Revisiting Hepatitis B Virus: Challenges of Curative Therapies

Abstract

With a yearly death toll of 880,000, hepatitis B virus (HBV) remains a major health problem worldwide, despite an effective prophylactic vaccine and well-tolerated, effective antivirals. HBV causes chronic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma. The viral genome persists in infected hepatocytes even after long-term antiviral therapy, and its integration, though no longer able to support viral replication, destabilizes the host genome. HBV is a DNA virus that utilizes a virus-encoded reverse transcriptase to convert an RNA intermediate, termed pregenomic RNA, into the relaxed circular DNA genome, which is subsequently converted into a covalently closed circular DNA (cccDNA) in the host cell nucleus. cccDNA is maintained in the nucleus of the infected hepatocyte as a stable minichromosome and functions as the viral transcriptional template for the production of all viral gene products, and thus, it is the molecular basis of HBV persistence. The nuclear cccDNA pool can be replenished through recycling of newly synthesized, DNA-containing HBV capsids. Licensed antivirals target the HBV reverse transcriptase activity but fail to eliminate cccDNA, which would be required to cure HBV infection. Elimination of HBV cccDNA is so far only achieved by antiviral immune responses. Thus, this review will focus on possible curative strategies aimed at eliminating or crippling the viral cccDNA. Newer insights into the HBV life cycle and host immune response provide novel, potentially curative therapeutic opportunities and targets.

Keywords: Hepadnaviridae; cccDNA; hepatitis B virus; hepatocellular carcinoma; interferons; reverse transcriptase.

FIG 1

Schematic of hepatitis B virus (HBV) replication cycle. 1. Virus binding and entry into the host cell (large rectangle). 2. Intracellular trafficking and delivery of relaxed circular DNA (rcDNA [rc]) to the nucleus (large circle). 3 and 3a. Conversion of rcDNA to cccDNA (CCC) (3) or integration of the double-stranded linear DNA (dslDNA [dsl]) into host DNA (3a). 4, 4a, and 4*. Transcription to synthesize viral RNAs (wavy lines), including the following: C mRNA for both the core and polymerase (reverse transcriptase [RT]) proteins; LS mRNA for the L envelope protein; S mRNA for the M and S envelope proteins; X mRNA for the X protein, which is transported to the nucleus to stimulate cccDNA transcription (4*); and precore mRNA (PreC) for the precore protein. The C mRNA is also the pgRNA. 5. Translation to synthesize viral proteins. 6 and 6a. Assembly of the pgRNA- (and RT)-containing capsid (6) or, alternatively, empty capsids (6a). 7. Reverse transcription of pgRNA to synthesize the minus-strand ssDNA and then rcDNA. 8. Nuclear recycling of capsids containing progeny rcDNA to form more cccDNA (intracellular cccDNA amplification). 9, 9a, 9b, 9c, and 9?. Envelopment of the rcDNA-containing capsid and secretion of complete virions (9), or alternatively, secretion of empty virions (9b) or spheric and filamentous subviral particles containing HBsAg only (9a). Processing of the precore protein and secretion of HBe (9c). The secretion of putative RNA virions is not yet resolved (9?). The different viral particles outside the cell are depicted schematically as follows, with their approximate concentrations in the blood of infected persons indicated: the complete, empty, or RNA virions are depicted as large circles (outer envelope) with an inner hexagonal shell (capsid) with or without rcDNA (unclosed, double concentric circle) or RNA (wavy line) inside the capsid, respectively, and subviral spheres and filaments as small circles and a cylinder. It is important to point out that the concentrations of all these particles can vary widely between different patients and over time in the same patient. Intracellular capsids are depicted as hexagons, either containing viral pgRNA, minus-strand ssDNA (straight line), or rcDNA or empty. The letters “P” denote phosphorylated residues on the immature capsids (containing ssDNA or pgRNA) or empty capsid. The dashed line of the diamond in the rcDNA-containing mature capsid signifies the destabilization of the mature capsid, which is dephosphorylated. The empty capsids, like mature capsids, are also less stable than immature capsids but, unlike mature capsids, are phosphorylated. The soluble, dimeric HBeAg is depicted as gray double bars. The thin dashed line and arrow denote the fact that HBeAg is frequently decreased or lost late in infection. Boxed letters denote the viral proteins translated from the mRNAs. The filled circle on rcDNA denotes the RT protein attached to the 5′ end of the minus strand (outer circle) of rcDNA, and the arrow denotes the 3′ end of the plus strand (inner circle) of rcDNA. For simplicity, synthesis of the minor dslDNA in the mature capsid, its secretion in virions, and infection of dslDNA-containing virions are not depicted here. Potential therapeutic strategies targeting cccDNA are highlighted in yellow boxes. i, inhibitor. See text for details. (Adapted from Viruses [180]).

FIG 2

Productive and nonproductive pathways of HBV cccDNA formation. The closed minus-strand rcDNA (cM-RC DNA) (middle row) is considered to be a likely intermediate during cccDNA (CCC DNA) formation. Other protein-free-rcDNA (PF-RC DNA) species (top and bottom rows) are considered to be off-pathway products for cccDNA formation. Putative host factors involved in generating the various PF-rcDNA species and cccDNA are indicated. The question mark in the diamond (bottom) denotes the unknown nature of the 5' end of the minus strand of this PF-RC DNA species. See text for details. (Adapted from reference 97).