HBV cccDNA Generated Using Single-Stranded M13 Phage DNA for Authentic Minichromosome Functionality

The cccDNA pool, characterized by a low copy number, poses challenges for cccDNA research. To accurately mimic the full-length sequences and structure of HBV cccDNA, we developed a novel method using an M13 phage-based system and deoxyribozyme to produce full-length single-strand DNA (ssDNA) corresponding to both the positive and negative strands of cccDNA.

This method involves the cyclization of the ssDNA followed by the addition of T4 DNA ligase, thereby producing the cccDNA. The in vitro cyclized cccDNA, formed by M13 phage-derived single-stranded DNA (McccDNA), allows the formation of minichromosomes in transiently transfected hepatic cells, recapitulates key stages of the HBV life cycle, and effectively responds to multiple antiviral drugs. Additionally, McccDNA was observed to more accurately transcribe major viral RNAs than recombinant cccDNAs containing exogenous sequences, and was also able to organize nucleosomes in a sequence-specific manner, resembling those events during natural viral infection.