Therapeutic interventions aimed at cccDNA: unveiling mechanisms and evaluating the potency of natural products

Abstract

Hepatitis B virus (HBV) infection persists as a formidable global health predicament, imposing a substantial burden on public health. It not only elevates the risk of cirrhosis but also significantly heightens the incidence of hepatocellular carcinoma (HCC), thereby exacerbating the complexity of managing this disease. Central to the intractability of chronic hepatitis B is the tenacious persistence of covalently closed circular DNA (cccDNA) within the nuclei of infected hepatocytes. This cccDNA serves as a stable transcriptional template, continuously fueling the production of viral components and rendering the virus refractory to current antiviral interventions.​ The attainment of a definitive cure for HBV infection hinges upon the development of innovative antiviral strategies that can precisely and effectively target and eliminate cccDNA from the infected liver cells. In this regard, natural products have emerged as a promising source of potential therapeutics. This comprehensive review delves into the natural products that have shown promise in specifically targeting cccDNA. It meticulously elucidates the intricate molecular mechanisms through which these natural compounds modulate cccDNA activity, such as interfering with cccDNA formation, disrupting its epigenetic regulation, or inhibiting its transcriptional output. Developing innovative strategies to target and eliminate cccDNA is crucial for curing HBV infection, and natural products hold great promise. This review details several natural products with cccDNA-targeting potential, supported by clear mechanisms and data. Dehydrocheilanthifolin (DHCH) from Corydalis saxicola inhibits HBsAg and HBeAg secretion in HepG2.2.15 cells. It may disrupt viral processes like pgRNA packaging or DNA polymerase activity, with IC50 values for reducing extracellular, intracellular DNA, and cccDNA at 15.08 μM, 7.62 μM, and 8.25 μM respectively. Methyl helicterate from Helicteres angustifolia decreases HBsAg, HBeAg, HBV DNA, and cccDNA in HepG2.2.15 cells. 15.8 μM reduces intracellular cccDNA. Curcumin from turmeric reduces viral load and cccDNA in d-imHCs; 30µM halves cccDNA levels. Epigallocatechin gallate (EGCG) from green tea hinders viral transcription and replication. 22.9μg/ml EGCG lowers cccDNA by about 60%. Asiaticoside from Hydrocotyle sibthorpioides inhibits HBsAg, HBeAg, and cccDNA in HepG2.2.15 cells. Notably, despite extensive research, no natural product has yet obtained clinical validation for cccDNA clearance, highlighting the significant translational gap between pre-clinical research and clinical application. By elucidating these molecular mechanisms, this review aims to contribute to the development of HBV-targeted therapies, offering valuable insights for designing novel therapeutic agents and optimizing existing treatment regimens, ultimately advancing the quest for an effective cure for HBV infection.

Keywords: HBV; cccDNA; covalently closed circular DNA; hepatitis B virus; natural product.

Figure 1

Mechanisms and structural differences in rcDNA-to-cccDNA transformation. The conversion of rcDNA to cccDNA involves the following sequential steps:(1) The viral polymerase attached to the 5’-end of the negative strand is removed, resulting in protein-free rcDNA (pf-rcDNA). (2) The RNA primer at the 5’-end of the positive strand is removed. (3) Using the negative strand as a template, the positive strand of viral DNA is completed. (4) The ends of both the positive and negative strands are ligated. (5) The DNA undergoes hyper - helical coiling and associates with histones to form the cccDNA minichromosome. cccDNA consists of two fully formed strands: the minus - strand on the outer part and the plus-strand on the inner part. At nucleotide (nt) positions 1826 and 1592, there are two direct repeats (DRs). Moreover, the location of the origin is at the EcoRI site.

Figure 2

Regulatory factors of cccDNA transcription in HBV. The HBV life cycle begins with HBV binding to the NTCP receptor on hepatocytes. The viral nucleocapsid then enters the nucleus, where rcDNA is converted to cccDNA. Using cccDNA as a template, transcription generates different RNAs that synthesize specific viral proteins. PgRNA interacts with polymerase, gets packaged into capsids, and through reverse transcription, a complete viral genome is formed. Post-replication, HBV particles and non-infectious SVPs are assembled and secreted, potentially with multivesicular bodies (MVBs) involved in transport. Various host and viral factors that are involved in cccDNA regulation. These include transcription factors (e.g., TBP, AP-1, Sp1), DNA methyltransferases (DNMT1, DNMT2, DNMT3), protein methyltransferases (PRMT1, PRMT5), histone deacetylases (SIRT3, HDAC1, HDAC11), and host restriction factors (Smc5/6, ZEB2, STIM1, PSME4).

Figure 3

HBV life cycle and the role of natural products in modulating the process. The life cycle of HBV involves the following key steps: HBV initiates the infection process of hepatocytes by specifically binding to the NTCP receptor, which is the crucial starting event for the virus to invade host cells. Subsequently, the viral nucleocapsid is transported into the nucleus and enters the nucleus. Within the nucleus, rcDNA is converted into cccDNA under the action of host-related factors, forming a stable cccDNA molecule. Using cccDNA as a template, the transcription process is initiated, generating RNAs of different lengths (mainly 3.5kb, 2.4kb, 2.1kb, and 0.7kb). A 3.5kb RNA produces the protein product from HBcAg and polymerase; a 2.4kb RNA is translated into L-HBsAg and a 2.1kb RNA synthesizes the other two surface antigens, M-HBsAg and S-HBsAg; and a 0.7kb RNA produces HBxAg. The pgRNA interacting to HBV polymerase is selectively packaged into capsid particles. Inside the capsid particles, a DNA negative strand is synthesized using pgRNA as a template through the reverse transcription process. Subsequently, a DNA positive strand is synthesized, ultimately forming a complete viral genome. After genome replication, HBV viral particles and non-infectious SVPs are formed and secreted extracellularly. SVPs mainly refer to particles composed of HBsAg. Their self-assembled is independent of viral genome replication. For example, HBsAg can be highly expressed and self-assemble into these particles, with their secretion amount often exceeding that of infectious viral particles. Also shown in the figure are other types of secreted particles, in addition to virions and SVPs. Additionally, structures like MVBs, which may play a role in transporting. Various natural products, including Ciliatoside A, Dicoumarol, Sphondin, EGCG, Methyl helicterate, DHCH, Asiaticoside, Curcumin, Isochlorogenic acid A, Cimicifuga foetida L. and Furanocoumarins (Tyagi et al., 2024), play important inhibitory roles in the HBV life cycle.