Regulatory roles of G-quadruplexes and G-quadruplex-binding proteins across the enhancer and promoter of the HBV genome

Hepatitis B virus (HBV) infection poses a global threat to human health due to the limited availability of effective treatment options. Recent studies have shown that the structures of G-quadruplex (G4) are related to the pathogenesis of the virus and potential targets for antiviral therapy. G-quadruplex-binding proteins (G4BPs) play a key role in regulating the G4 landscape and its associated functions by anchoring, stabilizing, or unwinding G4 structures. Researchers have actively pursued the identification of G4 structures within the HBV genome, yet a comprehensive, genome-wide analysis revealing their regulatory role and that of G4BPs has remained largely elusive. Consequently, our understanding of the intricate interactions between HBV G4 and G4BPs remains quite limited. Biophysical and molecular biology approaches were employed to investigate HBV G4, focusing on core promoter and enhancer (CP/EN) activities. Mass spectrometry, DNA pull-down, and surface plasmon resonance (SPR) characterized interactions between G4 and G4BPs. Employing the dual luciferase reporter system and HBV infection model, this study utilized binding assays designed for the G4 mutant, alongside overexpression and knockdown experiments, to delve into the regulatory functions of G4 and G4BPs. We identified two functional G4 elements located at positions 1204 and 1732 within the HBV CP/EN regulatory region. Reporter assays containing the CP/EN sequences showed that disruption of either the 1204 or 1732 G4 structure significantly reduced promoter activity. In the HBV 1.3-mer system, disruption of the 1732 G4 markedly decreased HBsAg, HBeAg and HBcAg expression, whereas total HBV RNA and 3.5 kb RNA levels were not significantly affected. Both G4 elements selectively recruited multiple host proteins in vitro, among which the 1732 displayed stronger binding to HNF4A. Functional assays further demonstrated that HNF4A enhanced CP/EN-driven reporter activity in a G4-dependent manner, and stabilization of G4 structures by BRACO-19 further promoted the interaction between G4 and HNF4A. In addition, CNBP, the most highly enriched G4-binding protein, positively regulated HBV antigen and RNA expression; however, CNBP did not enhance CP/EN promoter activity, indicating a regulatory mechanism independent of CP/EN activation. This study reveals that two G4 structures within the HBV CP/EN regulatory region function as structural platforms for host factor recruitment and play distinct roles in regulating viral gene expression. In particular, the 1732 G4 facilitates HNF4A-dependent activation of CP/EN activity, thereby supporting efficient viral protein production, whereas CNBP promotes HBV expression through a CP/EN-independent mechanism.