The life cycle of hepatitis B virus and antiviral targets

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Hepatitis B virus (HBV) is one of the most dangerous human pathogens. Indeed, accord-ing to the WHO, approximately 2 billion people worldwide have been infected and more than 350 million are chronically infected. Chronic HBV infection predisposes to severe liver dis-ease, including liver cirrhosis and hepatocellular carcinoma. Approximately 600,000 people die each year due to the acute or chronic conse-quences of hepatitis B (WHO estimations in 2008) [201].

Our knowledge of the molecular biology of HBV has increased considerably over the past decades, leading to the development of very effective prophylactic vaccines and to the development of therapeutic approaches against HBV. Among these, two are currently approved to treat chronically HBV-infected patients. Pegylated IFN-a is used as an antiviral as well as to enhance the host’s immune defence sys-tem. However, only 30% of pegylated-IFN-a-treated patients achieve a sustained antiviral response [1]. Alternatively, oral administration of nucleoside/nucleotide analogs (NUCs), which specif ically inhibit viral polymerase (VP) activity and thus suppress HBV repli-cation, significantly improves liver histology and the clinical outcomes of the disease after 1 year of treatment [2]. Unfortunately, NUCs act at a late stage of the HBV life cycle and do not prevent the formation and activity of the

HBV transcription template, the so-called cir-cular covalently closed DNA (cccDNA). Since cccDNA has a long half-life, long-term treat-ments with NUCs are necessary to cure HBV-infected cells, but also lead to the inevitable selection of HBV drug-resistant strains [3]. New therapeutic approaches that target other viral proteins besides VP are required to decrease viral drug resistance and improve treatments against HBV.

HBV structure & proteins Hepatitis B virus is a small, enveloped DNA virus that replicates via an RNA intermedi-ate and belongs to the Hepadnaviridae fam-ily. HBV particles, commonly termed Dane particles, are spherical lipid-containing struc-tures with a diameter of approximately 42 nm (Figure 1). The inner shell of the virus consists of an icosahedral nucleocapsid, which is assem-bled from 120 dimers of the core protein. The nucleocapsid is covered with a membrane made up of three forms of the viral envelope protein: large (L), middle (M) and small (S), which are acquired together with the host’s lipids dur-ing budding into the endoplasmic reticulum (ER). The three surface proteins are commonly defined as hepatitis B surface antigens. They are translated from their own start codons but share the same C-terminal amino acids, called the S domain. As a consequence, the

The life cycle of hepatitis B virus and antiviral targets

Julie Lucifora1 & Fabien Zoulim†2,3,4,5

1Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Trogerstrasse, 30, 81675 Munich, Germany 2INSERM, U1052, 151 Cours Albert Thomas, 69003 Lyon, France3Université de Lyon, 69003 Lyon, France4Hepatology Department, Hospices Civils de Lyon, 69004 Lyon, France5Institut Universitaire de France, 103, bd Saint-Michel, 75005 Paris, France†Author for correspondence: [email protected]

Hepatitis B virus (HBV) remains a major public health issue with more than 350 million people chronically infected worldwide. Therapies using IFN-a or nucleos(t)ide analogs, which are currently approved for the treatment of chronic HBV infection, have failed to completely eliminate the virus. By describing the HBV life cycle, this article focuses on potential HBV targets and recent advances in the development of alternative antiviral strategies to fight chronic HBV infection. The targets that are currently under investigation to develop inhibitors are viral entry, circular covalently closed DNA formation and its regulation, nucleocapsid assembly and viral morphogenesis, as well as the innate response of infected cells against HBV infection. Other steps in the viral life cycle are potential targets; however, the development of novel antivirals remains challenging. The development of new HBV inhibitors represents a major advance in the field of antiviral therapy, to prevent antiviral drug resistance and to achieve long-term control of viral replication.

Keywords

n antiviral targets n hepatitis B virus n inhibitory drugs n life cycle

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M protein contains an extra domain, terned the preS2 domain, compared with the S pro-tein, and the L protein contains two extra domains compared with the S protein: the preS2 and preS1 domains. Nucleocapsids con-tain a single copy of the HBV genome con-sisting of a 3.2-kb partially double-stranded relaxed circular (rc) DNA molecule. This rcDNA is covalently linked to the VP at the 5´ end of the complete strand, also called viral (-) strand DNA. Besides the Dane particles, HBV infection also leads to the secretion of subviral particles, which consist of empty viral envelopes with filamentous or spheri-cal shapes (Figure 1). Subviral particles are the most abundant HBV structures released into the blood, and are thought to facilitate virus spread and persistence in the host by adsorbing virus-neutralizing antibodies.

In addition to polymerase and the structural proteins, the HBV genome also encodes for two nonstructural proteins, which, currently, have less-well defined functions. Secreted HBeAg may have immunoregulatory functions [4–7], whereas the X protein (HBx) seems to have multiple functions. HBx interacts with vari-ous cellular partners and modifies several cel-lular processes, including transcription, cell-cycle progression, DNA-damage repair and apoptosis [8,9]. Using in vitro HBV infection

models, we recently demonstrated that HBx is essential to initiating and maintaining HBV transcription [10].

