Hepatitis B virus cccDNA is formed through distinct repair processes of each strand

Lei Wei,Alexander Ploss

doi:10.1038/s41467-021-21850-9

Abstract

Hepatitis B virus (HBV) is a highly contagious pathogen that afflicts over a third of the world’s population, resulting in close to a million deaths annually. The formation and persistence of the HBV covalently closed circular DNA (cccDNA) is the root cause of HBV chronicity. However, the detailed molecular mechanism of cccDNA formation from relaxed circular DNA (rcDNA) remains opaque. Here we show that the minus and plus-strand lesions of HBV rcDNA require different sets of human repair factors in biochemical repair systems. We demonstrate that the plus-strand repair resembles DNA lagging strand synthesis, and requires proliferating cell nuclear antigen (PCNA), the replication factor C (RFC) complex, DNA polymerase delta (POLδ), flap endonuclease 1 (FEN-1), and DNA ligase 1 (LIG1). Only FEN-1 and LIG1 are required for the repair of the minus strand. Our findings provide a detailed mechanistic view of how HBV rcDNA is repaired to form cccDNA in biochemical repair systems.

Highlights

Hepatitis B virus (HBV) is a highly contagious pathogen that afflicts over a third of the world’s population, resulting in close to a million deaths annually
The covalently linked HBV polymerase and a 10 nt DNA flap are on the minus strand, whereas a 5ʹ-capped RNA primer and single-stranded DNA gap are on the plus strand (Fig. 1a)
These results indicate that all five factors are essential for the repair of the plus strand, whereas only flap endonuclease 1 (FEN-1) and ligase 1 (LIG1) are required for repairing the minus strand

Summary

Introduction

Hepatitis B virus (HBV) is a highly contagious pathogen that afflicts over a third of the world’s population, resulting in close to a million deaths annually. We previously established a biochemical reconstitution system, and identified five human factors—proliferating cell nuclear antigen (PCNA), the replication factor C (RFC) complex, DNA polymerase delta (POLδ), flap endonuclease 1 (FEN-1), and DNA ligase 1 (LIG1)—that are core components of Okazaki fragment synthesis as a minimal set of factors essential for repairing HBV rcDNA to form HBV cccDNA20. The detailed molecular mechanisms by which these factors repair the four lesions on HBV rcDNA have not been determined It is not known whether the repair of lesions on the plus and minus strands of HBV rcDNA require different sets of factors, whether the repair of both strands is dependent on each other, and what the repair steps and kinetics involved in repair of all the lesions are. We developed a novel strategy for monitoring the repair process of all a b

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