Fact-checked by Grok 2 weeks ago

cccDNA

Covalently closed circular DNA (cccDNA) is the stable, episomal form of the hepatitis B virus (HBV) genome that persists in the nucleus of infected hepatocytes, serving as the primary template for viral transcription and replication.^[1] This ~3.2 kb double-stranded circular DNA molecule is derived from the virus's relaxed-circular DNA (rcDNA) and is organized into a chromatin-like minichromosome structure associated with host histones H2A, H2B, H3, and H4, which regulates its transcriptional activity through epigenetic modifications such as methylation and acetylation.^[1]^[2] The formation of cccDNA occurs shortly after HBV entry into hepatocytes via the sodium taurocholate cotransporting polypeptide (NTCP) receptor, where rcDNA undergoes host-mediated repair processes to resolve its structural lesions: the covalently attached viral polymerase and RNA oligomer on the minus strand are removed (e.g., by tyrosyl-DNA phosphodiesterase 2, TDP2), gaps are filled using DNA polymerase δ and other factors, and nicks are ligated by DNA ligase 1, resulting in a fully covalently closed circle within approximately 16 hours post-infection.^[3]^[2] These repair mechanisms, involving proteins like PCNA, RFC, FEN-1, and those resembling Okazaki fragment maturation for the plus strand, ensure the stability of cccDNA, which is independent for each strand and rate-limited by protein adduct removal.^[3] cccDNA's persistence is central to HBV chronicity, as it remains replication-competent for decades even in individuals who have cleared acute infection or seroconverted to hepatitis B surface antigen (HBsAg)-negative status, enabling viral reactivation under immunosuppression or stress.^[1] In quiescent hepatocytes, cccDNA levels in chronic hepatitis B patients range from 0.0032 to 0.032 copies per cell, varying by disease phase; approximately 2 copies per cell are observed in in vitro infection models, diluted during cell division but replenished through nuclear reimport of progeny rcDNA; its half-life is estimated at 33–57 days in animal models and around 6 months in humans, rendering it resistant to current antiviral therapies like nucleos(t)ide analogs (e.g., tenofovir) that target reverse transcription but spare the cccDNA pool.^[2]^[1] Transcription from cccDNA is driven by RNA polymerase II via four promoters and two enhancers, modulated by the viral HBx protein that disrupts host restriction factors like Smc5/6 to boost expression of pregenomic RNA and subgenomic mRNAs encoding core, polymerase, surface, and X proteins.^[2] As the molecular reservoir of HBV persistence, cccDNA underlies the development of chronic hepatitis, cirrhosis, and hepatocellular carcinoma, representing the primary obstacle to achieving a functional cure for the approximately 254 million people living with chronic hepatitis B infection worldwide, as of 2022.^[2]^[4] Therapeutic strategies targeting cccDNA—such as interferon-α to reduce its transcription, APOBEC3-mediated deamination, or inhibitors of formation and maintenance—hold promise but face challenges due to its integration with host chromatin and off-target risks.^[2]^[1]

Introduction

Definition and Discovery

Covalently closed circular DNA (cccDNA) is a stable, double-stranded circular form of viral DNA that is fully covalently closed, lacking gaps, nicks, or protein attachments, distinguishing it from the relaxed circular DNA (rcDNA) found in mature virions. This structure serves as an episomal minichromosome in the nucleus of infected cells and is the primary transcriptional template for viral gene expression. cccDNA is characteristically associated with hepadnaviruses, including the human hepatitis B virus (HBV), as well as related viruses like duck hepatitis B virus (DHBV) and woodchuck hepatitis virus (WHV), and plant caulimoviruses such as cauliflower mosaic virus (CaMV).^[1]^[5] The concept of covalently closed circular DNA emerged in the 1960s through studies of viral and bacterial genomes, where it was first described in bacteriophages and plasmids as a supercoiled or relaxed closed form resistant to certain exonucleases. In the context of animal viruses, investigations into HBV DNA intermediates during the 1970s revealed the presence of circular viral DNA species in infected livers and serum. Pioneering work by Robinson et al. in 1974 characterized the HBV genome as a partially double-stranded relaxed circular DNA of approximately 3.2 kilobases, with endogenous polymerase activity completing the strands in vitro, laying the groundwork for identifying closed forms.^[5] A landmark milestone in understanding cccDNA came from research on DHBV as a model for hepadnavirus replication. In 1982, Summers and Mason demonstrated that DHBV generates cccDNA from rcDNA shortly after infection, establishing it as a persistent nuclear reservoir that serves as the template for viral transcription, enabling ongoing replication through reverse transcription of an RNA intermediate. This study not only confirmed cccDNA's covalently closed structure via resistance to exonuclease digestion but also highlighted its amplification in infected duck liver cells, providing critical insights into HBV persistence that were unattainable with human samples due to ethical constraints.^[6]^[7]

Significance in Viral Infections

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) plays a pivotal role in establishing chronic infections, contributing to a significant global health burden. According to the latest WHO estimates (2024 report, data for 2022), approximately 254 million people worldwide live with chronic HBV infection, with the virus causing an estimated 1.1 million deaths annually, primarily from cirrhosis and hepatocellular carcinoma.^[4] This persistence driven by cccDNA underscores HBV's status as a major public health challenge, particularly in regions with limited access to vaccination and antiviral therapies. In virology, cccDNA represents a unique mechanism for viral longevity, enabling lifelong persistence within hepatocytes by serving as a stable nuclear reservoir that evades complete clearance by the host immune system or current nucleoside/nucleotide analog treatments.^[8] Unlike many other DNA viruses, such as adenoviruses, which typically cause acute infections without forming a durable episomal template, HBV's cccDNA allows for continuous low-level viral transcription and replication, fostering immune tolerance and chronicity.^[8] This feature of cccDNA is characteristic across the Hepadnaviridae family, including related viruses like woodchuck hepatitis virus, where it similarly promotes persistent infections in their respective hosts, distinguishing hepadnaviruses from non-persistent DNA viruses and complicating efforts toward viral eradication.^[9]