Overview of the HBV life cycle Hepatitis B virus is thought to be internalized into cells through receptor-mediated endo-cytosis [11]. Proteolytic cleavage of the surface protein occurs within the endosomal compart-ment, resulting in a conformational change that exposes some translocation motifs at the surface of the viral particle. The high density of translocation motifs allows endosomal escape of the nucleocapsid into the cytosol [12]. The naked nucleocapsid is then directed towards the nucleus, and the HBV genome is translocated to the nucleus [13]. At this stage, the rcDNA genome is converted into a cccDNA, the template for viral transcription. The 3.5-kb pregenomic RNA (pgRNA) serves as mRNA for the synthesis of polymerase and core proteins. Additionally, it is used as a template for reverse transcription. The 2.4/2.1-kb subgenomic RNAs encode for three viral envelope proteins. The pgRNA is reverse transcribed within the nucleocapsid in the cyto-plasm into new rcDNA. Mature nucleocapsids are then either directed to the secretory pathway for envelopment or are redirected towards the nucleus to establish a cccDNA pool [14]. An over-view of the HBV life cycle is depicted in Figure 2.

Dane particle

rcDNA

Core

Pol

M S L

FilamentSphere

HBeAg

HBsAg

HBV proteins

S Small surface protein

M Middle surface protein

L Large surface protein

Core Capsid protein

HBeAg Secreted e antigen

Pol Polymerase

HBx X protein (non-secreted)

Figure 1. HBV proteins and hepatitis B virus virion structure. (A) List of all HBV proteins. (B) Viral particles present in the serum of HBV-infected individuals. Dane particles consist of full infectious viral particles that contain the viral genome. Spheres and filaments consist of an empty viral envelope. Together, Dane particles, spheres and filaments are recognized as HBsAg. The precore protein is secreted as HBeAg. HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus; rcDNA: Relaxed circular DNA.

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The life cycle of hepatitis B virus & antiviral targets Review

D

F

G

J

Polymerase

pgRNA

Core

HBeAg

HBsAg

Hepatocyte

Surface proteins

Entry

Endosome

Trafficking

rcDNA

cccDNA

Integration into the host DNA

I

Transcription of viral RNA

Trafficking

H Morphogenesisand secretion

DNA synthesis

Nucleocapsid formation and pgRNA packaging

Nucleus

PreC/pgRNAPre-S1Pre-S2

X

AAAAAA

AAAAAA

cccDNA formation

HBx

Translation

I

Figure 2. Overview of the key steps of the hepatitis B virus life cycle. (A) Once internalized in hepatocytes by endocytosis, (B) the naked HBV nucleocapsid is directed towards the nucleus and the HBV genome is translocated into the nucleus. (C) The rcDNA genome is then converted into cccDNA, (D) the template for viral transcription. (E) After HBV-RNA transcription and subsequent HBV-protein synthesis, (F) the pgRNA is encapsidated and (G) reverse transcribed to result in a new rcDNA molecule. (H) Mature nucleocapsids are then either directed to the secretory pathway for envelopment and new virions are released, (I) or they are redirected towards the nucleus to amplify the cccDNA pool. (J) As an optional step, HBV can be integrated into the cellular host genome.cccDNA: Circular covalently closed DNA; HBeAg: Hepatitis B e antigen; HBsAg: Hepatitis B surface antigen; HBx: Hepatitis B virus X protein; pgRNA: Pregenomic RNA; rcDNA: Relaxed circular DNA.



HBV entryHepatitis B virus infection is restricted to hepato cytes, but the early steps of the HBV life cycle, including virus attachment to the cell surface, are poorly understood. However, recent developments from cell culture (HepaRG cells [15], primary tupaia hepatocytes [16]) and animal (severe combined immuno-deficiency–uPA mice, [17]) models, to study HBV infection, have significantly improved our understanding of mechanisms leading to HBV entry into hepatocytes.

The S domain of the surface protein crosses the lipid bilayer several times. The pre-S domain is located in the cytoplasmic side during the initial steps of assembly, and is located both outside and inside the virus after budding of the viral particle [18]. The L protein plays a major role in the HBV infection process. Indeed, the first clues to its involvement came from stud-ies that used synthetic peptides spanning the pre-S1 domain and from antibodies against this domain that strongly inhibited HBV binding to HepG2 cells [19,20]. Later mapping experiments showed that a domain that includes amino acids 2–48 of the L protein mediates attachment of the virus to hepatocytes, whereas the S pro-tein seems to be involved in other steps [21]. Moreover, a myristic acid residue, covalently attached to glycine 2 of the L protein dur-ing translation, appears to determine HBV infectivity [22,23].

