Hepatitis B virus (HBV) infects the liver and is commonly spread through sexual, blood, and vertical contact. It is a leading cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. Although several hundred million people in the world are infected by HBV, an effective vaccine exists, as well as non-curative treatment [1
]. Current antiviral drugs, except immunomodulatory drugs such as pegylated interferon α and thymalfasin, are all directed against the HBV polymerase (Pol) [2
], though therapeutics for novel targets are in development [5
]. Although there is no small animal model capable of infection with human HBV, informative studies have been performed using related HBV species that infect avian and mammalian hosts, such as duck HBV (DHBV) and woodchuck HBV [6
]. With the discovery of the receptor for HBV (human sodium taurocholate co-transporting polypeptide receptor (hNTCP or SLC10A1
], the ability to create transgenics in small animals and cell culture systems is also leading to advances in model systems [8
HBV contains a small circular DNA genome, reverse transcribed by the HBV Pol from viral RNA [9
]. The HBV Pol protein is a major focus of basic and translational research; indeed, it is the only current target of any HBV-specific antiviral and the only region commonly sequenced during treatment escape [10
]. Although the catalytic activity of HBV Pol occurs within the RT and RNase H domains (Figure 1
), the TP domain also shows considerable functional utility. The TP domain is unique to Hepadnaviridae
, and therefore, any drug cross-reactivity would likely be low.
Downstream of the TP domain are the spacer, reverse transcriptase (RT), and RNase H domains (Figure 1
). HBV Pol is similar in sequence and structure to the polymerase found in the human immunodeficiency virus (HIV,) which also contains RT and RNase H domains. Indeed, emtricitabine, lamivudine, and tenofovir are able to inhibit both HBV and HIV [9
]. Much more is known of the HIV Pol, including several high-resolution structures. Due to their similarity, models of HBV Pol borrow this knowledge about the HIV Pol [11
]. Despite similarities, definitive descriptions of the three-dimensional structure of HBV Pol do not yet exist. Additionally, HBV’s TP and spacer domains are unique to the Hepadnaviridae family. Currently, their structures can only be predicted.
TP proteins also exist in other microbes; however, they give clues to the function—not necessarily the structure—of HBV Pol. For example, bacteriophage, members of the Adenoviridae family, and Streptomyces
bacteria are groups of microbes that encode a TP protein for priming DNA synthesis [13
]. Typical among these TP proteins is the use of a tyrosine, serine, or threonine for initiating priming [16
]. Beyond these priming residues, little amino acid homology can be identified among TP proteins (Figure 1
). One commonality among TP proteins is the presence of a disordered priming loop in their protein structure, whose flexibility allows access to the active site of DNA synthesis proteins. However, other than in Hepadnaviridae, all TP proteins exist separately from the catalytically active polymerase protein. The polymerase in Hepadnaviruses synthesizes both DNA strands while still attached to the DNA [18
Determining the three-dimensional structure of the TP domain has thus far proved impossible. Reasons include the difficulty of purifying large amounts or truncated portions of HBV Pol for crystallography. Additionally, the structure is disordered in several places, and the protein may exist in several conformations [19
]. The conformation of HBV Pol varies during the several stages of DNA synthesis and is maintained by both host chaperone proteins and its association with an RNA secondary structure element called epsilon (ε RNA). An initial conformation is provided when the host chaperone proteins Hsc70, Hsp40, Hsp90, and Hop bind to HBV Pol [20
]. Only this chaperone-associated Pol protein is capable of binding ε RNA. The subsequent binding of ε RNA induces another conformational change in HBV Pol before DNA synthesis, allowing the delivery of the Y63 priming residue to the active site in the RT domain [22
]. The conformation changes after priming, as evidenced by protein cleavage assays in DHBV and the finding that continued synthesis of viral DNA does not continue along the 5′ ε RNA but is instead templated by the 3′-end of the pgRNA after template switching [23
]. These facts suggest that the structure of the polymerase may not be amenable to crystallography. Other means of determining structure have been performed, including epitope mapping with the analysis of antibody binding sites [25
]. Technologies such as high-resolution mass spectrometry, nuclear magnetic resonance spectroscopy, and cryogenic electron microscopy may prove useful in determining a structure.
