The hepatitis B virus establishes its persistence through a peculiar molecular intermediate known as covalently closed circular DNA, or cccDNA. After the virus enters a hepatocyte, its partially double-stranded circular genome travels to the nucleus, where host repair enzymes convert it into a fully double-stranded, covalently closed circle. This minichromosome behaves remarkably like a cellular chromosome: it is wrapped in histone proteins, carries epigenetic marks, and serves as the template for all of the viral RNA transcripts that drive the production of new virions and, critically, of hepatitis B surface antigen, the viral protein that accumulates to enormous levels in the blood of chronically infected patients and is thought to exhaust and dysregulate antiviral immune responses. Because cccDNA is extraordinarily stable and can persist in dividing and non-dividing hepatocytes alike, clearing it has been the elusive goal of hepatitis B research for decades.

Existing treatments manage the consequences of cccDNA activity rather than the reservoir itself. Nucleoside and nucleotide analogues, such as tenofovir and entecavir, potently inhibit the viral polymerase and prevent new rounds of replication, but they leave cccDNA untouched, which is why discontinuation almost universally leads to viral rebound. Pegylated interferon-alpha can produce functional control in a minority of patients, likely through a combination of direct antiviral and immunomodulatory effects, but its tolerability is poor and its cure rates remain low. The result is that most patients face lifelong daily therapy, and the dream of a finite treatment course that leaves the liver free of active viral transcription has remained out of reach. Epigenetic silencing offers a conceptual alternative: if the viral minichromosome can be rendered permanently or durably transcriptionally silent, the virus may be functionally inactivated even though its DNA physically remains.

The investigational therapy described in the new study is built on this premise. It is designed as an epigenetic silencer, a construct that targets the hepatitis B viral DNA and recruits repressive chromatin-modifying machinery to the viral genome, thereby converting the active cccDNA minichromosome into a heterochromatic, transcriptionally inert state. In practical terms, the therapy aims to deposit repressive histone marks and promote DNA methylation patterns at the viral regulatory elements that normally drive transcription of all the viral RNAs, including the pregenomic RNA that seeds new replication and the abundant subgeniral RNAs that encode surface and core proteins. By shutting down transcription at its source, the approach addresses both the production of infectious virus and the antigenemia that blunts immune recovery.

The engineering challenge at the heart of such a therapy is specificity. The human liver contains roughly two hundred billion hepatocytes, and the silencing machinery must find the small fraction of cells harboring transcriptionally active cccDNA while leaving the host genome and its epigenetic landscape untouched. The reported system couples a DNA-binding module that recognizes sequences within the hepatitis B genome to effector domains capable of recruiting repressive complexes. Because the viral regulatory region, including the core promoter and enhancer elements, contains sequence features that are conserved across hepatitis B genotypes but absent from the human genome in the same configuration, the design exploits a natural sequence boundary between pathogen and host. Delivery to hepatocytes is achieved with a liver-directed formulation intended to concentrate the payload in the organ where more than ninety percent of viral replication occurs.

Epigenetic silencing of viral genomes is not an entirely new idea, but translating it into a viable therapy has required overcoming several biological realities. The first is that cccDNA is not a static target: it exists in variable copy numbers per cell, ranges from fully active to partially silenced states even within a single liver, and can be replenished from integrated viral DNA in some patients. The second is reversibility. Epigenetic marks are, by definition, modifiable, and a silenced minichromosome could in principle be reactivated if repressive marks decay or if the cell encounters inflammatory or proliferative signals that remodel chromatin. The investigators therefore designed the therapy not merely to deposit transient repressive marks but to establish a self-reinforcing silenced state, in which repressive chromatin at the viral promoter recruits further repressive machinery, creating a memory of inactivation that persists across time and, in dividing cells, across cell generations.

The functional consequences of such silencing would extend beyond simply lowering viral load. Hepatitis B surface antigen is now understood to be a principal driver of immune dysfunction in chronic infection, with circulating subviral particles reaching concentrations of micrograms per milliliter and contributing to the exhaustion of natural killer cells, T cells, and B cells that would otherwise clear infected hepatocytes. A therapy that transcriptionally inactivates cccDNA would be expected to produce a steep and sustained decline in surface antigen, removing this immunosuppressive burden and creating the conditions under which the patient’s own immune system can recognize and eliminate residual infected cells. In this sense, epigenetic silencing is best understood not as a standalone cure but as the keystone of a combination strategy, in which silencing of the reservoir is followed or accompanied by therapeutic vaccination, immune checkpoint modulation, or other immunorestorative interventions that consolidate the gain.

Safety considerations loom large for any therapy that deliberately manipulates chromatin. Broadly acting epigenetic drugs, such as the histone deacetylase inhibitors developed in oncology, carry systemic toxicity profiles that make them unattractive for chronic liver disease. The investigational silencer is therefore designed to act locally and selectively, with its repressive activity confined to the viral sequences it is targeted to. Another concern is the fate of integrated hepatitis B DNA, which in many chronic infections is scrambled into host chromosomes and cannot be circularized or fully silenced by the same mechanism; integrated DNA is a known source of surface antigen production and, in some contexts, of insertional mutagenesis that contributes to hepatocellular carcinoma. The therapy’s developers and independent commentators alike emphasize that silencing cccDNA addresses the replicative reservoir but does not by itself excise integrated sequences, underscoring that long-term monitoring for surface antigen rebound and for liver cancer risk will remain necessary even in successfully treated patients.