Molecular Structure and Properties

Chemical Composition

cccDNA, or covalently closed circular DNA, is the stable episomal form of the hepatitis B virus (HBV) genome, consisting of a double-stranded DNA molecule approximately 3.2 kilobases (kb) in length. This structure arises from the repair of the viral precursor, relaxed circular DNA (rcDNA), which features a full-length minus strand and an incomplete plus strand with gaps and overlaps; host cell ligases and polymerases complete and covalently close these strands to form the mature cccDNA circle. Unlike rcDNA, cccDNA lacks the covalently attached viral polymerase at the 5' end of the minus strand, eliminating protein priming and enabling its persistence as a naked DNA template.^[8]^[3]^[10] A hallmark of cccDNA's topology is its supercoiled configuration, which facilitates compact packaging within the infected hepatocyte nucleus. This supercoiled DNA associates closely with host histones, such as H3 and H4, forming a minichromosome-like structure that mimics cellular chromatin and contributes to its epigenetic regulation. The absence of viral proteins covalently bound to the DNA distinguishes cccDNA from other hepadnaviral intermediates, underscoring its reliance on host factors for structural integrity.^[8]^[3]^[9] The cccDNA sequence encompasses the complete HBV genome, including critical regulatory elements such as the direct repeats DR1 and DR2, which function as signals for genome packaging. It also contains enhancers EnhI and EnhII, which modulate transcriptional activity, along with the core promoter that directs the initiation of pregenomic RNA synthesis. These elements are conserved across HBV genotypes, ensuring the structural foundation for viral gene expression.^[11]^[12]^[13]

Stability and Half-Life

The covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) exhibits remarkable biophysical stability within infected hepatocytes, primarily due to its organization into a minichromosome structure decorated with host histones, which shields it from cellular nucleases and degradation pathways.^[14] This nucleosomal packaging positions histones non-randomly along the cccDNA, forming a compact chromatin-like entity that resists enzymatic breakdown and contributes to its persistence during chronic infection.^[14] Epigenetic modifications further bolster cccDNA durability by modulating its chromatin accessibility and transcriptional activity without altering the underlying DNA sequence. Histone acetylation, such as at H3K9, H3K27, and H3K122 residues, promotes an open chromatin conformation that enhances stability and replication, while hypoacetylation by histone deacetylases (e.g., HDAC1) can repress activity but does not typically lead to degradation.^[14] Similarly, CpG methylation at specific islands on the cccDNA—particularly island 2 and 3—correlates with transcriptional silencing and long-term maintenance, protecting the molecule from host restriction factors during quiescence.^[14] Estimates of cccDNA half-life vary by model and context, highlighting its longevity as a barrier to viral clearance. In vitro studies using HBV-infected HepG2-NTCP cells report a half-life of approximately 40 days, with a projected full lifespan of 58 days under controlled conditions.^[15] In vivo data are more limited due to challenges in direct measurement, but analogous studies in duck hepatitis B virus (DHBV)-infected models indicate a half-life of 35–57 days, while inferences from nucleoside reverse transcriptase inhibitor (NRTI)-suppressed chronic HBV patients suggest an average of 9.2 months, underscoring persistence over months to years in humans.^[8] Viral and host factors dynamically influence cccDNA half-life and stability. The HBV X protein (HBx) promotes longevity by recruiting histone acetyltransferases like p300 and CBP to the minichromosome, countering repressive modifications and enhancing resistance to turnover.^[14] Conversely, host APOBEC3A, upregulated by high-dose interferon-α, can accelerate degradation through cytidine deamination of the cccDNA, though this requires additional immune activation for efficacy and does not occur spontaneously.^[14]

