G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes

TY - JOUR

T1 - G-quadruplexes control hepatitis B virus replication by promoting cccDNA transcription and phase separation in hepatocytes

AU - Giraud, Guillaume

AU - Rodà, Mélanie

AU - Huchon, Pélagie

AU - Michelet, Maud

AU - Maadadi, Sarah

AU - Jutzi, Daniel

AU - Montserret, Roland

AU - Ruepp, Marc David

AU - Parent, Romain

AU - Combet, Christophe

AU - Zoulim, Fabien

AU - Testoni, Barbara

N1 - Publisher Copyright: © The Author(s) 2023.

PY - 2024/3/21

Y1 - 2024/3/21

N2 - Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.

AB - Phase separation regulates fundamental processes in gene expression and is mediated by the local concentration of proteins and nucleic acids, as well as nucleic acid secondary structures such as G-quadruplexes (G4s). These structures play fundamental roles in both host gene expression and in viral replication due to their peculiar localisation in regulatory sequences. Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is an episomal minichromosome whose persistence is at the basis of chronic infection. Identifying the mechanisms controlling its transcriptional activity is indispensable to develop new therapeutic strategies against chronic hepatitis B. The aim of this study was to determine whether G4s are formed in cccDNA and regulate viral replication. Combining biochemistry and functional studies, we demonstrate that cccDNA indeed contains ten G4s structures. Furthermore, mutations disrupting two G4s located in the enhancer I HBV regulatory region altered cccDNA transcription and viral replication. Finally, we showed for the first time that cccDNA undergoes phase separation in a G4-dependent manner to promote its transcription in infected hepatocytes. Altogether, our data give new insight in the transcriptional regulation of the HBV minichromosome that might pave the way for the identification of novel targets to destabilize or silence cccDNA.

UR - http://www.scopus.com/inward/record.url?scp=85188308662&partnerID=8YFLogxK

U2 - 10.1093/nar/gkad1200

DO - 10.1093/nar/gkad1200

M3 - Article

C2 - 38113270

AN - SCOPUS:85188308662

SN - 0305-1048

VL - 52

SP - 2290

EP - 2305

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 5

ER -