The publication arrives at a moment of unusual momentum in hepatitis B cure research. Dozens of candidates are advancing through clinical pipelines, including entry inhibitors, capsid assembly modulators, RNA interference therapeutics that degrade viral transcripts, surface antigen secretion inhibitors, and therapeutic vaccines. RNA interference drugs in particular have demonstrated that reducing viral antigen is feasible and well tolerated, and several have advanced to late-stage trials, though rebound after discontinuation has highlighted the persistence of the cccDNA reservoir that RNA-based approaches cannot touch. An epigenetic silencer occupies a distinctive position in this landscape because it acts on the reservoir itself, at the level of the minichromosome, rather than on the RNA or protein products that flow from it. If its silenced state proves durable, it could provide the reservoir control that RNA interference and direct-acting antivirals lack, while complementing the immune-directed agents needed to finish the job.

Much work remains before such a therapy could reach patients. The path from proof of concept in laboratory and animal models of hepatitis B infection to demonstrated safety and efficacy in humans is long, and epigenetic therapies will face scrutiny from regulators accustomed to evaluating small molecules and antibodies. Questions about the completeness of silencing across the enormous heterogeneity of cccDNA populations, the behavior of the therapy in patients with high viral loads and advanced fibrosis, the potential for rare escape variants in the targeted viral sequences, and the reversibility of the silenced state over years rather than weeks will all need rigorous answers. Nevertheless, the development of an investigational therapy designed to transcriptionally inactivate viral DNA marks a conceptual milestone: it treats the hepatitis B minichromosome not as an untouchable fixture of chronic infection but as an epigenetic state that can be rewritten. For the hundreds of millions of people living with chronic hepatitis B, the prospect of a finite course of treatment that leaves the virus silent, the immune system restored, and the liver protected represents a goal that has never been closer to serious experimental pursuit.

Subject of Research: Development of an investigational epigenetic silencer therapy that transcriptionally inactivates hepatitis B viral DNA in chronic hepatitis B

Article Title: Development of an investigational epigenetic silencer therapy to transcriptionally inactivate viral DNA in chronic hepatitis B

Article References: Anglero-Rodriguez, Y., Xiong, Q., Cappelluti, M. A., Voytek, S. B., Acosta, G., Choo-Wing, R., Hoffman, L., Khadka, A., Medina, J. A., Mugambwa, C., Pantano, C., Harel, S., DiPiazza, A., Abraham, S., Hildebrand, E. M., Ramirez, R. N., Zhai, A., Guo, X., Abubucker, S., … Marlowe, J. L. (2026). Development of an investigational epigenetic silencer therapy to transcriptionally inactivate viral DNA in chronic hepatitis B. Nature Biomedical Engineering. https://doi.org/10.1038/s41551-026-01802-8

Image Credits: AI Generated

DOI: 10.1038/s41551-026-01802-8

Keywords: chronic hepatitis B, hepatitis B virus, cccDNA, epigenetic silencing, viral DNA, hepatitis B surface antigen, antiviral therapy, chromatin modification, functional cure, liver disease, RNA interference, transcriptional repression

Yet the numbers remain sobering. According to the review, even with the best currently available regimens, the functional cure rate among patients with chronic hepatitis B stays below 30 percent, and in many treatment settings it is far lower. Nucleos(t)ide analogues such as entecavir and tenofovir suppress viral replication with remarkable potency and an excellent safety profile, but they act on the reverse transcriptase step of the hepatitis B virus life cycle and leave the intracellular reservoirs of viral genetic material untouched. Pegylated interferon-alpha, the other pillar of standard therapy, can reinvigorate antiviral immune responses in a subset of patients, but response rates are limited and tolerability issues frequently force discontinuation. The result is a therapeutic plateau: viral suppression is easy to achieve, but genuine immune-mediated control of the infection remains the exception rather than the rule.

At the heart of this plateau, the authors identify the persistence of covalently closed circular DNA, or cccDNA, as the central virological obstacle. cccDNA is the circular, episodic form of the hepatitis B virus genome that resides in the nucleus of infected hepatocytes and serves as the transcriptional template for all viral RNAs, including the pregenomic RNA from which new viral particles are assembled. Because cccDNA is stable, long-lived and present in minute quantities per cell, it evades both the cytoplasmic degradation pathways triggered by nucleos(t)ide analogues and the surveillance of the immune system, which struggles to recognize infected hepatocytes that produce little or no viral antigen. Clearing cccDNA from the liver would require either eliminating every infected hepatocyte or inducing selective degradation of the minichromosome itself, and neither approach is currently achievable with acceptable safety in the overwhelming majority of patients.

