Abstract
G-quadruplex (G4) structures are found in eukaryotic cell replication origins, but their role in origin function remains unclear. In this study G4 motifs are found in the lytic DNA replication origin (oriLyt) of human cytomegalovirus (HCMV) and recombinant viruses show that a G4 motif in oriLyt essential region I (ER-I) is necessary for viral growth. Replication assays of oriLyt-containing plasmids and biochemical/biophysical analyses show that G4 formation in ER-I is crucial for viral DNA replication. G4 pull-down analysis identifies viral DNA replication factors, such as IE2, UL84, and UL44, as G4-binding proteins. In enzyme-linked immunosorbent assays, specific G4-binding ligands inhibit G4 binding by the viral proteins. The Epstein-Barr virus oriLyt core element also forms a stable G4 that could substitute for the oriLyt ER-I G4 in HCMV. These results demonstrate that viral G4s in replication origins represent an essential structural element in recruiting replication factors and might be a therapeutic target against viral infections.