Mutational analysis of GQ8 and GQ18 for G4 stability and reporter gene suppression.

(A and B) Biophysical characterization of G4 mutant oligonucleotides for GQ8 and GQ18. CD spectroscopy (A) and melting analysis (B) of GQ8 and GQ18 oligos (wild-type and G4-disrupting mutant sequences) associated with UL35 and UL75/UL76 genes, respectively, are shown. (C to E) Luciferase reporter assays demonstrating the effects of GQ8 and GQ18 mutations on promoter activities. HF cells were transfected via electroporation with reporter plasmids that expressed luciferase from the promoter regions of UL35, UL75, and UL76 containing wild-type or mutant G4 sequences. At 24 h after transfection, cells were infected with HCMV (Towne) with treatment of DMSO (as a control) or 5 μM NMM or TMPyP2 for 24 h prior to luciferase assays, which were performed at 32 h (for UL35) or 48 h (for UL75 and UL76) after virus infection. The resulting luminescence values are plotted as luciferase units.