Compounding the cccDNA problem is a second form of viral persistence that the review highlights in detail: the integration of hepatitis B virus DNA into the genome of the host hepatocyte. Integration occurs frequently during the chronic phase of infection, driven by the_error-prone nature of viral replication and the continual turnover of infected cells. Integrated viral sequences are typically rearranged and replication-defective, but they can act as persistent factories for hepatitis B surface antigen, sustaining high circulating levels of HBsAg even when cccDNA transcription has been effectively silenced. This has two major consequences. First, integrated DNA means that surface antigen loss cannot automatically be equated with the elimination of the viral reservoir, complicating the interpretation of treatment endpoints. Second, chromosomal integration is a recognized mutagenic event, and insertional disruption of host genes such as TERT has been implicated in the development of hepatocellular carcinoma, linking viral persistence directly to the most feared clinical outcome of chronic infection.

The review then turns to the immunological face of the problem, describing how prolonged exposure to elevated levels of viral antigens — above all hepatitis B surface antigen and hepatitis B e antigen — induces a state of immune tolerance that is sculpted from the very earliest stages of infection. In patients infected at birth or in early childhood, the developing immune system encounters enormous quantities of circulating antigen during the critical windows in which tolerance mechanisms are established. High antigen loads drive the deletion or functional silencing of HBV-specific T cells, promote the differentiation of regulatory T cells and exhaust antigen-presenting dendritic cells, so that by the time chronicity is established the immune system has been trained, in effect, to ignore the virus. Maternal antigen passage and the unique tolerogenic environment of the liver, which is continuously exposed to gut-derived material and normally biased toward immune non-responsiveness, reinforce this state and help explain why infection acquired early in life so rarely resolves spontaneously.

Even in patients whose T cells do persist, the review documents a landscape of immune exhaustion and dysfunction operating at multiple regulatory levels. HBV-specific CD8 T cells in chronic hepatitis B display the classic molecular signature of exhaustion: sustained expression of inhibitory receptors such as PD-1, CTLA-4, TIM-3 and LAG-3, shortened telomeres, impaired cytotoxic function and a shift toward a terminally differentiated, senescent phenotype. CD4 helper function is similarly compromised, weakening the support that CD8 cells require for sustained antiviral activity. Beyond the T cell compartment, natural killer cell function is dysregulated, B cell responses are blunted with defective production of neutralizing anti-HBs antibodies, and the intrahepatic cytokine milieu — enriched in suppressive mediators such as interleukin-10 and indoleamine 2,3-dioxygenase — actively restrains inflammatory responses. Exhausted T cells, the authors emphasize, are not simply passive casualties; they represent a partially reversible differentiation state, which is precisely why they have become the focal point of new immunotherapeutic strategies.

It is against this mechanistic backdrop that the review surveys the emerging arsenal of immunotherapy. Therapeutic vaccines aim to present HBV antigens in an immunogenic context that can break tolerance and prime new antiviral T cell responses. Checkpoint inhibitors, most notably antibodies directed against PD-1 or PD-L1, seek to reverse the exhausted state of HBV-specific T cells, an approach supported by incidental observations of HBsAg decline in cancer patients treated with such agents. Agonists of pattern-recognition receptors, including Toll-like receptor 7 and 8 agonists, are designed to reawaken innate antiviral defenses and restore interferon production within the liver. Additional strategies under investigation include engineered T cell technologies, cytokine modulation, and combinations that pair direct-acting antivirals with immune interventions to first reduce antigen load and then rebuild antiviral immunity. The authors stress, however, that no single agent has yet demonstrated the capacity to induce functional cure reliably, and that the field’s most promising direction lies in rational combination: reducing the antigenic burden, releasing the brakes on T cell function, and providing the innate cytokine signals needed to coordinate a durable multi-layered response.

The review also underscores why the liver’s unique immunobiology makes this task so difficult. The hepatic environment is constitutively tolerogenic, populated by liver sinusoidal endothelial cells, Kupffer cells and stellate cells that favor the induction of regulatory rather than effector T cell responses. Any curative strategy must therefore overcome not only the viral reservoirs and the exhaustion of the adaptive immune system, but also an organ-specific architecture that has evolved to suppress inflammation. Biomarker development is identified as an equally pressing need: quantitative HBsAg levels, hepatitis B RNA signatures and emerging measures of immune restoration are all being explored as tools to identify which patients are most likely to benefit from which combination, and to shorten the lengthy trials that current endpoints demand.

By synthesizing virological persistence mechanisms, the layered failure of antiviral immunity and the emerging immunotherapeutic toolkit into a single framework, Lin, Yu and Sun aim to provide both a theoretical foundation and practical critical perspective for the clinical translation of functional cure strategies. Their central message is one of mechanistic realism: the obstacles to curing chronic hepatitis B functionally are not a single barrier but an interlocking system of viral reservoirs, integrated DNA, antigen-driven tolerance and multi-level immune exhaustion. Understanding how these pieces reinforce one another — and designing combinations that dismantle them in the correct sequence — is, the authors conclude, the surest path toward turning a goal achieved by fewer than a third of patients today into a routine clinical outcome tomorrow.

