Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation

Area of Science:

• Hepatology
• Virology
• Molecular Biology

Background:

• Chronic hepatitis B virus (HBV) infections cause significant mortality.
• Eradicating HBV requires eliminating the stable covalently closed circular DNA (cccDNA) form.
• A complete understanding of cccDNA formation factors is lacking due to inadequate biochemical systems.

Purpose of the Study:

• To identify and characterize the host factors essential for HBV cccDNA formation.
• To establish a biochemical system for reconstituting cccDNA formation.
• To explore potential therapeutic interventions targeting cccDNA formation.

Main Methods:

• Developed experimental systems using cell extracts and purified human proteins to study HBV cccDNA formation.
• Employed yeast and human extract screenings to identify key host factors.
• Utilized biochemical assays and HBV-infected human cells to validate findings and test inhibitors.

Main Results:

• Identified five core components of lagging-strand synthesis as essential: proliferating cell nuclear antigen, replication factor C complex, DNA polymerase δ, flap endonuclease 1, and DNA ligase 1.
• Successfully reconstituted cccDNA formation using purified human proteins, defining a minimal set of factors.
• Demonstrated that the DNA polymerase inhibitor aphidicolin reduces cccDNA formation in vitro and in infected cells.

Conclusions:

• Defined the minimal set of host factors required for HBV cccDNA formation.
• Established a reconstituted system for studying cccDNA biogenesis.
• Provided evidence for targeting DNA polymerase activity as a strategy to inhibit cccDNA formation.