Responsible for nearly half of all cases of hepatocellular carcinoma, chronic hepatitis B virus (HBV) is a worldwide concern and a major public health problem. Through rigorous research, investigators have identified 2 key aspects of the HBV lifecycle essential to the development of chronic infections: the establishment of a viral minichromosome in the form of covalently closed circular (ccc) DNA and the expression of the regulatory hepatitis B virus X (HBx) protein.

Researchers penned an overview of recent advances in the scientific understanding of cccDNA and the mechanistic and functional roles of HBx in a recent article in ACS Infectious Diseases.

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cccDNA Overview

According to researchers, the successful establishment of cccDNA is “critical” for HBV replication. Despite this, the specific host mechanisms that act to convert relaxed circular (rc) DNA into cccDNA are poorly understood.

Scientific advances have allowed researchers to begin identification of the roles played by various host enzymes in cccDNA generation, either through rationally testing the enzymes involved in DNA metabolism or by RNA silencing. Additional research has identified similarities between the antisense strand of rcDNA structure and 5′ flap structures, formed during Okazaki fragment maturation, as a contributor to the formation of cccDNA, and RNA silencing screening studies also identified several DNA polymerases — α, η, κ, and λ — that mediate cccDNA establishment.

Taken together with the recent confirmation of DNA ligases I and III as crucial in converting rcDNA to cccDNA and the identification the role of topoisomerase 1 and 2 in cccDNA synthesis, these factors may represent a novel therapeutic avenue for the treatment of chronic HBV infection, according to the researchers.

Further biochemical and biophysical studies are required to define the role of cccDNA-bound hepatitis B core antigen, which would prove “invaluable” for the study of both cccDNA and HBx.

HBx Overview

HBx is the primary effector protein encoded by HBV. Although numerous human proteins have been identified as potential interactors with HBx, “relatively few” of these interactions have known functional outcomes, representative of the ambiguity that surrounds the poorly understood structure of the protein.

Within the nucleus, HBx performs dual roles: redirecting host transcription factors, including p53, NF-κB, and CREB, to change the expression of a “wide variety of gene families,” and initiating virus replication via the stimulating transcription of cccDNA. Although HBx is critical for both cccDNA transcription and the development of viremia in vivo, the HBx protein itself is not packaged in the mature virion. According to the researchers, this raises the question of how, during initial HBV infection, HBx can be expressed without being already present in the cell.

“Current theories propose that HBx may be transcribed from rcDNA or dslDNA before or during conversion into cccDNA, though one recent report identified HBx mRNA in both cell culture derived virus preparations and HBV patient plasma, suggesting that it may indeed be packaged into the virion,” the researchers noted.

Yael David, PhD, and colleagues went on to describe the more well-known HBx functions and regulatory mechanisms.

HBx and CRL4. Until recently, the structural basis and functional significance for the interaction between HBx and damaged DNA-binding protein 1 (DDB1) was unknown; however, research has found that DDB1 works as an obligate adaptor protein for the cullin-RING ligase 4 (CRL4) E3 ligase complex. Later proteomic studies identified the HBx-mediated degradation of the structural maintenance of chromosomes. More targeted in vitro and cell-based studies are needed.

HBx and chromatin modifications. According to the researchers, decades of study have illustrated that HBx is able to redirect the cellular machinery responsible for the erasure or deposition of histone posttranslational modifications, to produce the active chromatin landscape on cccDNA. Specific chromatin immunoprecipitation-based studies found that, within HBx-deficient infections, there was an “increased occupancy of the histone deacetylases Sirt1 and HDAC1” on cccDNA. This suggests that HBx may either outcompete for binding sites or mediate their degradation. Ultimately, the ability of HBx to recruit diverse proteins to cccDNA “raises questions about the mechanism by which it mediates such interactions.”

Posttranslational modifications of HBx. To modulate its functions, HBx itself is posttranslationally modified, adding an additional layer of complexity to HBx biology and regulation. Recent studies have described the biochemical roles for both HBx posttranslational modifications and the enzymes that deposit them, which may eventually provide insight into so-called novel pathways to indirectly target the HBx function. In the future, a more detailed analysis of HBx structure and posttranslational modifications in vivo will be required, allowing future in vitrostudies to use more physiologically relevant conditions.