In vitro studies conducted either in HepaRG cells or in primary tupaia hepatocytes have shown that peptides, consisting of authen-tically myristoylated amino acids of the N-terminal part of the L protein, specifically inhibit HBV infection [15,21,24]. These peptides offer new perspectives in the development of strategies to block an essential step in the HBV life cycle. The prototype of these peptides (HBVpreS2/2–48myr) inhibits HBV infection, with an IC

50 of approximately 8 nM [24]. Their

ability to prevent HBV infection in vivo has been shown using a severe combined immuno-deficiency–uPA murine model [25]. Indeed, accumulation of peptides in the liver and spe-cific inhibitory potency after subcutaneous delivery at low doses have both been demon-strated [25]. These peptides are now entering the first phases of clinical evaluation for the treatment of chronic hepatitis B.

Immunoadhesins that contain the pre-S1 domain have also been noted for their inhibitory effect on HBV entry into primary human hepa-tocytes [26]. They do not seem to be as efficient

as myristoylated peptides in vitro, but might offer the advantage of having higher stability in vivo [26].

Hepatitis B virus entry into hepatocytes is now thought to be a multistep process. Dane particles are the first to be trapped at the surface of the cell by heparan sulfate proteo-glycans [27], and can then bind to a high-affinity receptor that confers uptake of the virus into the cells [28]. The identification of the high-affinity receptor(s) has remained one of the most puzzling questions in the field of HBV research. Several potential HBV-binding factors (e.g., immunoglobulin A receptor, asialoglyco-protein receptor, apoH and lipoprotein lipase) have been described over the past few years, but none have been convincingly shown to be essen-tial in HBV entry [29–42]. The development of molecules that can compete with HBV for bind-ing to heparan sulfate, or to the yet unknown, high-affinity receptor(s), would lead to very promising antiviral strategies.

HBV capsid transport towards the nucleus & genome release

Hepatitis B virus uses the cellular machinery located within the nucleus for transcription of its RNA. As a consequence, the HBV genome within the capsid has to be transported across the cytoplasm to reach and enter the nucleus.

As in vitro infection models are rather in efficient, most likely because of a defect(s) in the entry process, lipofection assays have been performed to study the behavior of nucleo capsids after their release from the endosomal compart-ment into the cytoplasm. Capsid lipofection into HuH7 cells, consisting of the replacement of the HBV envelope by a lipid shell, has led to highly productive HBV replication and has demon-strated capsid accumulation at the nuclear envelope within 15 min [13]. Further experi-ments using nocodazole – a depolymerizing microtubule drug – together with binding and co-immunoprecipitation assays, have revealed active microtubule-dependent capsid transfer towards the nucleus [13]. Upon phosphorylation within the cytoplasm, capsids undergo struc-tural changes that lead to exposure of increasing numbers of the C-terminal part of the core pro-teins, which contain a nuclear localization sig-nal [43,44]. Exposure of the nuclear localization signal increases the probability of an interaction with the importin a/b proteins and binding to the nuclear-pore complexes [43]. Using permea-bilized cells, it has been further demonstrated that nucleocapsids are imported in an intact



form through the nuclear-pore complexes into the nuclear basket where they interact with nucleoporin 153. Once inside the nuclear-pore complex, mature capsids (containing dsDNA and, thus, less stable than immature capsids containing RNA) disassemble and release HBV genomes within the karyoplasm [44,45].

Viral capsids released from the endosomal pathway and newly synthesized progeny cap-sids have the same structure and, thus, can both be directed to the nucleus via the same mecha-nisms. It was shown that immature capsids (from newly synthesized progeny capsids) are also transported through the nuclear-pore com-plexes, but remain arrested within the nuclear basket and do not show any release of the imma-ture genome [44,46], thus preventing disruption of the viral life cycle. Immature capsids may complete genome maturation while they are arrested in the nuclear basket and then lead to HBV genome release within the karyoplasm to increase the number of cccDNA molecules.

So far, no inhibitor of HBV nucleocapsid transport toward the nucleus has been described, and efforts should be made in this direction as it could prevent the establishment of HBV rep-lication in de novo infected cells, as well as the amplification of the cccDNA pool in cells that are already infected with HBV.

cccDNA formation As shown in Figure 3, conversion of rcDNA into cccDNA requires several steps: removal of the VP linked to the 5́ end of the (-) strand DNA as well as one of the redundant sequences (either in the 3´ or in 5́ end); removal of the RNA primer linked to the 5´ end of the (+) strand; comple-tion of the viral (+) strand DNA; and ligations of DNA extremities for both (+) and (-) strands.

Using transient transfection of the enveloped deficient HBV genome or stable cell lines sup-porting replication of the enveloped deficient HBV genome in an inducible manner, it was demonstrated that surface proteins negatively regulate HBV cccDNA formation before the removal of the VP linked to the 5´ end of the rcDNA (-) strand [47,48]. Furthermore, this rcDNA deproteinization appears to occur within the capsid before translocation to the nucleus, by cleaving the phosphodiester bound between the tyrosine of the polymerase and the 5´ phosphoryl group of the (-) strand DNA [48]. It was also observed that deproteinized- rcDNA contains only full-length (+) strand DNA, and completion of this strand has to occur earlier [47,48]. These results and the recent finding that in vitro deproteinization is inhibited by viral DNA polymerase inhibi-tors [49] suggest that completion of (+) strand