Formation and Maintenance

Biosynthesis from rcDNA

The biosynthesis of covalently closed circular DNA (cccDNA) begins with the delivery of relaxed circular DNA (rcDNA), the mature viral genome packaged within the nucleocapsid, to the host cell nucleus following capsid uncoating during hepatitis B virus (HBV) infection.^[10] The rcDNA precursor features an incomplete plus strand with a single-stranded gap and a 5'-capped RNA oligonucleotide serving as the primer for its synthesis, while the minus strand contains a nick and is covalently linked at its 5' end to the viral polymerase protein.^[3] This nuclear import is facilitated by host karyopherins interacting with nuclear localization signals on the capsid and polymerase, enabling the rcDNA to access cellular repair machinery.^[10] The conversion of rcDNA to cccDNA is exclusively mediated by host cell enzymes, with no direct involvement of viral proteins in the repair steps.^[3] Deproteination of the minus strand 5' end is primarily achieved by tyrosyl-DNA phosphodiesterase 2 (TDP2), which cleaves the phosphodiester bond linking the polymerase to the DNA, although flap endonuclease 1 (FEN1) and certain proteases can contribute.^[10] Removal of the 8–9-nucleotide DNA flap from the minus strand terminal redundancy and the RNA primer from the plus strand gap is performed by FEN1 and the 3' flap endonuclease Mus81, with the latter playing a critical role in trimming the 3' flap structure, generating nicks that are sealed by DNA ligase 1 (LIG1).^[3]^[16] Completion of the plus strand involves DNA polymerase δ (POLδ), loaded via replication factor C (RFC) onto proliferating cell nuclear antigen (PCNA), which extends the 3'-OH end to fill the gap while displacing the RNA primer for subsequent FEN1 cleavage; DNA polymerase κ (POLκ) can also participate in this extension.^[17] These processes resemble host DNA repair mechanisms for Okazaki fragments and lesion repair, ensuring the formation of a stable, covalently closed circular molecule.^[3] In infected cell models, such as HepG2-NTCP or HepaRG lines, rcDNA to cccDNA conversion initiates within hours post-infection and becomes detectable by 24-48 hours via Southern blot analysis.^[10] Efficiency varies but typically ranges from 10-50% of input rcDNA in human hepatocyte models, lower for HBV compared to related duck HBV, and is modulated by cellular DNA repair pathways including non-homologous end joining, which may influence strand ligation under suboptimal conditions.^[18] In vitro reconstitution with nuclear extracts achieves up to 45-80% repair, highlighting the potential of host factors but also rate-limiting steps like protein adduct removal in vivo.^[3] The resulting cccDNA serves as the mature episomal template (as detailed in Chemical Composition).

Nuclear Localization and Persistence

Upon infection of hepatocytes, hepatitis B virus (HBV) capsids containing relaxed circular DNA (rcDNA) are transported through the cytoplasm along microtubules and imported into the nucleus via the nuclear pore complex (NPC).^[19] This process involves recognition of nuclear localization signals on the capsid by importin α1, facilitating docking and translocation across the NPC, where the capsid interacts with nucleoporin 153 (Nup153) in the nuclear basket.^[20]^[21] Once inside the nucleus, the rcDNA is released and repaired to form covalently closed circular DNA (cccDNA), which persists as an extrachromosomal episome, organized into a minichromosome with host histones, independent of chromosomal integration.^[22] The persistence of cccDNA in the hepatocyte nucleus is maintained through multiple mechanisms that ensure its long-term stability during chronic HBV infection. Intracellular recycling of HBV genomes from progeny virions contributes to amplifying and replenishing the cccDNA pool, allowing de novo formation without relying solely on initial infection.^[23] Epigenetic modifications, including histone acetylation and methylation on the cccDNA minichromosome, enable silencing or activation to evade host immune clearance, with factors like nuclear HBx protein modulating these changes to sustain transcriptional activity.^[24] In chronic infection, the cccDNA pool typically stabilizes at 3-50 copies per infected hepatocyte, reflecting a balance between formation, dilution during cell division, and rare natural depletion.^[25] Regulation of cccDNA levels and function involves interplay between host transcription factors and viral elements. Hepatocyte nuclear factors HNF1α and HNF4α bind to the HBV core promoter on cccDNA, driving transcription of pregenomic RNA and maintaining episomal stability.^[26] Viral proteins, such as HBx, further recruit host chromatin remodelers to prevent excessive silencing, while the absence of robust host interventions like nucleoside analogs rarely leads to cccDNA depletion, underscoring its role in viral persistence.^[3]

Role in HBV Lifecycle

Transcriptional Template

cccDNA functions as the central transcriptional template in the hepatitis B virus (HBV) lifecycle, organized as an episomal minichromosome within the nucleus of infected hepatocytes. This structure recruits host RNA polymerase II and associated transcription factors to synthesize all viral RNAs, including the pregenomic RNA (pgRNA) and subgenomic messenger RNAs (mRNAs) that encode key viral antigens such as hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Unlike integrated viral DNA, cccDNA persists independently of the host genome, enabling sustained transcription and continuous production of viral proteins essential for immune evasion and chronic infection.^[8]^[27]^[28] The transcription process is precisely regulated by viral regulatory elements embedded within the cccDNA sequence. Four distinct promoters—preS1, preS2, core (also known as preC/C), and X—direct the initiation of specific transcripts, while two enhancers (EnhI and EnhII) amplify promoter activity. The core promoter, in particular, drives the synthesis of the 3.5 kb pgRNA, which serves dual roles in reverse transcription for genome replication and as a template for translating viral core and polymerase proteins. Enhancers and promoters interact with host factors to confer liver-specific expression, ensuring efficient viral gene activation in hepatocytes.^[29]^[12]^[28] Subgenomic mRNAs, transcribed from the preS1 (2.4 kb, encoding large HBsAg), preS2 (2.1 kb, encoding middle HBsAg), and X (0.7 kb, encoding HBx) promoters, facilitate the production of envelope proteins and regulatory factors. The precore transcript, also 3.5 kb but distinct from pgRNA, is processed to yield secreted HBeAg, contributing to viral persistence by modulating host immunity. This non-integrated, minichromosome-like organization of cccDNA allows for long-term transcriptional activity without reliance on host chromosomal machinery, underscoring its role as a stable reservoir for viral RNA output.^[30]^[8]^[22]