A functional cure for HBV would likely require combinations of drugs that target multiple non-redundant targets, perhaps including the TP domain of HBV Pol. Chronic HBV infections are currently treated with pegylated interferon-α, which increases immune activity, and/or with nucleoside analogs that block the RT domain from replicating viral DNA. Both of these main classes of drugs achieve hepatitis B surface antigen (HBsAg) loss only rarely; interferon is poorly tolerated, and nucleoside analogs are administered for life to achieve a reduction in viral load [2
Several drugs are in clinical trials, and drug development against HBV is an exciting realm of possibility. Techniques for drug development include 3D in silico modeling that attempts to dock libraries of chemicals to viral proteins. Candidates may be chosen for cell-based or cell-free assays [27
]. Cell based assays are more physiologically relevant, have the ability to test toxicity, and usually involve cells permissive to HBV (or transfected HBV DNA) such as primary human hepatocytes, HepG2, Huh7, HepaRG, or others [8
]. Cell-free assays, on the other hand, require purified cellular and/or viral components but provide a higher throughput capacity, such as using purified HBV Pol to measure elongation activity in vitro [32
]. Other specialized techniques such as split luciferase or other two-factor interaction tests may reveal molecular partnerships [33
Functionally essential and chemically unique, the TP domain’s potential as a therapeutic target is high. Therefore, an analysis of current research was performed to map the TP domain of HBV Pol. These studies evaluate the role of specific amino acid residues in four of the primary functions of the polymerase: RNA binding, protein priming, RNA packaging, and DNA synthesis. In addition, methods for evaluating these four main functions are described. The functional mapping of specific regions within HBV Pol’s TP domain is discussed, namely, conservation analysis, secondary structure prediction, and targeted mutational studies. With no solved structure for HBV Pol, this analysis provides a valuable map of the TP domain. However, this mapping may be superseded once a suitable high-resolution three-dimensional structure is solved alongside its molecular interactions.
Although current drugs exist that target HBV Pol, they are not curative. Therefore, knowledge about the structure and function of the virus’ only catalytic protein, HBV Pol, will inform future treatment development. The structure of HBV Pol includes four domains: the TP, spacer, RT, and RNase H domains (Figure 1
). Much information is already available about the structure and function of the RT and RNase H domains of HBV Pol due to its homology with the well-studied HIV. Indeed, all current HBV-specific drugs, except immuno-based drugs like interferon and thymalfasin, target the RT domain. However, the TP domain has not previously been studied to the same degree. The TP domain is known to be essential for HBV Pol function and has no structural homologs in humans, organisms, or other known viruses. Thus, the TP domain is an ideal target for novel therapeutics. A coordinated effort is needed to take candidate drugs from models, to in vitro tests, to cell culture or animal model systems, and on to clinical trials. It is hoped that a hypothesis-driven targeting of the HBV TP domain can begin, utilizing the mapping provided in this review.
The TP domain is essential for most known polymerase functions, which are (in order of occurrence): binding to the ε RNA, packaging of viral pgRNA into the nucleocapsid, priming the initial synthesis of DNA through the unique process of protein priming, and yjr continued DNA synthesis of both the minus and plus strands (Figure 2
). Although the exact structure of the TP domain is still unknown, recent advances and standard assays have been able to map specific functions onto the structure. The TP domain contains seven subdomains, with three conserved loops that control RNA binding (T3 loop), RNA packaging (L2 loop), and protein priming (priming loop). The helical subdomains are less well conserved and less functionally important (Figure 3
c and Figure 4
The findings summarized herein, which describe and map the functions of the TP domain of HBV Pol, will inform therapeutic drug development and basic HBV biology. Additional studies of the TP domain are essential to uncovering the overall functionality of the HBV Pol, its three-dimensional structure, and the ability to target critical regions such as individual TP domain loops.