SUNGKYUNKWAN UNIVERSITY SCHOOL OF MEDICINE
CLOSE HEADER
Research Highlights
G-quadruplex as an essential structural element in cytomegalovirus replication origin
Daegyu Park, Woo-Chang Chung, Shuang Gong, Subramaniyam Ravichandran, Gwang Myeong Lee, Minji Han, Kyeong Kyu Kim, Jin-Hyun Ahn
Nature Communications
Infection of human cytomegalovirus (HCMV), a member of betaherpesvirus, leads to persistent or latent infection in healthy people but can cause several diseases during reactivation in immunocompromised individuals. HCMV is also the most frequent causative virus of congenital infection in fetuses and can lead to neurologic disorders. Although the origin of DNA replication in lytic HCMV infection was identified decades ago, the mechanism by which the lytic origin is activated remains unclear.
We found several putative G4-forming sequences (G4 motifs) in the lytic DNA replication origin (oriLyt) of HCMV and studied their G4 formation and its role in oriLyt function. By producing recombinant viruses, we show that a G4 motif in oriLyt essential region I (ER-I) is necessary for viral growth. Replication assays of oriLyt-containing plasmids and a series of biochemical/biophysical analyses showed that G4 formation in ER-I is crucial for viral DNA replication. We determined through G4 pull-down assays that viral DNA replication factors, such as IE2, UL84 (initiators of HCMV DNA replication), and UL44 (a viral DNA polymerase processivity factor), bind to the G4. ELISA using purified viral proteins also showed that these proteins bind to the G4 with high affinity, and specific G4-binding ligands can inhibit the G4 binding by UL84 and IE2. Furthermore, we show that the essential core element of the oriLyt of Epstein-Barr virus, a gammaherpesvirus, also formed a stable G4 that could substitute for the G4 in HCMV oriLyt ER-I. These results demonstrate that viral G4s in replication origins are an essential structural element that recruits replication factors and can be a therapeutic target against viral infections.
We found several putative G4-forming sequences (G4 motifs) in the lytic DNA replication origin (oriLyt) of HCMV and studied their G4 formation and its role in oriLyt function. By producing recombinant viruses, we show that a G4 motif in oriLyt essential region I (ER-I) is necessary for viral growth. Replication assays of oriLyt-containing plasmids and a series of biochemical/biophysical analyses showed that G4 formation in ER-I is crucial for viral DNA replication. We determined through G4 pull-down assays that viral DNA replication factors, such as IE2, UL84 (initiators of HCMV DNA replication), and UL44 (a viral DNA polymerase processivity factor), bind to the G4. ELISA using purified viral proteins also showed that these proteins bind to the G4 with high affinity, and specific G4-binding ligands can inhibit the G4 binding by UL84 and IE2. Furthermore, we show that the essential core element of the oriLyt of Epstein-Barr virus, a gammaherpesvirus, also formed a stable G4 that could substitute for the G4 in HCMV oriLyt ER-I. These results demonstrate that viral G4s in replication origins are an essential structural element that recruits replication factors and can be a therapeutic target against viral infections.