-

+3´

3´

3´3´

5´

5´

5´

5´

(+) strand

(-) strand(-) strand

CGG

Relaxed circular DNA cccDNA

Removal of the polymerase and one of the two redundant sequences

Removal of the RNA primer

Completion of the viral (+) DNA strand

Ligations of DNA extremities

Association with cellular proteins

HistonePolymerase Redundant sequence RNA primer

Figure 3. Representation of relaxed circular DNA conversion to circular covalently closed DNA. (A) Removal of viral polymerase linked to the 5’ end of the (-) DNA strand and of one of the redundant sequences, (B) removal of the RNA primer linked to the 5’ end of the (+) strand, (C) completion of the viral (+) DNA strand and (D) ligations of DNA extremities for both (+) and (-) strands. (E) Once formed, cccDNA associates with proteins, such as histones, resulting in being supercoiled and forming the so-called cccDNA minichromosome. cccDNA: Circular covalently closed DNA.



DNA and removal of polymerase linked to the (-) strand DNA are probably catalyzed by the VP itself and/or by cellular proteins packaged into the nucleocapsid. Two different groups have identified cellular tyrosyl phosphodieste-rase-2 as a molecule able to specifically cleave VP from rcDNA: its exact role in cccDNA formation is currently under investigation [50,51]. Moreover, consistent with the aforemen-tioned mechanisms for DNA entry into the nucleus, it has been shown that deproteiniza-tion of rcDNA is tightly linked with nucleo-capsid disassembly and HBV DNA transloca-tion into the karyoplasm [49]. A recent study that precisely analyzed rcDNA and cccDNA sequences showed that, once in the nucleus, the redundant sequence at the 5´ end of the (-) strand is removed and that both DNA strands then undergo DNA-repair reactions, which are probably carried out by cellular DNA-repair machinery [52]. The hypothesis that the initial cccDNA formation may at least partially depend on cellular enzymes is consistent with data showing that the initial formation of cccDNA from incoming virions cannot be prevented using potent NUCs that inhibit VP or (+) strand DNA synthesis [53–55]. However, a recent study reported that genera-tion of cccDNA via intracellular recycling is also probably regulated in a virus-specific man-ner. Indeed, it was shown that, whereas HBV and duck HBV (DHBV) cccDNA formation were not cell-type dependent, DHBV cccDNA conversion was much more efficient than that of HBV [56].

The recycling of the neo-formed nucleo-capsids, together with the long half-life of the cccDNA molecule, ensures HBV persistence in cells. Thus, the discovery of compounds that specifically inhibit cccDNA formation remains one of the major challenges to cur-ing chronic HBV infections. Recent data on cccDNA stability in liver regeneration suggest that therapeutic strategies that aim to induce a certain amount of cell injury and encourage compensatory hepatocyte regeneration may be necessary to significantly reduce cccDNA loads in HBV chronically infected patients and for the possible long-term immunological control of HBV infection [57]. Furthermore, the observation that efficient DHBV cccDNA for-mation is taking place in human hepatocytes [56] could facilitate experimental identification of the human cell ular factors involved in the cccDNA process, which may then suggest a potential target for drug development.

Control of HBV RNA transcription: HBV transcripts

Once formed, the cccDNA serves as a tran-scriptional template for viral RNA synthesis by cellular RNA polymerase II (the enzyme also responsible for cellular mRNA synthesis).

Histones and nonhistone proteins are bound to cccDNA, resulting in a chromatin-like structure named viral minichromosome [58,59]. Epigenetic modification of the cccDNA mol-ecule, which leads to the control of HBV RNA transcription, has been recently reported. For instance, it was shown that HBV transcrip-tion is regulated by the acetylation status of the cccDNA-bound H3/H4 histones [60]. Recent studies have highlighted HBx as a key regulator of transcription from cccDNA, either by favor-ing histone acetylation or by preventing histone deacetylation [10,61]. As HBx plays a central role in HBV infection [10], it would be a very interest-ing target for new therapies that prevent HBV viral replication. By contrast, methylation of the cccDNA CpG island was shown to inhibit HBV transcription in vitro [62], and the ratios of rcDNA to cccDNA molecules in HBeAg-positive individuals have revealed that cccDNA methylation correlates with impaired virion pro-ductivity [63]. Theoretically, finding drugs that could specifically increase cccDNA methylation would also help to inhibit HBV transcription.

Efficient transcription of HBV genes also requires a number of ubiquitous and hepato-cyte-specific transcription factors (for a review see [64]). Several studies have described com-pounds that can inhibit HBV transcription by targeting transcription factors. For instance, it has been shown that cytokine IL-6 controls expression of HNF1a and HNF4a (the two major transcription factors that allow HBV transcription [65]) by activating JNK and ERK kinases [66]. The helioxanthin analog 8–1 and the plant Phyllanthus amarus can also sup-press HBV RNA transcription by, respectively, decreasing levels of HNF4a and HNF3b [67] and repressing the HBV enhancer I [68]. Zinc-finger proteins have also shown activity against HBV, likely owing to competition for DNA binding sites with transcription factors, thus interfering with read-through transcription by RNA polymerase across the zinc-finger protein-binding region [69].

