Amino acid residues at core protein dimer-dimer interface modulate multiple steps of hepatitis B virus replication and HBeAg biogenesis

Abstract

The core protein (Cp) of hepatitis B virus (HBV) assembles pregenomic RNA (pgRNA) and viral DNA polymerase to form nucleocapsids where the reverse transcriptional viral DNA replication takes place. Core protein allosteric modulators (CpAMs) inhibit HBV replication by binding to a hydrophobic “HAP” pocket at Cp dimer-dimer interfaces to misdirect the assembly of Cp dimers into aberrant or morphologically “normal” capsids devoid of pgRNA. We report herein that a panel of CpAM-resistant Cp with single amino acid substitution of residues at the dimer-dimer interface not only disrupted pgRNA packaging, but also compromised nucleocapsid envelopment, virion infectivity and covalently closed circular (ccc) DNA biosynthesis. Interestingly, these mutations also significantly reduced the secretion of HBeAg. Biochemical analysis revealed that the CpAM-resistant mutations in the context of precore protein (p25) did not affect the levels of p22 produced by signal peptidase removal of N-terminal 19 amino acid residues, but significantly reduced p17, which is produced by furin cleavage of C-terminal arginine-rich domain of p22 and secreted as HBeAg. Interestingly, p22 existed as both unphosphorylated and phosphorylated forms. While the unphosphorylated p22 is in the membranous secretary organelles and the precursor of HBeAg, p22 in the cytosol and nuclei is hyperphosphorylated at the C-terminal arginine-rich domain and interacts with Cp to disrupt capsid assembly and viral DNA replication. The results thus indicate that in addition to nucleocapsid assembly, interaction of Cp at dimer-dimer interface also plays important roles in the production and infectivity of progeny virions through modulation of nucleocapsid envelopment and uncoating. Similar interaction at reduced p17 dimer-dimer interface appears to be important for its metabolic stability and sensitivity to CpAM suppression of HBeAg secretion. Author summary Binding of CpAMs at a HAP pocket between Cp dimer-dimer interfaces misdirects HBV capsid assembly. We demonstrated herein that CpAM-resistant mutations with single amino acid substitution of Cp residues at the HAP pocket not only confer resistance to CpAM suppression of pgRNA packaging, but also to the induction of capsid structure alteration and mature nucleocapsid uncoating as well as the inhibition of cccDNA synthesis by GLS4, a type I CpAM currently in phase 2 clinical trials. Moreover, these mutations in the context of precore protein also conferred resistance to CpAM inhibition of HBeAg secretion. We have thus obtained genetic evidence suggesting that CpAMs disrupt the multiple steps of HBV replication and inhibit HBeAg secretion by misdirecting the interaction of Cp or reduced p17 dimers at the HAP pocket. The mechanistic insights gained from this study should facilitate the development of Cp/p17-targeting antiviral drugs for the treatment of chronic hepatitis B.