Contribution to Viral Replication

cccDNA serves as the central template for sustaining hepatitis B virus (HBV) replication through an intracellular amplification loop that recycles newly synthesized viral genomes back to the nucleus. Pregenomic RNA (pgRNA), transcribed from the nuclear cccDNA, is packaged into immature capsids in the cytoplasm where it undergoes reverse transcription by the viral polymerase to form relaxed circular DNA (rcDNA). A portion of these rcDNA-containing nucleocapsids is then transported back to the nucleus, where the rcDNA is repaired and converted into new cccDNA molecules, thereby amplifying and maintaining the pool of viral templates without relying on host DNA synthesis mechanisms. The efficiency of this recycling process is limited, with approximately 5-10% of progeny rcDNA successfully returning to the nucleus and contributing to new cccDNA formation in human hepatoma cells, as evidenced by a 10-20:1 ratio of rcDNA to cccDNA observed in experimental models. This low efficiency is facilitated by the viral polymerase, which is efficiently removed from about 70% of nuclear rcDNA, and the core protein, which modulates capsid stability and uncoating but can impose a bottleneck by limiting complete disassembly. These viral factors ensure selective nuclear import and repair, supporting persistent replication despite the inefficient recycling rate. HBV replication, driven by this cccDNA-dependent loop, is inherently non-cytopathic, meaning it does not directly cause hepatocyte death and can persist indefinitely within infected cells, which is a key enabler of chronic infection. This non-lytic nature allows continuous viral production without triggering immediate cell destruction, contrasting with more aggressive viruses and contributing to the long-term establishment of HBV persistence in the liver.00137-5/fulltext)

Pathogenesis and Clinical Implications

Chronic Infection and Disease

The persistence of covalently closed circular DNA (cccDNA) in hepatocytes is a primary driver of chronic hepatitis B virus (HBV) infection, as its stability enables continuous viral transcription and replication despite host immune responses.^[31] This nuclear reservoir sustains lifelong production of viral antigens, such as hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), resulting in persistent antigenemia that overwhelms the immune system.^[31] Prolonged exposure to these antigens induces T-cell exhaustion, characterized by diminished virus-specific T-cell proliferation, cytokine secretion, and cytotoxicity, thereby establishing immune tolerance and preventing viral clearance.^[31] Elevated intrahepatic cccDNA levels directly correlate with increased HBsAg production and the severity of liver inflammation in chronic HBV patients, as evidenced by positive associations between cccDNA copy numbers and histological activity indices (r = 0.312, p = 0.033 for HBsAg).^[32] This chronic inflammatory state promotes progressive liver damage, including fibrosis, with approximately 20-30% of untreated chronic HBV carriers developing cirrhosis over 20-30 years.^[33] Cirrhosis further elevates the risk of hepatocellular carcinoma (HCC), with an annual incidence of 2-5% among cirrhotic patients, underscoring cccDNA's role in oncogenic pathways through sustained inflammation and genomic instability.^[34] The likelihood of chronicity varies markedly by age at infection and geographic region, reflecting differences in transmission modes and immune maturity. Vertical transmission from mother to child results in chronic infection in about 90% of neonates, compared to only 5-10% in immunocompetent adults infected horizontally.^[35] Globally, chronic HBV prevalence disproportionately affects low- and middle-income regions, with 254 million people living with the infection in 2022, including 97 million in the WHO Western Pacific Region and 65 million in the African Region, driven by higher rates of perinatal and early childhood exposure.^[4]

Challenges in Treatment

The persistence of covalently closed circular DNA (cccDNA) in hepatocytes poses significant barriers to achieving a cure for chronic hepatitis B virus (HBV) infection, as current antiviral therapies primarily target viral replication rather than the viral reservoir itself. Nucleoside analogs such as entecavir and tenofovir, introduced in the 2000s, effectively suppress HBV DNA replication by inhibiting the viral polymerase, leading to profound reductions in serum HBV DNA levels (often >4 log10 IU/mL). However, these agents do not directly eliminate cccDNA, resulting in only modest decay rates, typically less than 1 log10 reduction after one year of therapy and even slower clearance over subsequent years despite continuous suppression.^[36] This limited impact on cccDNA underscores the inefficacy of nucleoside analogs in eradicating the infection, as the stable minichromosome continues to serve as a template for viral transcription.^[37] Intrahepatic cccDNA levels at the end of therapy serve as a reliable predictor of post-treatment relapse, outperforming serum HBV DNA measurements in forecasting sustained virologic response. Elevated residual cccDNA correlates with higher risks of viral reactivation, with studies showing that even undetectable serum HBV DNA does not guarantee clearance if cccDNA persists. Furthermore, HBsAg seroclearance, a marker of functional cure, remains rare under nucleoside analog therapy, occurring at annual rates of approximately 1-3% in treated chronic HBV patients, depending on factors like genotype and baseline antigen levels.^[38]^[39] Additional challenges include the potential for drug resistance, although entecavir and tenofovir exhibit low resistance rates (<1% after 5-6 years in nucleoside-naive patients), necessitating vigilant monitoring and potential switches in therapy. The inability to eliminate cccDNA often requires lifelong treatment to maintain viral suppression, increasing the burden of long-term adherence and associated costs. Upon cessation of therapy, rebound viremia is common due to residual cccDNA, with virological relapse occurring in 43-47% of patients, highlighting the critical need for strategies that target cccDNA directly to enable finite treatment durations.^[40]^[41]^[36]