Other compounds have been described in the past few months that can inhibit HBV tran-scription such as Curcuma longa extract [70], lutein [71], cinobufacini [72] and ethanol extracts of Hypericum perforatum [73]. Although these



data are preliminary and the exact mechanisms of their actions remain to be determined, these compounds could represent novel candidates to be further evaluated as therapies against HBV.

Several other antiviral approaches, such as RNA interference, have been evaluated to target the viral transcripts themselves [74,75]. However, some challenges, including poor siRNA stabil-ity, inefficient cellular uptake, widespread bio-distribution and nonspecific effects, need to be overcome. Different strategies to improve the therapeutic efficacy of siRNAs, while avoiding their off-target effects, are under investigation (for a review, see [76,77] and more recently [78–82]). Antisense oligo nucleotides [83,84] and ribozymes [85–89] have also been described for their efficient targeting of HBV RNA. However, their use in therapeutic strategies against HBV also requires that their delivery to infected cells is improved. Finally, IFN-a, via MxA activity, was shown to inhibit the nucleocytoplasmic export of viral mRNA [90].

Capsid formation & maturationOnce synthesized, pgRNA is encapsidated together with VP. The HBV capsid spontane-ously self-assembles from many copies of core dimers present in the cytoplasm. It has been shown that the formation of a trimeric nucleus and the subsequent elongation reactions occur by adding one dimeric subunit at a time until it is complete [91]. Three components are involved in specific packaging of pgRNA into the capsid: VP, the nucleic acid-binding domain of the core protein and the e stem–loop in the 5´ region of pgRNA [92–95]. A recent study has reported that core protein dimers can bind and encapsidate both pgRNA and heterologous RNA molecules with a high level of cooperation, irrespective of the phosphorylation status [96]. This strongly suggests that VP is probably the factor required for specific packaging of pgRNA.

Phenylpropenamide derivatives [97–99], includ-ing compounds named AT-61 and AT-130, and oxymatrine [100], a type of alkaloid extracted from the herb Sophora alopecuroides, have been shown to inhibit pgRNA packaging. A recent study suggested that phenylpropen amides are, in fact, accelerators of HBV capsid assem-bly, and their actions result in the formation of empty capsids [101]. These very interesting results illustrate the importance of the kinetic pathway in successful virus assembly.

Heteroaryldihydropyrimidines, including compounds named Bay 41–4109, Bay 38–7690 and Bay 39–5493, were discovered to prevent

the proper formation of viral core particles and to increase degradation of the core protein [102–105]. It was also shown that peptide aptam-ers, which bind to the HBV core protein, inhibit capsid formation by specific sequestration of bound proteins into aggresomes [106] and bis-ANS, a small molecule that acts as a molecular ‘wedge’ and interferes with normal capsid–protein geometry and capsid formation [107]. Stability of the capsid may also be disrupted by cytokines, such as IFN-a [108] and TNF-a [109].

Maturation of the nucleocapsid consists of synthesis of the (-) DNA strand by reverse-transcriptase activity of VP, followed by pgRNA degradation by RNase H activity of VP and synthesis of the (+) DNA strand by the DNA polymerase activity of VP. In viral DNA synthe-sis, the corresponding inhibitors, such as NUCs, and our understanding of the mechanisms of resistance to NUCs, have been extensively stud-ied over the past few years (for review, see [110]). Of note, different NUCs, such as lamivudine, adefovir, entecavir, telbivudine and tenofovir, have been approved to treat chronic HBV infec-tion, and continuing efforts are being made to maximize their therapeutic benefits and to minimize their resistance rates. For instance, after 5 years of treatment, 80–90% of patients receiving lamivudine (which was the first NUC available on the market) showed the emergence of drug-resistant mutants, whereas no resistance has been observed with tenofovir, so far [110].

All stages of viral DNA synthesis can be found within the cells, but only mature cap-sids, containing rcDNA, are secreted [111,112]. This suggests that the RNA-containing capsids cannot be incorporated into virions and that DNA synthesis is associated with morphological changes. Structural differences between capsids containing RNA or DNA have, indeed, been observed [113], as well as differences in the phos-phorylation state of the arginine-rich domain of the core protein [114]. It has also been shown that capsids from various sources encapsidated active PKC and inhibition of PKC phospho-rylation does not affect genome maturation, but results in capsid accumulation and inhibits virion release [115].

Morphogenesis & virus releaseHepatitis B virus budding has been shown to be strictly dependent on the L protein [116], and when the ratio between L proteins and nucleo-capsids is not optimal, the latter are preferentially recycled to the nucleus to amplify the cccDNA molecule pool [117]. Moreover, the interaction of



L protein with capsids is critical for HBV assem-bly as it can be disrupted by peptides interact-ing with the core [118]. Based on data obtained recently in different studies, it has been proposed that HBV virions can bud into late endosomes or multivesicular bodies, and exit the cell via the exosome pathway (for a review, see [119]).

