D e novo synthesis of hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA

Thomas Tu; Benno Zehnder; Bingqian Qu; Stephan Urban

doi:10.1016/j.jhepr.2020.100195

D e novo synthesis of hepatitis B virus nucleocapsids is dispensable for the maintenance and transcriptional regulation of cccDNA

JHEP Rep. 2020 Oct 14;3(1):100195. doi: 10.1016/j.jhepr.2020.100195. eCollection 2021 Feb.

Authors

Thomas Tu^{1

2}, Benno Zehnder¹, Bingqian Qu^{1

3}, Stephan Urban^{1

3}

Affiliations

¹ Department of Infectious Diseases, Molecular Virology, Heidelberg University Hospital, Heidelberg, Germany.
² Storr Liver Centre, Westmead Clinical School and Westmead Institute for Medical Research, Faculty of Medicine and Health, The University of Sydney, Westmead, NSW, Australia.
³ German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany.

Abstract

Background & aims: Chronic HBV infection cannot be cured by current therapeutics owing to their limited ability to reduce covalently closed circular (ccc)DNA levels in the livers of infected individuals. Therefore, greater understanding of the molecular determinants of cccDNA formation and persistence is required. One key issue is the extent to which de novo nucleocapsid-mediated replenishment (reimport) contributes to cccDNA levels in an infected hepatocyte.

Methods: We engineered an infectious HBV mutant with a genome encoding a stop codon at position T67 in the HBV core open reading frame (ΔHBc HBV). Importantly, ΔHBc HBV virions cannot initiate nucleocapsid synthesis upon infection. Long-term in vitro HBV infection markers were followed for up for 9 weeks in HepG2-NTCP cells (A3 clone) and HBV DNA was quantified using a newly-developed, highly-precise PCR assay (cccDNA inversion quantitative PCR).

Results: ΔHBc and wild-type (WT) HBV resulted in comparable expression of HBV surface antigen (HBsAg), which could be blocked using the entry inhibitor Myrcludex B, confirming bona fide infection via the receptor sodium taurocholate cotransporting polypeptide (NTCP). In primary human hepatocytes, Huh7-NTCP, HepG2-NTCP, and HepaRG-NTCP cells, comparable copy numbers of cccDNA were formed. cccDNA levels, transcription of viral RNA, and HBsAg secretion remained comparably stable in WT and ΔHBc HBV-infected cells for at least 9 weeks.

Conclusions: Our results imply that de novo synthesised HBc plays a minor role in transcriptional regulation of cccDNA. Importantly, we show that initially-formed cccDNA is stable in hepatocytes without requiring continuous replenishment in in vitro infection systems and contribution from de novo DNA-containing nucleocapsids is not required. Thus, short-term therapeutic targeting of capsid-reimport is likely an inefficient strategy in eliminating cccDNA in chronically infected hepatocytes.

Lay summary: The hepatitis B virus can maintain itself in the liver for a patient's lifetime, causing liver injury and cancer. We have clarified exactly how it maintains itself in an infected cell. This now means we have a better idea at how to target the virus and cure a chronic infection.

Keywords: ALT, alanine aminotransferase; Antivirals; Bulevirtide; CIs, capsid inhibitors; Capsid inhibitors; Core protein; Covalently closed circular DNA; DHBV, duck hepatitis B virus; HBV DNA integration; HBV persistence; HBV, hepatitis B virus; HBcAg; HBsAg, hepatitis B virus surface antigen; Hepcludex; Myrcludex B; NC, naked capsids; NTCP, sodium taurocholate cotransporting polypeptide; NUCs, nucleos(t)ide analogues; ORF, open reading frame; PEG, polyethylene glycol; PHH, primary human hepatocytes; SN, supernatant; VP, virions; WT, wild-type; cccDNA, covalently closed circular DNA; dpi, days post inoculation; mge, multiplicity of genomic equivalent; pgRNA, pregenomic RNA; rcDNA, relaxed circular DNA; vge, viral genome equivalents.