Research and Therapeutic Strategies

Current Approaches

Current approaches to targeting cccDNA in chronic hepatitis B virus (HBV) infection primarily focus on indirect suppression of its formation and maintenance through antiviral agents, entry inhibition, and immune modulation, as direct eradication remains challenging. Nucleotide analogs, such as entecavir and tenofovir, inhibit HBV reverse transcriptase, thereby blocking the conversion of pregenomic RNA to relaxed circular DNA (rcDNA) and indirectly reducing de novo cccDNA synthesis by limiting the pool of rcDNA available for nuclear conversion.^[42] Clinical studies have shown that one year of entecavir or lamivudine therapy reduces intrahepatic cccDNA levels by approximately 1 log, though existing cccDNA pools persist due to the drugs' lack of direct activity on the minichromosome.^[42] Similarly, interferon-alpha (IFN-α) therapy modulates the epigenetic landscape of cccDNA, promoting histone modifications that lead to transcriptional silencing and partial reduction in its activity.^[43] IFN-α induces a long-lasting suppression of cccDNA transcription through alterations in chromatin structure, including reduced histone acetylation, achieving HBsAg seroclearance in about 3-5% of treated patients after extended therapy.^[43]^[44] Entry inhibitors represent another strategy to prevent new cccDNA formation by blocking viral uptake into hepatocytes. Bulevirtide (formerly Myrcludex B), a synthetic lipopeptide mimicking the preS1 domain of HBV surface antigen, binds to the sodium taurocholate cotransporting polypeptide (NTCP) receptor, inhibiting virion attachment and subsequent rcDNA delivery to the nucleus.^[45] In preclinical models, bulevirtide efficiently prevents intrahepatic HBV spreading and amplification of cccDNA in infected human hepatocytes, completely blocking de novo cccDNA establishment in cell culture systems.^[45]^[46] Although approved in Europe for chronic hepatitis delta virus (HDV) co-infection with HBV since 2020, its investigational use in HBV monotherapy or combination regimens has demonstrated safety and potential to limit cccDNA replenishment in ongoing phase II/III trials as of 2025.^[47]^[48] Immune-based therapies aim to enhance host clearance of cccDNA-harboring hepatocytes by restoring HBV-specific T-cell responses. Therapeutic vaccines, such as GS-4774—a yeast-based platform expressing HBV core, surface, and X antigens—seek to stimulate cytotoxic T lymphocytes (CTLs) capable of targeting and eliminating infected cells; however, development was halted after phase II trials showed it was well-tolerated but did not achieve significant HBsAg reductions alone.^[49]^[50] In a phase II randomized trial involving virally suppressed chronic HBV patients, GS-4774 induced HBV-specific T-cell proliferation and interferon-gamma production, though combination with tenofovir showed improved immune activation but limited impact on cccDNA clearance markers.^[49]^[51] Complementing this, immune checkpoint inhibitors like anti-PD-1/PD-L1 antibodies (e.g., nivolumab) block inhibitory signals on exhausted T cells, potentiating their antiviral activity against cccDNA-persisting hepatocytes.^[52] Early clinical trials in HBV patients, often combined with vaccines, have reported enhanced T-cell functionality and modest HBsAg declines, with phase II studies (e.g., incorporating ChAdOx1-HBV/MVA-HBV with checkpoint blockade, ongoing as of 2025) to evaluate functional cure rates.^[52]^[53] These approaches underscore the need for combination regimens to address cccDNA persistence, as referenced in sections on treatment challenges.

Future Directions

Emerging research in gene editing holds significant promise for directly targeting and eliminating cccDNA in chronic hepatitis B virus (HBV) infection. CRISPR/Cas9 systems have been engineered to cleave cccDNA sequences, disrupting the viral minichromosome and inhibiting replication in preclinical models. For instance, studies in the 2020s have demonstrated efficient editing of HBV cccDNA in cell lines and mouse models, reducing viral markers such as HBsAg and HBV DNA levels by up to 90% without significant off-target effects. As of late 2025, several programs have advanced to clinical trials, including Precision BioSciences' PBGENE-HBV (phase 1 initiated October 2025), Beam Therapeutics' ARCUS-based ELIMINATE-B (phase 1/2a September 2025), and Excision BioTherapeutics' EBT-107 (positive preclinical data September 2025 supporting clinical entry), all designed to target cccDNA and integrated HBV DNA. Additionally, CRISPR approaches targeting host factors like the NTCP receptor, which facilitates HBV entry, have shown preclinical success in preventing new infections while complementing cccDNA clearance efforts in humanized mouse models. These advancements, including lipid nanoparticle delivery of Cas9 mRNA and guide RNAs, have proven safe and effective in vivo, advancing toward broader clinical evaluation.^[54]^[55]^[56]^[57]^[58]^[59] Epigenetic modulation represents another frontier for silencing or degrading cccDNA, focusing on its chromatin structure to prevent transcriptional activity. Histone deacetylase (HDAC) inhibitors, such as trichostatin A, have been shown to alter cccDNA histone modifications, reducing viral transcription in infected hepatocytes by promoting an open chromatin state that enhances immune recognition. Small interfering RNAs (siRNAs) targeting HBV transcripts or host epigenetic regulators further contribute by inducing cccDNA decay through RNA interference pathways, with preclinical data indicating up to 80% reduction in cccDNA pools in vitro. However, challenges persist, including limited specificity that risks off-target effects on host genes and incomplete eradication in quiescent hepatocytes, necessitating refined delivery systems like AAV vectors for liver-specific action.^[60]^[61]^[62] Advancements in in vivo modeling are critical to bridging gaps in understanding cccDNA dynamics and testing eradication strategies. Humanized mouse models, such as those with Fah-/- Rag2-/- Il2rg-/- livers repopulated with human hepatocytes, enable robust HBV infection and replication, allowing precise measurement of cccDNA half-life—estimated at 40-50 days in non-dividing cells—and its response to interventions. Organoid models derived from human liver tissue further recapitulate cccDNA persistence and epigenetic regulation, filling data voids in traditional rodent systems that lack human-specific receptors. These models support ongoing preclinical trials of combination therapies, including CRISPR with immune modulators, aiming for functional cure defined by sustained HBsAg loss, with early results showing enhanced cccDNA clearance when paired with existing nucleoside analogs.^[63]^[64]^[65] Despite over 30 years of research, complete cccDNA eradication remains elusive, underscoring the urgent need for integrated strategies that address its stability and hepatic reservoir. Current gaps include limited insights into cccDNA turnover in vivo and the lack of therapies achieving sterilizing cure, with functional cure rates in recent 2025 trials reaching 10-50% in select patient subgroups (e.g., low baseline HBsAg) using combinations like siRNA inhibitors with interferon, though overall rates remain below 20% across broader populations. Future efforts must prioritize multidisciplinary approaches, combining gene editing, epigenetics, and advanced models to develop scalable, safe interventions that eliminate the viral reservoir and prevent reactivation.^[66]^[44]^[67]^[68]^[69]

