Generation of HBV cccDNA using single-stranded M13 phage DNA for authentic minichromosome functionality

Area of Science:

• Virology
• Molecular Biology
• Hepatology

Background:

• Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as nuclear minichromosomes, sustaining viral RNA transcription and persistent infection.
• The low copy number and lack of authentic in vitro models for HBV cccDNA impede research and therapeutic development.

Purpose of the Study:

• To develop a novel method for generating sequence-authentic HBV cccDNA in vitro.
• To establish an authentic model for studying HBV cccDNA minichromosome functionality and host-virus interactions.

Main Methods:

• A novel M13 phage-based system and deoxyribozyme were used to produce full-length single-stranded DNA (ssDNA) corresponding to HBV cccDNA strands.
• ssDNA was cyclized and ligated to produce HBV cccDNA (McccDNA) in vitro.
• McccDNA was transfected into hepatic cells to form minichromosomes and assess biological activity.

Main Results:

• McccDNA successfully formed minichromosomes in transfected hepatic cells, recapitulating key HBV life cycle stages.
• McccDNA accurately transcribed viral RNAs, mimicking infection-derived transcripts more faithfully than recombinant cccDNA.
• The McccDNA model demonstrated sequence-specific nucleosome organization and responded to antiviral drugs, allowing for targeted mutagenesis studies.

Conclusions:

• McccDNA generated from M13 phage DNA serves as an authentic in vitro model for HBV cccDNA.
• This model recapitulates natural HBV cccDNA properties, facilitating the study of minichromosome functionality and antiviral strategies.