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.
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.
Hepatitis B virus (HBV) is a small DNA virus that chronically infects 240 million people worldwide and causes approximately 686,000 deaths annually due to various severe liver diseases, including cirrhosis, hepatocellular carcinoma (HCC) and liver failure. Currently approved direct-acting antiviral agents against HBV are six nucleos(t)ide analogues that inhibit viral DNA polymerase with varying potency and barriers to drug resistance. Although those viral DNA polymerase inhibitors significantly reduce viral load and prevent liver disease progression, they rarely cure HBV infection due to their inability to eradicate cccDNA.
HBV core protein is a small polypeptide of 183 amino acid residues. It exists in infected hepatocytes as several distinct quaternary structures and plays multiple roles in the viral replication cycle. The best characterized function of core protein is the assembly of pre-genomic (pg) RNA and viral DNA polymerase complex into nucleocapsids where HBV DNA synthesis takes place. Moreover, temporally and spatially regulated disassembly (or uncoating) of nucleocapsids is essential for delivery of viral relaxed circular (rc) genomic DNA into the nuclei of infected hepatocytes, where it is converted to covalently closed circular (ccc) DNA. Furthermore, it has also been suggested that core proteins may associate with cccDNA minichromosomes, in an as-yet undefined structural manner, to regulate its transcription. Interestingly, it was also reported that core protein can be hijacked by host immune responses to recruit cytokine-induced DNA cytosine deaminase APOBEC3A to cccDNA minichromosomes, which results in cytosine deamination and decay of cccDNA.
Due to their unique structures and essential roles in viral replication, disruption of, or interference with, nucleocapsid assembly and/or disassembly with small molecular core protein allosteric modulators (CpAMs) represents a new frontier in development of novel antiviral agents against HBV. Over the last two decades, at least five chemotypes of CpAMs have been reported. Those compounds bind to a hydrophobic pocket, designated as the HAP pocket, at the dimer-dimer interface near the C-termini of core protein subunits. Binding of these molecules in the HAP pocket induces large scale allosteric conformational changes in core protein subunits and alters the capsid assembly kinetics and pathways. While heteroaryldihydropyrimidines (HAPs), such as Bay 41–4109 and GLS4, misdirect capsid assembly to form non-capsid polymers of core proteins, all other chemotypes of CpAMs, including sulfamoylbenzamides (SBAs) and phenylpropenamides (PPAs), represented by ENAN-34017 and AT-61, respectively, induce the formation of morphologically “normal” empty capsids with distinct quaternary and/or tertiary structural changes and thus, preclude viral DNA replication. Thus far, several HAPs and SBAs have been shown to inhibit HBV replication in animal models and are currently under preclinical o
