Evidence for G-quadruplex-mediated transactivation by the immediate-early 2 protein of human cytomegalovirus

G-quadruplex (G4) formation in gene promoters regulates promoter activity either positively or negatively. Although G4s have been shown to enhance promoter activation by serving as structural elements that recruit transcription factors, similar information on viral promoters is limited. We recently found that the human cytomegalovirus (HCMV) 86 kDa immediate-early 2 (IE2) protein, which acts as a viral transactivator, binds to a parallel G4 structure in the viral lytic replication origin, oriLyt, playing a role in initiating viral DNA replication. Here, we show that IE2 can transactivate a viral promoter by targeting G4. In HCMV-infected cells, the transcript for the UL146-UL132 locus, known to be upregulated by IE2, was significantly suppressed by treatment with N-methyl mesoporphyrin IX (NMM), a parallel G4-binding ligand. A putative G4-forming sequence, also called G4 motif, was identified in the UL146 promoter. Circular dichroism, fluorescence turn-on, and gel electrophoresis analyses confirmed the stable formation of a parallel G4 from this motif. In G4 pull-down assays, IE2 bound to G4, and NMM inhibited the G4 binding of IE2. In reporter assays, the G4 motif was necessary for activity of the UL146 promoter, and NMM treatment suppressed IE2-mediated transactivation of the UL146 promoter. UL146 transcription was significantly reduced in a recombinant virus with G4-disrupting mutations in the UL146 promoter, especially at low multiplicity of infection, along with decreased G4 formation and IE2 binding. Our findings demonstrate that G4 can act as a positive structural element in viral promoters, uncovering a novel mechanism of IE2-mediated transactivation.

G4-quadruplex (G4) is a noncanonical nucleic acid secondary structure that forms in single-stranded DNA or RNA. G4 structures are involved in various cellular processes, including transcription, translation, and DNA replication. It has been shown that G4 formation in gene promoters can inhibit promoter activation. Recently, a positive role of G4 in promoters has also been discovered, but evidence in viral promoters remains limited. In this study, we demonstrate that a G4 structure forms in the human cytomegalovirus UL146 promoter and acts as a binding site for the viral transactivator IE2, which plays a key role in viral gene expression. Using recombinant viruses, we confirm that IE2 binding to G4 is necessary for efficient UL146 transcription during virus infection. This study provides evidence that G4 structures in promoters can positively regulate viral gene expression and uncovers a novel mechanism by which IE2 activates gene expression.