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, IMPORTANCE: 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.