Advances in HBx and cccDNA-Targeting Treatments

To date, a significant body of work has highlighted the relative importance of both cccDNA and HBx in HBV replication and in the persistence of chronic infection. In this vein, recent studies have reported a variety of approaches that can target viral elements through the use of small molecule or biologic tools. Methods range from rationally targeted approaches, backed by established and recently developed methodologies, to “target-agnostic high-throughput screens.”

Current genome editing methods allow for the “relatively facile, locus-specific” generation of double-stranded DNA breaks. This generation led to theories suggesting that a similar approach could be used to target cccDNA for degradation. Several independent groups reported relative successes, but barriers still remain that prevent the clinical use of this approach — specifically, the lack of clinical trials surrounding the practice of gene-editing therapy.

Hepatitis B virus has a 10-fold higher mutation rate compared with conventional DNA viruses; as a result, resistance mutations may rapidly rise in conjunction with strong selective pressure. In addition, an approximately 8% sequence divergence at the DNA level may make it difficult for researchers to development a therapy capable of targeting each HBV genotype. Perhaps most importantly, though, is that the generation of a double-stranded DNA break in cccDNA would result in the generation of dslDNA, which could lead to genome integration by host DNA and the promotion of oncogenesis.

Despite these potential pitfalls, genome editing remains an attractive approach, useful in both research and in the clinic setting.

The lifecycle of HBV is complex, creating a challenging environment for study; however, this complexity also opens up several pathways that may be adapted for use within high-throughput screening approaches. Multiple recent studies have reported successes through promising screening approaches, several of which specifically targeted HBx functions or restricted cccDNA expression.

One example was the identification of a high-throughput screening that advantageously leveraged the documented role of HBx in inhibiting certain RNA silencing pathways. Using a computational model to generate a 3-dimensional structural model of HBx, investigators identified potential binders, which were then used to screen for the reversal of HBx-mediated RNA silencing suppression. One compound, dubbed “IR415,” was generated; additional studies will be necessary to determine whether IR415 disrupts any other aspects of HBx function.

Even more recently, researchers identified another HBx targeting molecule using a split luciferase screening assay. Nitazoxanide, a broad-spectrum, anti-infective agent used typically to treat parasitic infections was identified as a potential disruptor. Future studies should examine this interaction.

Most recently, investigators reported a chemical screen used to identify inhibitors of episomal DNA expression. Dicoumarol, a small molecule, was shown to decrease episomal DNA expression, which was validated using HBV-infected primary human hepatocytes. A dose-dependent depletion of cccDNA was observed, suggesting that dicoumarol may be the basis for future drug development.

Looking Forward: Future Perspectives in HBV

The use of interdisciplinary techniques, in particular, have led to significant advances in the understanding of cccDNA establishment and regulation, as well as the mechanisms of HBx function; however, questions still remain regarding these “key components” of HBV.

One difficulty in the study of HBx is the scarcity of methods used to manipulate or modulate native HBx within the context of an active HBV infection; however, the use of small molecules as described may represent a unique opportunity to chemically address these methodological challenges. Similarly, the use of protein engineering strategies to better study HBx both in vivo and in vitro should be developed; protein fusion methods have been reported in the literature, but efforts must continue to build on this process.

Despite significant progress in identifying the elements involved in the establishment and repair of cccDNA, structural and regulatory component details remain “enigmatic,” according to the authors. Recent efforts have been made to characterize the landscape of post-translational modifications of cccDNA, but many of these efforts rely on chromatin immunoprecipitationplus next-generation DNA sequencing, which has several shortcomings — availability, inherent target bias, and relatively low throughput among them.

Finally, the development of increasingly sophisticated methods to study chromatin biology in HBV-susceptible cell lines may establish a platform allowing for the interrogation of key biochemical questions regarding cccDNA. Specifically, the development of a method for the reconstruction of a cccDNA model for use in in vitro biochemical and biophysical studies might provide “crucial insight into the mechanisms behind recognition and recruitment of host factors onto cccDNA,” the researchers noted.

“Continued efforts in recent years have revealed critical details about the cccDNA life cycle and the HBx function that may potentially serve as the basis for novel therapeutic approaches,” David and colleagues concluded. “[O]ngoing research is needed to further build upon these advances. Chemical biologic and protein engineering techniques are perfectly poised to fully illuminate these two cryptic yet indispensable components of chronic HBV infection.”

Reference

Prescott NA, Bram Y, Schwartz RE, David Y. Targeting hepatitis B virus covalently closed circular DNA and hepatitis B virus X protein: recent advances and new approaches. ACS Infect Dis. 2018;5(10):1657-1667.