; antiparallel BOM17; mixed gp054a and LTR-III; non G4 LTR-IIIc; ICP4 consensus binding sequence: IE3). Infected cell nuclear extracts (5 µg) were incubated with formaldehyde-activated G4-folded oligonucleotides; proteins bound to the G4s were eluted after high stringency washes. ICP4 was detected using the anti-ICP4 antibody. MW molecular weight (Marker VI, Applichem). Below the gel image, the 260/290 ratio for each biotinylated oligonucleotide is displayed. c Quantification of ICP4 bound to the G4s in (b) is indicated in the bar chart (average of two independent experiments, each oligo was tested in one replicate per experiment, SD is displayed). Band quantification was normalised for the fraction of ICP4 bound to its consensus sequence (IE3). d First derivative…

• 

  Fig. 2. ICP4 and un2 G4 colocalization in the nucleus of HSV-1 infected cells. a Representative images of immuno-DNA-FISH in U-2 OS cells infected with HSV-1, strain F, MOI of 3, fixed at 8 hpi. The green and red signals correspond to un2 G4 and anti-G4 Ab signals, respectively; the merged signals are in yellow. Scale bars 10 µm. Controls are shown in Supplementary Fig. S5; b Representative images of proximity ligation assay (PLA) in U-2 OS cells infected with HSV-1, strain F, MOI of 3, fixed at 8 hpi. The green dots (PLA signal) correspond to G4-ICP4 interaction (one or two dots per nucleus). The blue signal corresponds to TOTO3 nuclear staining. Scale bars 10 µm. Controls are shown in Supplementary Fig. S6. Both Immuno-DNA-FISH and PLA experiments were performed in three independent assays per tested condition.

• 

  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 sequence42, 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…

• 

  Fig. 4. G4-mediated ICP4 promoter regulation by ICP4 in cells. a Western blot analysis of ICP4 expression in the presence of increasing amounts of ICP4 expression plasmid in transfected U-2 OS cells. ICP4 and TK promoters were cloned in the promoterless pGL-4.10 plasmid upstream of the luciferase gene and transfected into U-2 OS cells. β-actin was used as housekeeping control for cell amount. Two independent experiments were performed with one replicate per condition in each experiment. b Luciferase reporter assay of ICP4 and TK promoter activity in the presence of increasing amounts of ICP4 protein from ICP4 expression plasmid. Two independent experiments were performed with three replicates per condition in each experiment. P-values and SD are reported.

• 

  Fig. 5. ICP4 expression in the presence of the G4-ligand B19. a Luciferase reporter assay of ICP4 promoter and control TK promoter activity in the presence of increasing B19 concentrations. ICP4 and TK promoters were cloned in the promoterless pGL-4.10 plasmid upstream of the luciferase gene and transfected into U-2 OS cells. U-2 OS cell viability in the presence of increasing concentrations of B19 was also evaluated. Two independent experiments with three replicates per condition were performed. Significant P-values and SD are reported. b Luciferase reporter assay of ICP4 and TK promoter activity in the presence of ICP4 expression plasmid and in the presence/absence of B19 added 2 h after (AT) or before (BT) transfection of reporter plasmid. Mean average values of two independent experiments with three replicates per condition are reported. SD is indicated. c Representative confocal microscopy images of U-2 OS cells transfected with ICP4-EYFP plasmid that express EYFP under the ICP4 promoter, in the presence of increasing concentrations of B19 (0, 5, 10, 20 µM, as indicated). Scale bars 20 µm. d Quantification expressed as corrected total cell fluorescence (CTCF) of cell images,…

• 

  Fig. 6. Model of ICP4 activity at G4s in viral IE promoters in HSV-1 infected cells. Schematic of the herpesvirus transcription factor ICP4 regulating viral gene expression and boosting its own expression through binding to parallel DNA G-quadruplexes (G4s).