Strategies that interfere with the correct fold-ing of the surface protein decrease virus secre-tion and its resulting infectivity. For example, a-glucosidase inhibitors are potent inhibitors of HBV-particle secretion [120,121] and the use of N-butyldeoxynojirimycin has been shown to induce secretion of HBV particles that con-tain an envelope with an altered composition of disulfide-linked oligomers and no detectable M protein. As a consequence, HBV infectiv-ity is reduced by 80% [122]. As an alternative strategy to targeting the surface proteins, single-domain antibodies that recognize the S protein were shown to reduce HBsAg secretion when expressed and retained in the ER as intra bodies. Moreover, in a hydrodynamics-based HBV mouse model, these intrabodies caused a ten- to >100-fold reduction in the concentration of HBV virions in plasma [123]. It was also demon-strated that treatments of HBV replicating cells with two human monoclonal anti-HBs antibod-ies (e.g., HepeX-B™; XTL Biopharmaceuticals, Israel) resulted in cellular uptake of the anti-bodies and intracellular accumulation of HBV particles, and prolonged blocking of their secretion [124].

The presence of cholesterol within the viral envelope is indispensable for HBV entry into hepatocytes. Indeed, infectivity of HBV pro-duced from cell culture in the presence of inhibitors of cholesterol synthesis is severely impaired, whereas cholesterol extraction from cellular membranes showed no effect on HBV infection, excluding a role of lipid rafts [125]. Cholesterol biosynthesis within the infected cell is thus a promising target to interfere with HBV morphogenesis and to lower infectivity of neo-formed virions. Confirming this hypothesis, it was recently shown that polyunsaturated ER liposomes, which can lower cholesterol, led to an approximately 40% decrease in HBV secre-tion and to an approximately 70% decrease in the resulting infectivity [126].

Other different compounds can inhibit HBV secretion, such as HBF-0259 (an aromatically substituted tetrahydro-tetrazolo-[1,5-a]-pyri-midine) [127] or the root extract of Boehmeria nivea [128], although their exact mechanisms of action remain unclear.

Virus–host-cell interactionsAlthough it is not essential for the HBV life cycle, the viral genome may integrate into the host genome. This is probably a result of inte-gration of viral double-stranded linear DNA by cellular enzymes such as topoisomerase I [129]. As mentioned earlier, chronic HBV infection is a major risk factor for the development of hepatocellular carcinoma, and HBV genome insertion into the host genome may contrib-ute to mechanisms leading to tumorigenesis. Integration of HBV DNA into the genome of hepatocytes was found in 85–90% of hepato-cellular carcinoma related to HBV infection, and no specific genes have been identified in humans that are preferentially targeted [130]. The insertion is thought to induce general genomic alterations, which may result in the loss of tumor-suppressor gene functions and/or activation of tumor-promoting genes. Blocking HBV DNA replication may also interfere with viral genome integration, as the generation of double-stranded linear DNA is a result of defects caused during replication. In this regard, it was shown that lamivudine treatment was associated with a decrease in hepatocellu-lar carcinoma development [131]. Early antiviral intervention is thus needed not only for chroni-cally infected patients to decrease HBV spread, but also to reduce insertional mutagenesis that can lead to HBV pathogenesis.

Long-term expression of viral proteins has also been suggested to play an important role in hepatocarcinogenesis. For example, it was shown that a truncated form of the L protein or of the M protein increases hepatocyte prolifera-tion by upregulating expression of cyclin A or activating the c-Raf-1/Erk2 signaling pathway, respectively [132]. Moreover, accumulation of surface proteins in the ER induces ER stress, which leads to oxidative stress and DNA dam-age [133], thus predisposing cells to transforma-tion. In addition to its crucial role in the control of HBV transcription, HBx has been shown to interact with various cellular partners and to modify diverse cellular processes, including transcription, cell-cycle progression, DNA-damage repair, and apoptosis (for a review, see [134]). HBx and the surface proteins are, thus, very interesting targets for the develop-ment of new therapies against chronic hepati-tis B infection by preventing viral replication. Indeed, by interfering with their functions, novel antiviral strategies could be designed to prevent viral replication as well long-term con-sequences of HBV infection.



Another interesting aspect of the interaction between the virus and the host cell is the capac-ity of the virus to induce an innate response in the hepatocyte and to then counteract this effect by developing a persistent infection. For many years, HBV was viewed as a stealth virus that did not induce an innate response when it spread throughout the liver [135,136]. However, recent observations have provided evidence that suggests that HBV can be sensed by liver cells and can elicit an antiviral response [66,137]. To reconcile these data, one can suggest that HBV may induce a transient innate response but may also rapidly inhibit it [138]. Characterization of cellular and viral factors involved in HBV rec-ognition, as well as in mechanisms employed to counteract the innate response, would pro-vide new insights into the design of therapies aimed at stimulating the antiviral response of infected cells.

