Identification and characterization of a G-quadruplex structure in the pre-core promoter region of hepatitis B virus covalently closed circular DNA

Approximately 250 million people worldwide are chronically infected with the hepatitis B virus (HBV) and at increased risk of developing cirrhosis hepatocellular carcinoma. The HBV genome persists as covalently closed circular DNA (cccDNA), which serves template for all mRNA transcripts. Current nucleos(t)ide analogs used to treat do not directly target cccDNA thus cannot eradicate infection. Here, we report discovery a unique G-quadruplex structure in pre-core promoter region that is conserved among nearly genotypes. This central critical steps viral life cycle, including generation pregenomic RNA, synthesis core polymerase proteins, encapsidation; thus, an understanding may lead identification novel anti-HBV targets. We utilized biophysical methods (circular dichroism small-angle X-ray scattering) characterize effect three distinct G A mutants. also microscale thermophoresis quantify binding affinity its mutants known quadruplex-binding protein (DHX36). To investigate physiological relevance G-quadruplex, employed assays using DHX36 pull-down compared infection HepG2 cells transfected wild-type mutant plasmids by monitoring levels genomic DNA, antigens. Further evaluation this host-protein interaction site facilitate development therapeutics against resilient template.