cccDNA: New Insights Into Its Formation and Role

Covalently closed circular DNA (cccDNA) is a key component in the life cycle of certain viruses, particularly hepatitis B virus (HBV). Its presence within infected cells enables persistent viral replication, making it a significant barrier to eradication. Understanding cccDNA has become crucial in virology and therapeutic research due to its role in chronic infections.

Recent studies have shed light on how cccDNA forms, functions, and interacts with host cellular mechanisms, shaping potential strategies to target it for better treatment outcomes.

Molecular Characteristics

Covalently closed circular DNA (cccDNA) is a highly stable, episomal form of viral DNA that serves as a genetic reservoir within infected cells. Structurally, it is a double-stranded DNA molecule with covalently linked ends, forming a continuous loop resistant to exonuclease degradation. Unlike linear or relaxed circular DNA intermediates, cccDNA lacks free DNA ends, preventing recognition by cellular repair mechanisms. Its supercoiled nature enhances stability, allowing it to persist in hepatocyte nuclei for extended periods, even without active viral replication.

cccDNA is regulated by nucleosomal organization, similar to host chromatin. Histone proteins, including H3 and H4, associate with cccDNA, forming a mini-chromosome that influences transcription. Post-translational histone modifications, such as acetylation and methylation, modulate viral promoter accessibility, controlling gene expression. Histone acetylation correlates with active transcription, while deacetylation and methylation contribute to silencing. This regulation enables cccDNA to switch between active and latent states, complicating eradication efforts.

Beyond histone modifications, cccDNA interacts with host and viral regulatory proteins. The hepatitis B virus X protein (HBx) maintains cccDNA transcription by counteracting host restriction factors. HBx facilitates the degradation of the structural maintenance of chromosomes complex 5/6 (SMC5/6), which represses cccDNA transcription. Additionally, host transcription factors such as hepatocyte nuclear factor 4 alpha (HNF4α) and cAMP response element-binding protein (CREB) bind to cccDNA, further regulating its transcriptional activity.

Formation Pathways

The conversion of hepatitis B virus (HBV) relaxed circular DNA (rcDNA) into covalently closed circular DNA (cccDNA) is a multi-step process involving viral and host cellular machinery. Upon entering hepatocytes, HBV virions release their partially double-stranded rcDNA into the cytoplasm, where it is transported to the nucleus. This rcDNA contains an incomplete positive strand and a covalently linked viral polymerase, features that must be resolved before cccDNA formation. The transition requires the removal of viral proteins, repair of strand discontinuities, and ligation to form a fully double-stranded, supercoiled genome capable of serving as a transcriptional template.

One of the earliest steps in cccDNA biogenesis is the removal of the viral polymerase, which remains attached to the 5′ end of the negative strand of rcDNA. Host nucleases, including tyrosyl-DNA phosphodiesterase 2 (TDP2), cleave the phosphotyrosyl bond between the polymerase and DNA, facilitating its release. The gaps in the viral genome must then be filled. Host DNA polymerases such as polymerase κ extend the incomplete strand to generate a fully double-stranded intermediate. This repair process also involves removing RNA primers from the 5′ ends of both strands, a step likely carried out by host RNase H and flap endonuclease 1 (FEN1).

The final step in cccDNA formation is the ligation of free DNA ends to generate a continuous, supercoiled molecule. DNA ligases, particularly DNA ligase I and DNA ligase III, mediate this process, ensuring the creation of a covalently closed structure. The efficiency of cccDNA formation depends on host repair enzymes, nuclear import mechanisms, and viral proteins that modulate these processes.

Role In Persistence

cccDNA persists in hepatocytes due to its structural resilience and ability to evade cellular degradation pathways. Unlike linear or partially double-stranded viral genomes, its supercoiled, episomal nature prevents recognition by DNA damage sensors. This stability allows it to remain intact, even without ongoing viral replication, sustaining hepatitis B virus (HBV) gene expression. Its persistence presents a major challenge for therapeutic strategies, as HBV can rebound once antiviral pressure is removed.

cccDNA undergoes dynamic transcriptional regulation rather than outright elimination. Unlike integrated viral DNA, which is often silenced by the host genome, cccDNA can switch between active and repressed states depending on intracellular conditions. Chromatin modifications, such as histone acetylation and methylation, influence whether cccDNA remains transcriptionally active or enters a low-expression phase. This plasticity means that even when viral replication is suppressed by nucleos(t)ide analogs, cccDNA can persist in a latent state, ready to reactivate under favorable conditions.

Hepatocyte longevity further complicates cccDNA elimination. These cells have a long lifespan, with some studies estimating half-lives ranging from months to years, allowing cccDNA to persist. Even during hepatocyte division, cccDNA is not efficiently diluted, as it does not integrate into the host genome. Instead, it partitions asymmetrically, ensuring that some daughter cells retain the viral reservoir. Additionally, non-cytolytic mechanisms, such as replenishment of cccDNA pools from newly infecting virions, contribute to long-term HBV maintenance despite antiviral treatment.

Detection Techniques

Detecting cccDNA is critical for assessing HBV persistence and evaluating therapeutic efficacy. Since cccDNA exists in low copy numbers, distinguishing it from other HBV DNA forms, such as integrated sequences or relaxed circular DNA (rcDNA), is challenging. Traditional polymerase chain reaction (PCR)-based methods struggle with specificity, necessitating advanced techniques.

Quantitative PCR (qPCR) with selective digestion improves specificity by using exonucleases such as Plasmid-Safe ATP-Dependent DNase, which degrades linear and rcDNA while sparing supercoiled cccDNA. However, contamination from partially closed DNA remains a limitation. Droplet digital PCR (ddPCR) enhances sensitivity by partitioning samples into thousands of micro-reactions, allowing for absolute quantification and improved discrimination between viral DNA species.

Host Interactions

cccDNA interacts with host cellular mechanisms that influence its transcriptional activity, stability, and potential clearance. These interactions determine whether the virus remains transcriptionally active, enters a low-replication state, or is suppressed by antiviral responses.

Epigenetic modifications play a significant role in cccDNA function. Histone deacetylases (HDACs) and histone methyltransferases (HMTs) contribute to transcriptional repression, reducing viral RNA production. Conversely, histone acetyltransferases (HATs) promote active transcription, sustaining viral protein synthesis. The interplay between these modifying enzymes allows HBV to adapt to changing cellular conditions. Small molecule inhibitors targeting these epigenetic regulators are being explored as potential therapies to silence cccDNA without directly eliminating it.

Cellular DNA repair pathways also impact cccDNA formation and maintenance. The host’s non-homologous end joining (NHEJ) and homologous recombination (HR) repair mechanisms help resolve gaps in the viral genome but may also stabilize cccDNA, inadvertently supporting its persistence. Modulating DNA repair enzyme activity could influence cccDNA levels, providing a potential therapeutic approach. Additionally, restriction factors such as the structural maintenance of chromosomes complex 5/6 (SMC5/6) suppress cccDNA transcription, though HBV counteracts this inhibition through the viral HBx protein. Understanding these host-virus interactions remains a focal point of research, as disrupting the factors that sustain cccDNA could offer new pathways to achieving an HBV cure.