Other interactions of the virus with the cellular machinery are also required in the viral life cycle and may represent potential new targets for drug discovery. Of note, it has been shown that chaperones are required for hepadna virus reverse-transcriptase bind-ing to e on pre genomic RNA and to activate viral-enzyme activity (for review see [139]). The specific inhibition of this interaction may rep-resent a novel target to prevent pgRNA encap-sidation and VP activity. The virus can also use the cellular machinery of the later steps during viral replication, as detailed earlier. It has also been shown that HBV exploits the multive-sicular machinery, with the aid of g2-adaptin, to assemble an egress [140].

Of note, full understanding of the inter-actions between HBV and the immune system is crucial for the development of new anti viral strategies. Indeed, patients who resolve an acute hepatitis B infection display strong poly-clonal and multispecific helper and cytotoxic T-cell responses against HBV core, polymerase and envelope proteins [141]. As these cellular responses are weak and often dysfunctional in patients with chronic HBV infection [141], the development of therapies aimed at boosting the immune system, such as therapeutic vac-cination [142] or adoptive T-cell transfer [143], is also very important.

ConclusionMajor advances in our understanding of HBV molecular biology have been made over the past few years. As a consequence, increasing num-bers of inhibitors that are involved in the HBV

life cycle are being investigated in experimen-tal models (Table 1). After being evaluated in relevant animal models and in clinical trials, these novel inhibitory molecules may provide new options to treat chronic HBV infection. In the meantime, efforts remain to be made to develop new inhibitory molecules that par-ticularly target the steps of the HBV life cycle without known inhibitors.

As described in this article, each step of the HBV life cycle has the potential to be developed into a therapeutic approach. However, inter-vention at specific steps may provide relative advantages. For instance, the development of molecules that inhibit cccDNA-molecule for-mation and stability is one of the most chal-lenging areas in the development of strategies against HBV. Such a therapeutic approach would, indeed, lead to an abortive HBV life cycle, could abolish HBV persistence and pre-vent the HBV rebound observed after the arrest of treatment with NUCs. The development of efficient HBV-attachment and -entry inhibi-tors is also a very attractive antiviral strategy from a therapeutic viewpoint, as it is the first opportunity to curtail the virus’ life cycle. This may lead to novel therapies that potentially pre-vent vertical transmission and inhibit reinfec-tion after liver transplant, and are extremely valuable for postexposure prophylaxis. By con-trast, although targeting the latter steps of the HBV life cycle, such as transcription, capsid formation and maturation, as well as assem-bly and release, has been proven to efficiently fight HBV infection, these approaches could be considered less attractive from a theoretical point of view as they do not cure infected cells of all HBV components. However, targeting these steps is still very valuable for the treat-ment of HBV chronic infection as it allows us to keep the viral load at a low level, avoids the spread of the virus and reduces liver damage. However, some issues remain to be addressed, such as the fate of pgRNA-containing nucleo-capsids, which may accumulate in the presence of NUC treatment (owing to the blockade of viral DNA synthesis).

It is important to keep in mind that antiviral molecules can be divided into two classes: those that directly target the viral components and those that target the host cell. The first class is likely to result in inhibitors with a high spe-cificity and potency, and with low toxicity, but potentially with a high risk of resistance devel-opment. By comparison, the latter approach may yield broad-spectrum antivirals that target



more than one virus and cause minimal or no resistance development, but have compa-rably lower potency and higher toxicity [144]. The optimal antiviral strategy would, thus, combine drugs with different mechanisms of action targeting both HBV and the cellular components necessary for a productive HBV life cycle. Such combinations could reduce the risk of selection of HBV drug-resistant strains. Strategies that combine specific molecules that target HBV and the molecules that induce an innate immune response have been recently described [145] [Ebert G et al., Unpublished Data] and should also be evaluated in clinical trials in order to assess their restoration and dura-tion in the specific immune response against HBV. Combined strategies may then prove use-ful to eradicate the viral genome or to induce long-term control of viral replication, thereby preventing the complications caused by the HBV disease.

Future perspectiveMajor challenges remain regarding our under-standing of the HBV life cycle. Answers to these key questions may not only improve our basic knowledge of the mechanism of viral

replication and pathogenesis, but will also help us develop innovative therapies beyond viral suppression that are induced by NUCs.

Deciphering viral entry should be achieved during the next decade, but may still be chal-lenging because of the difficulty in growing HBV in tissue culture when compared with other viruses such as HIV or HCV. Entry inhibitors, which represent a new treatment target, are currently being evaluated and may represent a new class of antivirals. Identifying the detailed molecular steps of cccDNA for-mation is also underway with major recent advances, which should pave the way for com-plete understanding of this process, as well as the epigenetic control of this DNA template for viral gene expression. This would represent a major breakthrough in studying the molecular biology of HBV as well as for drug develop-ment. Specific targeting of the formation and regulation of this viral genome species respon-sible for viral persistence may then become feasible. Obtaining a crystal structure of an enzymatically active VP represents one of the major challenges over the next decade. This could enable the development of novel inhibi-tors that could complement the classic NUCs.

Table 1. Hepatitis B virus potential targets and their main inhibitors.

Steps of the HBV life cycle

Targets Inhibitors Ref.