Subject of Research: Immunological mechanisms and barriers to achieving a functional cure for chronic hepatitis B

Article Title: The core obstacles and immunological mechanisms in the study of functional cure for Chronic Hepatitis B

Article References: Lin, L., Yu, D., & Sun, S. (2026). The core obstacles and immunological mechanisms in the study of functional cure for Chronic Hepatitis B. Virology Journal. https://doi.org/10.1186/s12985-026-03310-2

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03310-2

Keywords: chronic hepatitis B, functional cure, HBV, cccDNA, HBsAg loss, immune exhaustion, viral integration, immunotherapy, T cell dysfunction, immune tolerance, liver disease, antiviral therapy

The epidemiological picture painted by the review is strikingly heterogeneous. In the general population of low-endemicity countries, OBI prevalence can be as low as 0.8 percent. But in groups with concurrent viral infections the figure climbs steeply: more than 10 percent of individuals co-infected with hepatitis C virus or HIV carry occult HBV. The rate exceeds 40 percent among people with an isolated positive antibody to hepatitis B core antigen, a serological fingerprint of past exposure that persists long after surface antigen vanishes. A systematic review cited in the article found occult infection in adults across every region studied, and separate analyses in children show that babies born to surface-antigen-positive mothers can acquire OBI despite vaccination and hepatitis B immunoglobulin prophylaxis. These numbers matter because they define the size of the reservoir from which transfusion transmission, reactivation and, potentially, liver cancer can emerge.

Understanding how OBI arises requires a brief excursion into HBV biology. The virus maintains a persistent intermediate known as covalently closed circular DNA, or cccDNA, a minichromosome that resides in the nucleus of infected hepatocytes and serves as the transcriptional template for all viral RNAs. When surface antigen seroclearance occurs, whether spontaneously, after antiviral therapy or following recovery from acute infection, cccDNA is not eradicated. Instead it is transcriptionally silenced by epigenetic mechanisms, including histone deacetylation and methylation changes orchestrated by host factors such as SIRT3 and opposed by viral HBx protein recruits like LSD1 and Set1A. Integrated HBV DNA in the host genome can also persist independently of cccDNA, and both forms have been detected in the livers of patients classified as serologically recovered. The review emphasises that OBI therefore represents a spectrum of viral persistence rather than a single entity, arising either after HBsAg seroclearance in formerly chronically infected patients or after resolution of acute infection.

Why does surface antigen fall below detectable levels while viral DNA persists? The review details several converging mechanisms. Mutations in the PreS/S gene of the virus can alter or truncate the surface protein so that commercial assays fail to recognise it, a phenomenon known to account for a subset of occult infections. Viral variants carrying deletions in the X gene have been found in vaccinated individuals with OBI, suggesting immune selection pressures shape the hidden viral population. Host epigenetic silencing of cccDNA suppresses surface antigen expression to vanishingly small amounts, and newer ultrasensitive assays have shown that some patients previously labelled seronegative in fact have extremely low-level surface antigen detectable at concentrations below the standard cut-off of 0.05 IU/ml. This observation carries a conceptual twist: with sufficiently sensitive assays, some occult infections might be reclassified as overt infection, blurring the boundary between the two states.

Diagnosis remains the most technically demanding aspect of OBI. Serum HBV DNA levels in occult infection are typically extremely low and fluctuate over time, often dipping below the detection limits of routine commercial polymerase chain reaction assays. Hepatitis B core antibody, the single most useful serological marker, indicates prior exposure with a sensitivity of 77 percent and a specificity of 76 percent for seropositive occult infection, but it cannot by itself confirm active viral persistence. Liver tissue offers the definitive answer, since intrahepatic cccDNA is the hallmark of the condition, yet liver biopsy is rarely justifiable for diagnosis alone. The methodological frontier lies in droplet digital PCR, which partitions samples into thousands of nanolitre-scale reactions and enables absolute quantification of scarce DNA templates. Recent studies have validated high-sensitivity droplet digital PCR assays for both serum HBV DNA and intrahepatic cccDNA, and the review argues these platforms are reshaping what clinicians can detect. Complementary biomarkers such as hepatitis B core-related antigen and serum HBV RNA, which reflect cccDNA transcriptional activity, are being evaluated as non-invasive windows into occult viral replication.

The clinical consequences of undetected occult infection fall into three major categories. The first is transmission. Documented cases show HBV has been transmitted through transfusion of blood products and through solid organ transplantation from donors with occult infection, sometimes with devastating outcomes for immunosuppressed recipients. Analysis of transmission events has forced a revision of the minimal infectious dose, revealing that even very low viral loads in donated blood can establish infection. Nucleic acid testing of blood donations has reduced but not eliminated this risk, and the review discusses the ongoing international debate over whether anti-HBc screening of donors should be adopted more widely, weighing improved safety against the loss of otherwise suitable donors in endemic regions.