References

[1]
Persistence of hepatitis B virus covalently closed circular DNA ... - NIH
Sep 17, 2014 · Accumulating evidence has proven that the persistence of covalently closed circular DNA (cccDNA) is the major barrier to eradicating chronic HBV infection.
[2]
HBV cccDNA: The Molecular Reservoir of Hepatitis B Persistence ...
The cccDNA serves as the sole template for viral replication, as it generates the pregenomic RNA (pgRNA) necessary for producing new viral genomes. This stable ...Hbv Cccdna: The Molecular... · 2. Hbv Genome And Structure... · 5. Targeting Cccdna As A...
[3]
Hepatitis B virus cccDNA is formed through distinct repair processes ...
Mar 11, 2021 · The formation and persistence of the HBV covalently closed circular DNA (cccDNA) is the root cause of HBV chronicity.
[4]
Research progress in hepatitis B virus covalently closed circular DNA
Apr 15, 2022 · The key molecular basis of HBV persistence involves cccDNA, which plays a crucial role in the development of HCC. The HBV cccDNA minichromosome ...
[5]
Molecular biology of hepatitis B virus infection - ScienceDirect
With the help of the duck model, Summers, Mason and Taylor discovered that hepadnaviruses replicate via reverse transcription (Mason et al., 1982, Summers and ...
[6]
Replication of the genome of a hepatitis B--like virus by ... - PubMed
A pathway for the replication of the DNA genome of hepatitis B-like viruses by reverse transcription of an RNA intermediate is proposed.Missing: pdf | Show results with:pdf
[7]
Replication of the genome of a hepatitis B-like virus by reverse ...
Mason et al., 1982. W.S. Mason, C. Aldrich, J. Summers, J.M. Taylor. Asymmetric replication of duck hepatitis B virus DNA in liver cells (free minus-strand DNA).
[8]
Hepatitis B - World Health Organization (WHO)
Jul 23, 2025 · In 2022, hepatitis B resulted in an estimated 1.1 million deaths, mostly from cirrhosis and hepatocellular carcinoma (primary liver cancer).
[9]
Hepatitis B Virus cccDNA: Formation, Regulation and Therapeutic ...
Upon infection, HBV establishes a pool of covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. The cccDNA exists as a minichromosome ...
[10]
HBV cccDNA A Culprit and Stumbling Block for the Hepatitis B Virus ...
Jul 7, 2022 · Existing medicines are unable to disable covalently closed circular DNA (cccDNA), which may result in HBV persistence and recurrence.
[11]
Mechanism of Hepatitis B Virus cccDNA Formation - PMC
In this review, we will first briefly discuss the effects of viral proteins on cccDNA formation and then focus on the recent identification of host repair ...Missing: supercoiled 1970s
[12]
Pre- and Post-Transcriptional Control of HBV Gene Expression
As shown, the DNA genome contains two direct repeats (DR1 and DR2), two enhancers (EnhI, EnhII), and four promoter regions that regulate protein expression.
[13]
Episomal and integrated hepatitis B transcriptome mapping ... - Nature
Sep 26, 2025 · HBV cccDNA has four promoters and two enhancer regions that direct the synthesis of six major transcripts. The Core promoter regulates the ...
[14]
Core promoter: A critical region where the hepatitis B virus makes ...
The HBV genome contains four promoters (core, preS1, preS2 and X) and two enhancers I and II (ENI and ENII) as cis-acting elements to control HBV transcription ...
[15]
Epigenetic Regulation of Hepatitis B Virus Covalently Closed ... - NIH
cccDNA minichromosome is stably maintained in quiescent hepatocytes with a long half-life and is responsible for the persistence of infection during the ...
[16]
Hepatitis B virus genome recycling and de novo secondary infection ...
Aug 22, 2018 · ... cccDNA dynamics. HBV cccDNA decayed slowly with a half-life of about 40 days. Conclusions: After a slow infection process, HBV maintains a ...
[17]
DNA Polymerase κ Is a Key Cellular Factor for the Formation of ...
Together, the results indicate that cccDNA in HBV infected HepG2-NTCP cells were mainly synthesized from input viral rcDNA and that the intracellular ...
[18]
Generation of Covalently Closed Circular DNA of Hepatitis B Viruses ...
Initial generation of cccDNA occurs upon nuclear import of the virion-borne relaxed circular (rc) DNA to which the viral polymerase is covalently linked ...
[19]
Nuclear import of hepatitis B virus capsids and release of the ... - PNAS
We found that viral capsids were imported in intact form through the nuclear pore into the nuclear basket.Missing: rcDNA | Show results with:rcDNA
[20]
Structural basis for nuclear import of hepatitis B virus (HBV ... - Science
Jan 10, 2024 · This paper describes the mechanisms of HBV capsid recognition by importins. We found that importin α1 binds a nuclear localization signal (NLS) at the far end ...Missing: rcDNA | Show results with:rcDNA
[21]
Nucleoporin 153 Arrests the Nuclear Import of Hepatitis B Virus ...
We showed that HBV capsids directly interact with nucleoporin 153 (Nup153), an essential protein of the nuclear basket which participates in nuclear transport ...
