Disrupting HBV Chromatin Structure Blocks X Gene Transcription and Infection

Hepatitis B virus (HBV) relies on a unique DNA structure called cccDNA to initiate infection. This viral DNA forms a minichromosome by associating with host histone proteins, but the role of this structure in regulating viral gene expression has been unclear.

In a new study, researchers used a reconstituted HBV minichromosome system to show that nucleosome positioning on cccDNA directly controls transcription of the X gene, essential for degrading the host restriction factor Smc5/6 and triggering viral replication. Chromatin immunoprecipitation sequencing (ChIP-seq) revealed that nucleosome arrangement promotes a promoter architecture that supports transcription initiation. The study also found that CBL137, a chromatin-destabilizing compound, reduces full-length X gene transcription and inhibits HBV infection in primary human hepatocytes.

In an email Q&A with Yael David, PhD, associate member of the Chemical Biology Program at Memorial Sloan Kettering Cancer Center, she went into more detail about the study.

Contagion: Can you elaborate on the specific role of nucleosome occupancy in regulating the transcription of the HBV X gene? How does this process contribute to the initiation of HBV infection?

David: We have demonstrated that chromatinization enhances HBx transcription both in vitro and in cells. In the absence of histone occupancy, HBx transcription is minimal or undetectable, whereas proper nucleosome positioning markedly increases HBx expression. Although we are still investigating the underlying mechanisms, our ChIP-seq analyses suggest that chromatinization transforms the weak, TATA-less HBx promoter into a stronger, more human-like promoter by establishing a canonical -1/+1 nucleosome architecture that facilitates transcription machinery recruitment.

This chromatin-driven transcriptional activation is critical for early infection, as HBx is essential for the establishment of cccDNA. Specifically, early HBx expression enables the recruitment of the DDB1/CUL4 E3 ligase complex to the host restriction factor Smc5/6, leading to its degradation. In the absence of HBx, Smc5/6 sequesters the nascent cccDNA, blocking its transcriptional activity. Thus, early chromatin-dependent HBx transcription promotes productive infection by enabling efficient cccDNA establishment and viral gene expression.

Contagion: How does the drug CBL137 impact the chromatin structure of the HBV genome, and what implications might this have for broader antiviral strategies, especially for chronic HBV infections?

David: CBL137 destabilizes chromatin in both host and HBV contexts. Interestingly, at low doses, it appears to selectively disrupt HBV cccDNA chromatin while leaving host chromatin largely unaffected. This selective vulnerability highlights CBL137’s potential as an antiviral—not only for HBV but possibly for other viruses that rely on similar chromatin-based mechanisms to establish infection.

While we currently lack direct evidence that CBL137 is effective against chronic HBV infection, particularly due to the presence of integrated viral DNA, we hypothesize that it may still exert an effect by limiting the transcriptional activity of integrated HBV, disrupting the reactivation of latent cccDNA pools, or sensitizing infected cells to immune clearance through chromatin remodeling.

Contagion: Can you explain the potential clinical significance of CBL137 in human hepatocytes? How might this drug be developed into a treatment for chronic hepatitis B?

David: "While it is still early to fully evaluate the clinical potential of CBL137, our data suggest that it is effective at preventing the establishment of HBV infection in newly infected hepatocytes. This points to a potential role for CBL137 as a prophylactic or early-intervention antiviral agent by interfering with the formation or transcriptional activation of cccDNA, which is essential for persistent HBV infection."

"However, CBL137 also has limitations. As a DNA intercalator, it poses potential concerns related to toxicity, especially with long-term use, which may limit its application in the treatment of chronic hepatitis B. Despite these challenges, the mechanistic insights we are gaining from studying how cccDNA is established and maintained are opening up new opportunities for therapeutic development. CBL137 serves as a valuable tool compound to probe HBV chromatin biology and identify novel vulnerabilities in the viral life cycle. These insights could guide the design of more selective and safer epigenetic therapies targeting HBV persistence."

Contagion: What challenges remain in understanding the complete mechanism behind the chromatinization of HBV cccDNA, and are there other viral genes or pathways that may also be targeted using this approach?

David: "Significant challenges remain in fully understanding the mechanisms regulating HBV cccDNA, which may involve post-translational modifications, non-coding RNAs, chromatin-compacting factors, and complex transcriptional dynamics. However, our new platform has been transformative, enabling us to study the earliest events of HBV infection in unprecedented detail. We are now leveraging this system both to dissect the molecular mechanisms underlying cccDNA regulation and to identify novel therapeutic targets."

"We are just beginning to uncover the critical role that epigenetic mechanisms play in controlling cccDNA activity. Given these findings, it is both logical and likely that additional viral transcriptional events are also modulated by chromatinization and host chromatin architecture."

Contagion: How do your findings with CBL137 compare to current therapies for chronic hepatitis B, and could this represent a shift in how we approach treatment for this infection?

David: "Current therapies for chronic hepatitis B, such as nucleos(t)ide analogs, effectively suppress viral replication by inhibiting reverse transcription, but they do not target the viral reservoir—cccDNA—which remains transcriptionally active in infected hepatocytes. As a result, these therapies rarely achieve a functional cure, and treatment is often lifelong.

In contrast, our findings with CBL137 suggest a fundamentally different approach. CBL137 targets the epigenetic regulation of HBV by disrupting the chromatin structure of cccDNA, thereby silencing its transcriptional activity. This represents a shift from inhibiting viral replication to directly interfering with the persistence and transcriptional output of the viral reservoir itself."

"While CBL137 has limitations—including its role as a DNA intercalator, which may restrict its use in chronic settings—its selective action on HBV chromatin at low doses is both intriguing and promising. More importantly, this work provides a proof-of-concept that viral chromatin can be selectively targeted. This insight could open the door to a new class of therapeutics aimed at silencing or destabilizing cccDNA through epigenetic modulation, rather than direct antiviral inhibition."

"In that sense, CBL137 doesn’t just represent a potential therapeutic—it represents a conceptual shift in how we think about curing HBV: not only by suppressing the virus, but by reprogramming or disabling its chromatin to achieve long-term control or even clearance."

Reference

Prescott NA, Biaco T, Mansisidor A, et al. A nucleosome switch primes hepatitis B virus infection. Cell. February 20, 2025. Accessed April 17, 2025. doi:10.1016/j.cell.2025.01.033