The second category is reactivation. Patients with occult HBV who receive immunosuppressive therapy, particularly B-cell-depleting agents such as rituximab, but also corticosteroids, ibrutinib, abatacept, temozolomide and therapies used in haematopoietic stem cell transplantation, are at risk of reverse seroconversion, in which surface antigen reappears and viral replication surges, occasionally causing fulminant hepatic failure. Quantification of core antibody titres may help stratify risk among patients with resolved infection, and international guidelines from the AASLD, AGA and EASL converge on the recommendation that individuals with serological evidence of prior HBV exposure should receive prophylactic nucleoside analogue therapy before high-risk immunosuppression. The review stresses that occult carriers are precisely the patients in whom routine serological screening fails, underscoring the importance of anti-HBc testing before oncological and rheumatological treatments.

The third and most debated consequence is oncogenesis. In patients with cryptogenic cirrhosis and cryptogenic hepatocellular carcinoma, studies have repeatedly found occult HBV DNA, cccDNA and viral integration events within tumour and surrounding liver tissue. A high proportion of patients with undetectable surface antigen and hepatocellular carcinoma harbour HBV DNA integrated into hepatocyte genomes, sometimes without cirrhosis, and integrations that reshape genomic structure have been shown to promote carcinogenesis through insertional mutagenesis and dysregulation of oncogenes such as those on chromosome arms recurrently targeted in the cancer genome surveys. The review also describes associations between occult infection and accelerated fibrosis in chronic hepatitis C, more severe outcomes in non-alcoholic fatty liver disease, and a prediction of non-alcoholic steatohepatitis in severely obese individuals. Nevertheless, the authors are careful to note that routine OBI screening is not recommended for milder chronic liver disease of other causes, and that the oncogenic potential of occult infection requires further study before screening policies change.

Management of confirmed OBI follows a deliberately targeted approach. The review recommends that OBI be excluded in individuals presenting with cryptogenic cirrhosis or hepatocellular carcinoma, and that treatment with a nucleoside analogue be initiated if occult infection is confirmed. For transplant medicine, grafts from anti-HBc-positive donors can be used with appropriate antiviral prophylaxis and monitoring, a strategy supported by systematic reviews showing acceptable recipient outcomes in liver, kidney and heart transplantation. For blood services, nucleic acid testing in minipool or individual-donation formats, alongside cost-effectiveness modelling from centres such as Shandong Blood Center in China, informs the evolving balance between transfusion safety and blood supply. What remains conspicuously unresolved is the management of the millions of anti-HBc-positive individuals worldwide who have no evidence of active replication and no need for treatment, yet who carry a dormant viral archive whose clinical behaviour over a lifetime is only beginning to be characterised.

The review closes with a set of challenges that will define the next decade of occult HBV research. Ultrasensitive surface antigen assays may reclassify part of the occult reservoir, forcing a redefinition of the disease boundary. Standardisation of droplet digital PCR for cccDNA and serum DNA, harmonisation of HBV RNA and core-related antigen measurements, and the incorporation of viral integration mapping into clinical phenotyping are all identified as priorities. As functional cure strategies for chronic hepatitis B, built around HBsAg seroclearance, move into large trials, the review’s central message acquires added urgency: seroclearance is not viral eradication. Every patient whose surface antigen disappears may still harbour replication-competent virus, and the systems of blood banking, transplant medicine, oncology and hepatology must be designed with that hidden reservoir in mind.

Subject of Research: Occult hepatitis B virus infection, the persistence of replication-competent HBV DNA in liver or blood of hepatitis B surface antigen-seronegative individuals

Article Title: Occult hepatitis B virus infection

Article References: Mak, L.-Y., Hui, R. W.-H., Pollicino, T., Cornberg, M., Gish, R., Jacobson, I., Kennedy, P. T., Seto, W.-K., Raimondo, G., & Yuen, M.-F. (2026). Occult hepatitis B virus infection. Nature Reviews Gastroenterology & Hepatology. https://doi.org/10.1038/s41575-026-01257-x

Image Credits: AI Generated

DOI: 10.1038/s41575-026-01257-x

Keywords: occult hepatitis B, hepatitis B virus, HBV DNA, cccDNA, hepatitis B surface antigen, anti-HBc, hepatocellular carcinoma, HBV reactivation, droplet digital PCR, blood transfusion transmission, liver transplantation, nucleoside analogues

The structures in question are G-quadruplexes, or G4s, four-stranded nucleic acid architectures that form in guanine-rich sequences. In a G4, four guanine bases pair through Hoogsteen hydrogen bonding to create a planar G-quartet, and the stacking of at least two such quartets, stabilized by pi-pi interactions and by monovalent cations such as potassium, produces a compact and remarkably stable fold. Depending on the orientation of the four guanine tracts that form the core, G4s can adopt parallel, antiparallel or hybrid topologies. In human cells, G4s are known to regulate telomere maintenance, DNA replication, transcription, mRNA processing and chromatin remodeling, and conserved potential G-quadruplex sequences have been documented in the genomes of Epstein-Barr virus, hepatitis C virus, HIV and SARS-CoV-2, where they influence replication and infection.

