Genome-wide analysis of regulatory G-quadruplexes affecting gene expression in human cytomegalovirus
Fig 8
Analysis of the GQ8 mutant virus.
(A) Schematic representation of the regulatory region of the UL35 gene. Positions of the GQ8 (solid box) and putative TATA box (dashed box) for UL35 are indicated. The predicted polyadenylation signal for UL34 is underlined. Mutations introduced into GQ8 to disrupt G4 formation are indicated in red. The parts of UL34 and UL35 ORFs are indicated with amino acid sequences. (B) Graphical representation of various mutants and revertant viruses generated for the study of GQ8. The HCMV (Toledo) bacmid containing mutant GQ8 (mGQ8) and its revertant were produced using a counter-selection BAC modification kit (Gene Bridges) (see Materials and Methods). (C) Gel profiles showing restriction patterns of HCMV bacmid constructs. Wild-type (Wt), GQ8 mutant (mGQ8), and revertant (R) bacmids were digested with BamH1 and subjected to pulse-field gel electrophoresis. No apparent alteration of restriction fragment patterns was found in mGQ8 and revertant bacmids. (D) Effect of G4 ligands on UL35 transcription in wild-type, GQ8 mutant, and revertant viruses. HF cells were infected with recombinant HCMV [wild-type (Wt), GQ8 mutant (mGQ8), or revertant (R)] at an MOI of 1 and treated with DMSO, 5 μM of NMM, or TMPyP2 for 24 h prior to cell harvest at 32 h after HCMV infection. The mRNA levels of UL112 and UL35 were measured by qRT-PCR. The relative mRNA levels normalized by those of β-actin and IE1 are shown as graphs.