Unveiling the roles of HBV polymerase for new antiviral strategies

Abstract

Infection with HBV is common worldwide, with over 350 million chronic carriers. Chronic HBV infection is associated with cirrhosis and hepatocellular carcinoma. All currently available oral antivirals are directed against the HBV polymerase enzyme, a reverse transcriptase. HBV polymerase contains several important domains and motifs which define its functions and reveal ways to further target it. This enzyme executes many functions required for the HBV replication cycle, including viral RNA binding, RNA packaging, protein priming, template switching, DNA synthesis and RNA degradation. In addition, HBV polymerase must interact with host proteins for its functions. Future therapeutics may inhibit not only the DNA synthesis steps which are carried out by the reverse transcriptase domain (as all current antivirals do) but other domains, functions and interactions which are essential to the HBV replication cycle.

Keywords: HBV; antiviral targets; drug resistance; replication cycle; reverse transcriptase.

Figure 1. Polymerase-dependent events in the HBV replication cycle

Beginning at the cccDNA which is found in the nucleus of infected cells, HBV pgRNA is transcribed. It is exported to the cytoplasm and then translated. The translated polymerase binds to pgRNA at the 5′ ε stem-loop structure. This interaction facilitates the encapsidation, or packaging, of both polymerase and pgRNA. Priming may occur before, concurrent with or after nucleocapsid assembly. Using a free hydroxyl group on Y63 of the polymerase as primer, DNA is attached to HP using ε as a template. After the initiating GAA nucleotides are laid down, HP and these bases switch templates to the 3′ end and continue minus strand DNA synthesis. As the template pgRNA is copied, it is concurrently degraded by the RNase H activity of the polymerase. The resultant ssDNA-containing nucleocapsid is then further matured as the polymerase copies the second strand of DNA, yielding an rcDNA-containing nucleocapsid. This maturation of the nucleocapsid leads to its destabilization, which may facilitate disassembly (uncoating) to liberate the rcDNA for conversion to more cccDNA in the nucleus. The polymerase must be removed in order to allow cccDNA formation. cccDNA: Covalently-closed circular DNA; ε: Epsilon RNA secondary structure motif; HP: HBV polymerase; pgRNA: Pregenomic RNA; rcDNA: Relaxed circular DNA.

Figure 2. Sequence conservation and important domains, motifs and residues of HBV polymerase

(A) Genotype homology mapping. Amino acid residues of seven reference strains of HBV representing genotypes A–G were mapped according to differences among 11 amino acid regions (considering a single residue with five amino acids upstream and five downstream). Each point along the graph represents the percentage of amino acid differences within the corresponding 11 amino acid region. Higher graph values thus represent less homology in that region among genotypes. GenBank IDs are: X02763.1, D00329.1, X01587.1, V01460.1, X75657.1, X75658.1 and AF160501.1 for genotypes A–G, respectively. (B) The domains and conserved RT boxes A–G of HP are shown. Numbering is according to genotype D for the TP domain and spacer, and follows standardized numbering for the RT and RNase H domains [38]. The position of the surface gene, which is in a different reading frame, is also shown. (C) Functionally important motifs and residues revealed through in vitro mutant screens. Several regions are important for the functional roles of HP, including the T3 and RT1 motifs which facilitate ε RNA binding. See text for specific details. Drawn to scale. ORF: Open reading frame; RT: Reverse transcriptase; TP: Terminal protein. Reference genotype list is taken from Stuyver et al. [38].

Figure 3. Structure of the HBV polymerase and treatment-associated mutations in its reverse transcriptase domain

(A) Illustration of the four domains, including the three RT subdomains. Minus-strand DNA is attached at the TP, and DNA or RNA passes through the ‘hand’ which synthesizes DNA. Most nucleotide interactions are at the palm subdomain. (B) Subdomain organization of the RT domain of HBV polymerase. Boxes A–G are part of the enzyme's catalytic core, and they are homologous to other RT enzymes. Each nucleos(t)ide analog is shown with its treatment-associated resistance mutations. Drawn to scale. ^†Tenofovir mutations have not been shown to confer in vivo resistance (see text for details). AA: Amino acid; RT: Reverse transcriptase; TP: Terminal protein.