Researchers led by a team at Anhui Medical University systematically searched full-length hepatitis B virus genomes from genotypes A through H for sequences capable of forming G4s, using the QGRS Mapper algorithm to score the likelihood of quadruplex formation. High-scoring motifs clustered at positions 1204, 1732, 1886 and 3021 in a representative genotype B genome. Multiple sequence alignment and WebLogo analysis revealed that three of these motifs, at positions 1204, 1732 and 1886, are highly conserved across genotypes, with the exception of genotype G, while the 3021 motif is not evolutionarily conserved. Crucially, the 1204 motif lies within the region overlapping the X promoter and Enhancer I, the 1732 motif sits in the overlap between the core promoter and Enhancer II, and the 1886 motif falls within the epsilon RNA element of the precore and core gene.

To confirm that these predicted sequences actually fold, the team synthesized the corresponding oligonucleotides and subjected them to a battery of biophysical tests. Native polyacrylamide gel electrophoresis showed that the wild-type sequences migrated faster than guanine-mutated counterparts, consistent with the formation of compact intramolecular quadruplexes. Proton nuclear magnetic resonance spectroscopy detected the characteristic imino proton signals of Hoogsteen base pairing in the 10 to 12.5 parts per million range for all three sequences, signals that shifted toward Watson-Crick pairing signatures when guanines were mutated. Circular dichroism spectroscopy then resolved the topologies: the 1732 sequence adopts a parallel G4, while 1204 and 1886 fold into antiparallel conformations. Potassium ions proved preferred for stabilization, and the G4-stabilizing ligand BRACO-19 raised the melting temperatures of the structures, with 1732’s melting temperature exceeding the detectable range after ligand addition, underscoring the exceptional stability of these folds under physiological conditions.

Functional relevance was established with reporter systems. In a luciferase construct in which expression is driven by the hepatitis B virus core promoter together with Enhancer I and Enhancer II, disrupting the G4 at 1204, 1732 or 1886 significantly reduced reporter activity, with the 1732 mutation producing the strongest effect. In a more complete HBV 1.3-mer plasmid system, destroying the 1732 G4 markedly lowered the levels of hepatitis B surface antigen, e antigen and core antigen. Intriguingly, total viral RNA and the 3.5 kilobase pregenomic RNA were not significantly affected, pointing to a post-transcriptional role for the 1732 structure, possibly through modulation of translation efficiency, RNA conformation or transcript processing, rather than a direct effect on transcription itself. The 1204 mutation, constrained by the overlapping polymerase reading frame to a single G-to-A substitution that only partially weakened the fold, produced no significant changes in the 1.3-mer model, consistent with residual G4 signatures still visible in its circular dichroism spectra.

The next question was which host proteins these viral structures recruit. Using biotinylated 1204 and 1732 oligonucleotides as bait in pull-down experiments from HepG2.2.15 cell lysates, followed by data-independent acquisition mass spectrometry, the researchers identified 619 proteins enriched more than 1.5-fold over a non-G4 control, of which 34 participate in DNA repair, transcriptional regulation, RNA processing or G4 binding. The cellular nucleic acid-binding protein CNBP, a CCHC-type zinc finger protein, emerged as the most highly enriched G4-binding partner, while the hepatocyte nuclear factor 4 alpha, HNF4A, a master transcriptional activator of the hepatitis B virus core promoter, had never previously been reported to bind G4 structures. RNA interference against five candidate proteins, including CNBP and HNF4A, significantly reduced viral antigen and RNA levels, confirming their positive contribution to viral gene expression.

Surface plasmon resonance quantified the physical interactions. Both the 1204 and 1732 quadruplexes bound HNF4A directly, with equilibrium dissociation constants of 2.552 times ten to the minus five molar and 1.991 times ten to the minus six molar respectively, meaning the 1732 structure binds roughly an order of magnitude more tightly. Neither the mutated sequences nor a non-G4 control showed any detectable binding, and replacing potassium with lithium, which disrupts G4 folding, abolished the interaction entirely, demonstrating that the folded structure itself, not the primary sequence, is what HNF4A recognizes. BRACO-19, by further stabilizing the quadruplexes, actively enhanced HNF4A binding in pull-down assays. AlphaFold3-based structural predictions supported these findings, revealing hydrogen bonds between both G4 elements and specific HNF4A residues, with partially overlapping binding interfaces.

Functional assays then separated the two host factors mechanistically. Overexpression of HNF4A markedly increased activity of the core promoter-enhancer reporter, but when the 1732 G4 was destroyed, HNF4A could no longer fully restore reporter output, indicating that an intact 1732 quadruplex is required for optimal HNF4A-dependent activation. The authors propose a dual regulatory model in which HNF4A activates the core promoter-enhancer region primarily through its classical DNA binding sites, while a secondary, G4-dependent mechanism involving direct engagement of the 1732 structure potentiates maximal promoter output, likely facilitated by the genomic proximity of the two elements. CNBP, by contrast, boosted viral antigen and RNA levels when overexpressed and reduced them when knocked down, yet it did not enhance, and even slightly decreased, core promoter-enhancer reporter activity, indicating that CNBP promotes hepatitis B virus expression through a mechanism independent of this promoter region, perhaps by stabilizing or unwinding RNA G4s within viral transcripts, as it has been shown to do with SARS-CoV-2 genomic G4s.