[22]
Mapping of histone modifications in episomal HBV cccDNA ... - PNAS
We present a novel approach and first detailed analysis to our knowledge of cccDNA chromatin from de novo infected cells and infected liver tissue.
[23]
Hepatitis B virus genome recycling and de novo secondary infection ...
A recent analysis showed a cccDNA half-life of about 40 days in NTCP-overexpressing HepG2 cells (Ko et al., 2018). But that in a clinical setting is likely ...Research Article · Highlights · Introduction<|control11|><|separator|>
[24]
Nuclear HBx binds the HBV minichromosome and modifies ... - PNAS
HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into ...
[25]
HBV cccDNA and Its Potential as a Therapeutic Target
There are 3–50 copies of cccDNA per infected cell, and the number of copies decreases when the host cell divides. Thus, recycling of the new relaxed ...
[26]
Roles of hepatocyte nuclear factors in hepatitis B virus infection - PMC
Hepatocyte nuclear factors (HNFs) play the most important roles in cccDNA transcription, especially in the generation of viral genomic RNA, a template for HBV ...
[27]
Transcription of Hepatitis B Virus Covalently Closed Circular DNA Is ...
In the case of HBV, its cccDNA is organized into a viral minichromosome in the nucleus of the infected hepatocyte, persisting as a stable episome which serves ...<|control11|><|separator|>
[28]
Mechanisms of Hepatitis B Virus cccDNA and Minichromosome ...
Apr 15, 2024 · Summers, J.; Mason, W.S. Replication of the Genome of a Hepatitis B-like Virus by Reverse Transcription of an RNA Intermediate. Cell 1982 ...Missing: history | Show results with:history
[29]
Mapping the Interactions of HBV cccDNA with Host Factors - PMC
Transcription of viral pgRNA and mRNAs is regulated by four distinct promoters (preS1, preS2, core, and X) and two known enhancers (EnhI and EnhII).
[30]
Nonproductive Hepatitis B Virus Covalently Closed Circular DNA ...
Dec 7, 2022 · cccDNA serves as the template for four viral transcripts, known as the 3.5-kb, 2.4-kb, 2.1-kb, and 0.7-kb transcripts (originally characterized ...
[31]
Chronic Hepatitis B Virus Persistence: Mechanisms and Insights
Feb 13, 2025 · This review explores the formation and maintenance of cccDNA, highlighting host factors influencing cccDNA stability and viral replication. It ...
[32]
Association between intrahepatic cccDNA and the severity of liver ...
Jan 24, 2025 · This research aimed to examine the association between hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and liver inflammation in chronic ...
[33]
Point-of-Care Tests for Hepatitis B: An Overview - PubMed Central
Without treatment, one in every four persons infected with chronic hepatitis B will develop liver cirrhosis over 20–30 years, and 2–5% of people with cirrhosis ...
[34]
Hepatocellular carcinoma: epidemiology, screening, and ... - PMC
Overall, the annual incidence of hcc is 2%–5% in patients with cirrhosis from chronic hbv or hcv infection. Based on Canadian Liver Foundation data from 2013, ...
[35]
Vertical transmission of hepatitis B virus: challenges and solutions
In fact, the rate of chronicity is about 90% in infants infected at birth or during the first year of life, 30%–50% in children aged 1–6 years, and 5%–10 ...
[36]
Clinical relevance of the study of hepatitis B virus covalently closed ...
Jan 4, 2016 · Even with the most potent NA, intrahepatic cccDNA levels decrease slowly during NA therapy, that is, less than 1 log10 after 1 year of ...
[37]
HBV cccDNA: The Stumbling Block for Treatment of HBV Infection
Several studies have demonstrated that the CRISPR/Cas system can efficiently destroy the HBV cccDNA. Importantly, in this review, the authors pointed out some ...
[38]
Intrahepatic Hepatitis B Virus Covalently Closed Circular DNA Can ...
Intrahepatic HBV cccDNA and intrahepatic total HBV DNA levels at the end of therapy are superior to serum HBV DNA as surrogates of sustained virologic response.
[39]
Factors Associated With Rates of HBsAg Seroclearance in Adults ...
Oct 17, 2018 · In a meta-analysis of 42,588 patients in 34 studies, the pooled annual rate of HBsAg seroclearance was 1.02%, which increased to 17.99% after 15 ...
[40]
Nucleoside/nucleotide analogues in the treatment of chronic ...
Sep 29, 2011 · Entecavir has a high barrier to resistance. Therefore the development of drug resistance is rare, occurring at a rate of only 1.2% after 5 years ...
[41]
Rebound of HBV DNA after cessation of nucleos/tide analogues in ...
Mar 29, 2020 · Stopping treatment for patients with undetectable cccDNA resulted in rebound of serum HBV DNA, mostly within 20 weeks.
[42]
One-year entecavir or lamivudine therapy results in reduction of ...
Both nucleoside analogues reduced serum viral load, intrahepatic total HBV DNA, and cccDNA by about 4.8 logs, 2 logs, and 1 log respectively. An increase in the ...Missing: nucleotide inhibit<|separator|>
[43]
Alpha-Interferon Suppresses Hepadnavirus Transcription by Altering ...