Entry L protein Peptides consisting of the authentically myristoylated amino acids of the N-terminal part of the L protein

[15,21,24,25]

Nucleocapsid trafficking and DNA entry into the nucleus

Not determined None

cccDNA formation Not determined NoneControl of HBV transcription transcripts

HBx NoneTranscription factors IL-6, helioxanthin analog 8–1, the plant Phyllanthus amarus

and zinc finger proteins [66–69]

Unknown Curcuma longa Linn extract, lutein and ethanol extracts of Hypericum perforatum L

[70–73]

siRNA siRNA, antisense oligonucleotides, ribozyme and IFN-a [74,75,83–90]

Capsid formation and maturation

pgRNA packaging Phenylpropenamide derivates and oxymatrine [97–100]

Capsid assembly and stability Heteroaryldihydropyrimidines, peptide aptamers that bind to the core protein, bis-ANS, TNF-a and IFN-a

[102–109]

DNA synthesis Nucleos(t)ide analogs [110]

Virus assembly and release Interaction between capsid and the L protein

Peptides interacting with core proteins [118]

Surface proteins Antibodies against surface proteins [123,124]

Folding of surface proteins a-glucosidase inhibitors [120–122]

Cholesterol insertion within the viral envelope

Polyunsaturated ER liposomes [126]

Virus secretion HBF-0259, the root extract of Boehmeria nivea [127,128]

Integration of the DNA into the host genome

Not determined None

cccDNA: Circular covalently closed DNA; ER: Endoplasmic reticulum; HBV: Hepatitis B virus; HBx: Hepatitis B X protein nonsecreted; pgRNA: Pregenomic RNA.



The role of the X protein has been debated for 20 years. The availability of more effi-cient and relevant cell-culture models should help us to gain a precise understanding of its function in the replication cycle and the pathogenesis of virus-induced hepatocellular carcinoma. Greater understanding regarding the role of the innate and adaptive immune responses against HBV in controlling or eradi-cating infection is critical to developing new treatment- combination strategies based on direct antivirals and immune modulators.

Hepatitis B virus research in the next decade should result in major advances in our knowl-edge regarding the viral life cycle and the interactions between the virus and host cells. This new information will pave the way to the identification of new targets, to discoveries of

new drugs and to the development of effica-cious drug-combination strategies. These research efforts could help us reach the goal of finite-duration treatments, prevent resistance and prevent disease complications, including hepatocellular carcinoma.

Executive summary

Hepatitis B virus entry into hepatocytesnThe hepatitis B virus (HBV) L protein is the main viral factor involved in the process. Peptides consisting of authentically myristoylated

amino acids, with an N-terminal part, are potent inhibitors.nHBV particles are first trapped at the surface of the cell by heparan sulfate proteoglycans and then bind to a high-affinity receptor that

confers uptake of the virus into cells. No inhibitors have been described, so far, that can block the cellular factors involved.

Nucleocapsid trafficking & DNA entry into the nucleusnHBV nucleocapsids are transported across the cytoplasm toward the nucleus by an active microtubule-dependent mechanism. They are

then imported into the nuclear basket where they are disassembled and release HBV genomes within the karyoplasm. nNo inhibitors have been described, so far, to block these mechanisms.

Circular covalently closed DNA formationnCircular covalently closed DNA formation may depend on both viral and cellular enzymes.nNo inhibitors have been described, so far, to block this step.

Control of HBV transcription transcriptsnHBx controls epigenetic modification of the circular covalently closed DNA, which allows HBV-RNA transcription. No inhibitors of HBx

have been described so far.nEfficient transcription of HBV genes also requires a number of ubiquitous and hepatocyte-specific transcription factors. Different

molecules that inhibit these transcription factors have been described.nSeveral antiviral approaches, such as RNA interference, have been successfully evaluated for targeting viral transcripts.

Capsid formation & maturationnHBV capsids spontaneously self-assemble from many copies of core dimers present in the cytoplasm.nViral polymerase, the nucleic-acid-binding domain of the core protein, and the e stem–loop in the 5’ region of pregenomic RNA

(pgRNA) are involved in specific packaging of pgRNA into the capsid.nSeveral molecules inhibit capsid formation: pgRNA encapsidation, reverse transcription and DNA synthesis have been described.

Virus assembly & releasenInteraction of L protein with capsids is critical for HBV assembly. nStrategies that interfere with the correct folding of the surface protein decrease virus secretion and its resulting infectivity.nInhibition of cholesterol biosynthesis within the infected cell interferes with HBV morphogenesis, resulting in lower infectivity of

neo-formed virions.

Virus–host-cell interactionsnVirus genome integration into the host genome and the long-term expression of viral proteins contributes to the development of

hepatocellular carcinoma.nThese observations favor an early antiviral intervention for patients who are chronically infected with HBV.

ConclusionnThe optimal antiviral strategy would combine drugs with different mechanisms of action that inhibit the different steps within the

HBV life cycle.

Financial & competing interests disclosureJ Lucifora holds a stipend from the European Association for the Study of Liver disease (EASL): ‘Sheila Sherlock EASL Post-Doc Fellowship’. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the sub-ject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.



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The life cycle of hepatitis B virus and antiviral targets

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