The study’s implications extend beyond basic virology. Because properly folded G4s in the core promoter-enhancer region support, rather than repress, viral gene expression, these structures and the proteins that bind them represent candidate targets for new anti-hepatitis B strategies, complementing G4-directed approaches already explored against SARS-CoV-2 and other viruses. The authors caution that their experiments relied largely on plasmid-based reporter and 1.3-mer systems, and that future work using authentic infection models and fully chromatinized cccDNA templates will be needed to confirm whether HNF4A associates directly with viral cccDNA in living cells. Quantifying effects on nascent RNA synthesis, RNA polymerase II recruitment and liquid-liquid phase separation, a process recently implicated in G4-driven cccDNA transcription, could further clarify how these unusual DNA folds choreograph the life of a virus that has evaded elimination for millennia.

Subject of Research: Conserved G-quadruplex structures in the hepatitis B virus core promoter and enhancer regions and their host protein partners regulate viral gene expression

Article Title: Regulatory roles of G-quadruplexes and G-quadruplex-binding proteins across the enhancer and promoter of the HBV genome

Article References: He, L., Huang, B., Ma, H., Wang, L., Wu, Q., Zhang, J., Yu, L., & Lv, S. (2026). Regulatory roles of G-quadruplexes and G-quadruplex-binding proteins across the enhancer and promoter of the HBV genome. Virology Journal, 23(1), Article 218. https://doi.org/10.1186/s12985-026-03132-2

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03132-2

Keywords: hepatitis B virus, G-quadruplex, core promoter, enhancer, G4-binding proteins, HNF4A, CNBP, BRACO-19, viral gene expression, cccDNA, surface plasmon resonance, antiviral therapy

The central obstacle to a cure is the remarkable persistence architecture of the hepatitis B virus. After the virus enters hepatocytes through the NTCP receptor, its relaxed circular DNA genome is repaired by host factors into covalently closed circular DNA, or cccDNA, a stable episomal minichromosome that persists in the nucleus and serves as the transcriptional template for all viral RNAs and proteins. In parallel, fragments of viral DNA integrate at random sites into the host genome as integrated DNA, or intDNA, which does not produce new virus but continuously secretes surface antigen and, in some cases, the oncogenic HBx protein, fueling immune tolerance and carcinogenesis. Current interferon-alpha and nucleos(t)ide analog therapies leave both reservoirs untouched, so viral rebound is common once treatment stops, and long-term nucleos(t)ide therapy carries risks such as nephrotoxicity.

The Groningen team frames the therapeutic landscape around two epigenetic strategies. The first, broad-acting epidrugs, globally modulate histone- and DNA-modifying enzymes; nine such agents are already FDA-approved in oncology. The second, epigenetic editing, fuses effector enzymes to programmable DNA-binding domains — zinc-finger proteins, transcription activator-like effectors, or deactivated CRISPR-Cas9 — to write repressive marks at specific viral loci. Because HBV gene activity on cccDNA is governed by histone acetylation and methylation, with HBx recruiting p300 to boost H3 and H4 acetylation while SETDB1-mediated H3K9me3 and PRMT1-mediated H4R3 methylation repress transcription, the viral minichromosome is an unusually tractable epigenetic target.

Among epidrugs, sirtuin 2 inhibitors have produced the most compelling results. The SIRT2 inhibitor AGK2 reduced HBV DNA, RNA, HBsAg and HBeAg by 15 to 30 percent in vitro and in HBV-transgenic mice without hepatotoxicity, and recent work showed the drug acts by recruiting repressive histone lysine methyltransferases to cccDNA, enriching H4K20me1, H3K27me3 and H3K9me3 while reducing RNA polymerase II occupancy. A more specific allosteric inhibitor, FLS-359, blocked the conversion of rcDNA into cccDNA entirely in primary human hepatocytes when given before infection, cutting cccDNA formation by more than half — though it was ineffective after infection was established, positioning it as a preventive rather than curative agent. Meanwhile, the DNA methyltransferase inhibitor 5-azacytidine reactivated interferon-stimulated genes and sensitized otherwise unresponsive cells to interferon-alpha, but it also raised NTCP expression and HBsAg levels, highlighting the double-edged nature of globally acting agents.

That lack of locus specificity is precisely what epigenetic editing is designed to fix, and the review catalogs five preclinical editing studies organized around two approaches. Indirect editing directs a Krüppel-associated box, or KRAB, repressor domain to key HBV regulatory elements; KRAB acts as a scaffold recruiting histone deacetylases, lysine methyltransferases and heterochromatin proteins to shut down transcription. Direct editing instead guides DNA methyltransferases such as DNMT3a or the bacterial M.SssI enzyme to CpG islands overlapping viral promoters, writing de novo methylation that can be mitotically inherited. Both routes reduced HBV RNA, DNA and antigens from cccDNA and, in some contexts, from intDNA, with far better specificity than epidrugs.