IFN-α induced a profound and long-lasting suppression of cccDNA transcription, which required protein synthesis and was associated with the reduction of ...
[44]
Hepatitis B functional cure: Current and future perspective - PMC
Oct 27, 2025 · In summary, HBV persistence is maintained by stable cccDNA, immune evasion, sustained antigen exposure, and viral interference with host ...
[45]
The entry inhibitor Myrcludex-B efficiently blocks intrahepatic virus ...
Conclusions: Myrcludex-B efficiently not only prevented HBV spreading from infected human hepatocytes in vivo, but also hindered amplification of the cccDNA ...Missing: rcDNA nuclear
[46]
T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative ...
Finally, we demonstrate that entry inhibitors like Myrcludex B are able to block cccDNA formation completely. ... nuclear hepatitis B virus cccDNA.” Science 344: ...
[47]
Final Data From the Phase 3 MYR301 Study Demonstrated Longer ...
May 7, 2025 · Bulevirtide 2 mg remains the only approved treatment for adults with chronic HDV and compensated liver disease in the European Economic Area ( ...
[48]
Drug development for chronic hepatitis B functional cure - NIH
Apr 27, 2025 · Bulevirtide, formerly called Myrcludex B, is a linear lipopeptide derived from HBV PreS1 region that potently blocks this interaction by ...
[49]
Randomized phase II study of GS-4774 as a therapeutic vaccine in ...
Conclusions: GS-4774 was well tolerated, but did not provide significant reductions in serum HBsAg in virally suppressed patients with chronic hepatitis B.Missing: checkpoint inhibitors clearance cccDNA
[50]
Combined GS-4774 and Tenofovir Therapy Can Improve HBV ...
We performed a multicenter phase 2 study to evaluate the safety and efficacy of GS-4774, a yeast-based therapeutic vaccine engineered to express HBV antigens, ...Missing: checkpoint inhibitors clearance cccDNA
[51]
Checkpoint Inhibitors and Therapeutic Vaccines for the Treatment of ...
Mar 3, 2020 · In this review, we outline correlates of successful versus inadequate T cell responses to HBV, and discuss the rationale behind therapeutic ...
[52]
Therapeutic vaccination for treatment of chronic hepatitis B
A Phase II clinical trial has recently opened to recruitment to assess therapeutic HBV vaccines ChAdOx1-HBV and MVA-HBV in combination with check-point ...The Adaptive Immune Response... · Hbv-Specific T Cell... · Recombinant Vaccines
[53]
CRISPR/Cas9 for hepatitis B virus infection treatment
May 10, 2023 · This paper reviews the progress of different HBV CRISPR/cas9, vectors for delivering to the liver, and the current situation of preclinical and clinical ...
[54]
Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus ...
Our study demonstrated that SM-102-based CRISPR is safe and effective in targeting HBV episomal and integration DNA in vivo.
[55]
Recent Progress and Future Prospective in HBV Cure by CRISPR/Cas
Dec 21, 2021 · CRISPR-mediated genome editing has emerged as a promising therapeutic approach to specifically destroy persistent HBV genomes, both cccDNA and integrated DNA, ...2. Hbv Life Cycle And... · 5. Crispr-Cas9 In... · 6.1. Cas9-Be For Permanent...
[56]
Targeting HBV cccDNA through chromatin destabilization
Gene-editing technologies, such as CRISPR/Cas9, have demonstrated the potential for permanently disrupting HBV cccDNA, but critical challenges, including off- ...
[57]
Epigenetic regulation and its therapeutic potential in hepatitis B virus ...
This review summarizes the current knowledge of cccDNA biology, the regulatory mechanisms underlying cccDNA transcription, and novel anti-HBV approaches.
[58]
Epigenetic modulation in chronic hepatitis B virus infection
Mar 17, 2020 · This review summarizes major findings in HBV epigenetic research, focusing on the epigenetic mechanisms regulating cccDNA activity and the modifications ...Hbv Replication Cycle · Regulation Of Cccdna... · Histone Modifications Of The...<|control11|><|separator|>
[59]
In Vivo Mouse Models for Hepatitis B Virus Infection and Their ... - NIH
Oct 29, 2021 · This review summarizes the currently available mouse models for HBV research, including hydrodynamic injection, viral vector-mediated transfection, recombinant ...Missing: organoid half- dynamics
[60]
Forthcoming Developments in Models to Study the Hepatitis B Virus ...
Apr 14, 2023 · Mice containing livers repopulated with human hepatocytes would provide excellent in vivo models for studies on human liver diseases and ...
[61]
Estimating hepatitis B virus cccDNA persistence in chronic infection
We estimate that during HBe-antigen positive (HBeAg POS ) infection the cccDNA lifespan is 61 (36–236) days, whereas during the HBeAg NEG phase of infection it ...
[62]
https://link.springer.com/article/10.1007/s00281-020-00780-6
[63]
Targeting HBV cccDNA Levels: Key to Achieving Complete Cure of ...
cccDNA forms a highly stable minichromosome by integrating host histone and non-histone proteins, making it susceptible to epigenetic regulation. This provides ...