HBV core protein allosteric modulators differentially alter cccDNA biosynthesis from de novo infection and intracellular amplification pathways

Hepatitis B virus (HBV) core protein assembles viral pre-genomic (pg) RNA and DNA polymerase into nucleocapsids for reverse transcriptional DNA replication to take place. Several chemotypes of small molecules, including heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs), have been discovered to allosterically modulate core protein structure and consequentially alter the kinetics and pathway of core protein assembly, resulting in formation of irregularly-shaped core protein aggregates or “empty” capsids devoid of pre-genomic RNA and viral DNA polymerase. Interestingly, in addition to inhibiting nucleocapsid assembly and subsequent viral genome replication, we have now demonstrated that HAPs and SBAs differentially modulate the biosynthesis of covalently closed circular (ccc) DNA from de novo infection and intracellular amplification pathways by inducing disassembly of nucleocapsids derived from virions as well as double-stranded DNA-containing progeny nucleocapsids in the cytoplasm. Specifically, the mistimed cuing of nucleocapsid uncoating prevents cccDNA formation during de novo infection of hepatocytes, while transiently accelerating cccDNA synthesis from cytoplasmic progeny nucleocapsids. Our studies indicate that elongation of positive-stranded DNA induces structural changes of nucleocapsids, which confers ability of mature nucleocapsids to bind CpAMs and triggers its disassembly. Understanding the molecular mechanism underlying the dual effects of the core protein allosteric modulators on nucleocapsid assembly and disassembly will facilitate the discovery of novel core protein-targeting antiviral agents that can more efficiently suppress cccDNA synthesis and cure chronic hepatitis B.
Author summary Persistent HBV infection relies on stable maintenance of a nuclear episomal viral genome called covalently closed circular (ccc) DNA, the sole transcriptional template supporting viral replication. The currently available antiviral therapeutics fail to cure chronic HBV infection due to their failure to eradicate or inactivate cccDNA. In addition to packaging viral pregenomic (pg) RNA and DNA polymerase complex into nucleocapsids for reverse transcriptional DNA replication to take place, HBV core protein also participates in and regulates virion particle assembly, capsid uncoating and cccDNA formation. We report herein an intriguing observation that selected core protein allosteric modulators not only inhibit nucleocapsid assembly, but can also act on assembled, nucleus-bound nucleocapsids to promote their uncoating and consequentially interfere with cccDNA biosynthesis. This finding establishes molecular basis for development of novel core protein targeting antiviral agents with improved efficacy of suppressing cccDNA synthesis and curing chronic HBV infection.