The zinc-finger platform, the most rigorously studied to date, illustrates both the promise and the limits. Zhao and colleagues built a six-finger ZFP-KRAB artificial transcription factor targeting the X gene enhancer that cut HBx RNA by roughly 60 percent in Hep3B cells, a line carrying integrated viral DNA. Luo and colleagues achieved the most durable result, with HBV DNA falling to about one-third of control levels in transgenic mice by day seven and remaining significantly lower at day 28, although HBsAg was unchanged. The direct approach by Xirong and colleagues, fusing DNMT3a to a ZFP targeting the X promoter CpG island, lowered HBsAg by 58 percent in mice and 90 percent in cells, with confirmed methylation at seven CpG sites — but the effect reversed by day 20, likely through passive or TET-mediated demethylation. A single TALE-based study by Bloom and colleagues delivered the fastest repression, cutting HBsAg and HBV RNA by 80 percent in Huh7 cells within 48 hours and by 80 to 95 percent in mice within five days, though attribution to KRAB was complicated by the absence of a TALE-only control.

The dCas9 platform, which replaces costly protein engineering with inexpensive single-guide RNAs, is now moving fastest toward patients. The only peer-reviewed dCas9 data for HBV come from a doctoral thesis by Rendón, who fused M.SssI to dCas9 and targeted the conserved CpG islands adjacent to the C and S gene promoters, achieving 2 to 15 percent increases in methylation that downregulated C and S gene transcription but faded within 48 hours. Nevertheless, two clinical trials of dCas9 epigenetic editing for hepatitis B are already underway — NCT06745973 and NCT06671093 — and parallel successes silencing HIV provirus with dCas9-KRAB, plus the demonstration that combined KRAB and DNA methyltransferase editors such as CRISPRoff produce durable, heritable silencing, provide strong proof of concept. Notably, dCas9 editing avoids the genotoxicity risk that shadows nuclease-based CRISPR-Cas9 approaches, whose guide RNAs can partially match human genomic sequences and whose double-strand breaks at the many, variable intDNA loci raise off-target mutagenesis concerns.

Industry has now taken up the challenge with three programs. Tune Therapeutics’ Tune-401, a liver-targeting lipid nanoparticle carrying a guide RNA to CGI2 and mRNA encoding a dCas9 fused to a methyltransferase and an undisclosed repressor, reported 99.99 percent repression of cccDNA-derived HBV RNA in primary human hepatocytes, strong repression of RNA and HBsAg from intDNA in Hep3B cells, epigenetic marks persisting through 275 rounds of cell division over 550 days, minimal off-target effects by RNA-seq, and a favorable safety profile in non-human primates — though these results have not yet been peer reviewed. A Phase 1b trial is recruiting in Hong Kong, Moldova and New Zealand. EpiGENIC’s Epi-003, a similar LNP platform, has entered a Phase 1 trial in China, and nChroma Bio’s CRMA-1001 has shown greater than 99 percent HBsAg reduction sustained for six months in preclinical models. A related Omega Therapeutics trial using LNP-delivered ZFP editors in liver cancer further validates liver-directed epigenetic delivery.

Significant hurdles remain before any of this translates into a functional cure. HBV genomes differ by more than 8 percent at the nucleotide level across genotypes, and the error-prone reverse transcriptase plus the chaotic nature of integration create sequence variability that could blunt editing efficacy, demanding bioinformatic surveillance and possibly personalized designs. Heterochromatin spreading from KRAB editors could silence neighboring host genes at unpredictable intDNA sites, while complete silencing of all antigen sources raises the paradox that eliminating HBsAg might allow exhausted immune cells to miss residual infection, arguing for integration with immune-reactivation strategies. Model limitations compound the problem: primary human hepatocytes rarely form intDNA, Hep3B lacks infection dynamics, and the field needs 3D organoid and humanized mouse systems that capture both reservoirs. Cost and equity loom as well, since most patients live in Southeast Asia and Sub-Saharan Africa while comparable gene therapies have been priced near two million dollars. Still, the authors conclude that with optimized effector combinations, LNP-based hit-and-run delivery — already shown to sustain liver gene silencing for over a year in mice and over 90 days in non-human primates in PCSK9 studies — and clinically relevant models, precision epigenetic editing could deliver what nucleos(t)ide analogs never could: a stable, reversible, non-genotoxic functional cure for chronic hepatitis B.

Subject of Research: Epigenetic editing strategies to silence hepatitis B virus cccDNA and integrated DNA for a functional cure of chronic hepatitis B

Article Title: The promise of epigenetic editing strategies in functionally curing chronic hepatitis B virus infections

Article References: The promise of epigenetic editing strategies in functionally curing chronic hepatitis B virus infections. (n.d.). https://doi.org/10.1186/s43682-025-00041-3

Image Credits: AI Generated

DOI: 10.1186/s43682-025-00041-3

Keywords: epigenetic editing, chronic hepatitis B, hepatitis B virus, cccDNA, integrated HBV DNA, dCas9, zinc-finger proteins, TALEs, KRAB, epidrugs, lipid nanoparticles, functional cure