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

ABSTRACT Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists as minichromosomes in the nuclei of hepatocytes and serves as the transcriptional template for viral RNAs. The stable cccDNA pool, characterized by a very low copy number, poses challenges for studying its molecular characteristics and developing targeted therapeutics. 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-stranded 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, as it contains no exogenous sequences, was shown to accurately transcribe major viral RNAs and organize nucleosomes in a sequence-specific manner. Furthermore, the McccDNA model allows for targeted deletion of viral proteins, producing a loss-of-function phenotype useful for further minichromosome studies. In conclusion, McccDNA generated from single-stranded M13 phage DNA recapitulates the natural properties of HBV cccDNA and serves as an authentic model to study cccDNA minichromosomes functionality. IMPORTANCE HBV remains a major global health burden, with cccDNA serving as the transcriptional template for viral RNAs that sustain persistent infection. The low copy number of cccDNA in the nucleus and the lack of authentic in vitro models hinder its investigation and therapeutic development. Here, we established an M13 phage–derived single-stranded DNA approach to efficiently generate sequence-authentic HBV cccDNA in vitro . Upon transfection, the cccDNA molecules assembled into minichromosomes and were proven to be biologically active, producing viral RNAs more faithfully recapitulating infection-derived transcripts compared to recombinant cccDNA. The system also permits targeted mutagenesis for loss-of-function studies as well as site-specific labeling. These results provide insights into the nucleosome organization and transcriptional characteristics of the HBV cccDNA minichromosome, offering an authentic platform to investigate minichromosome functionality and host-virus interactions.