The human hepatitis B virus (HBV) is a small-enveloped DNA virus causing acute and chronic hepatitis. Despite the availability of a safe and effective vaccine, HBV infection still represents a major global health burden, with about 240 million people chronically infected worldwide ( Cornberg 2019 ). Many epidemiological and molecular studies have shown that chronic HBV infection represents the main risk factor for hepatocellular carcinoma development ( Pollicino 2011, Dandri 2016, Levrero 2016, WHO 2018). The rate for chronicity is approximately 5% in adult infections, but it reaches 90% in neonatal infections. HBV transmission occurs vertically and horizontally via exchange of body fluids. In serum, up to 1012 HBV genome equivalents per mL serum can be found. Although HBV does not induce direct cytopathic effects under normal infection conditions (Wieland 2004, Thimme 2003), liver damage and chronic inflammation are believed to be induced mainly by the ongoing attempts of the immune system to counteract the infection (McMahon 2009, Dandri 2012).
HBV is the prototype member of the Hepadnaviridae family, which are the smallest known DNA-containing, enveloped animal viruses. Characteristic of HBV is its high tissue- and species-specificity, as well as a unique genomic organization with asymmetric mechanism of replication (Nassal 2015). Since all hepadnaviruses use a reverse transcriptase to replicate their genome, they are considered distantly related to retroviruses. Despite the tremendous progresses made in understanding the molecular virology of HBV, some key steps of the infection and interrelations between HBV and host components are still poorly understood. Nevertheless, the discovery of the cellular receptor (Yan 2012) and the establishment of innovative infection models and molecular techniques have opened up new possibilities to investigate specific steps of the lifecycle as well as the organisation and activity of the covalently closed circular DNA (cccDNA), the viral minichromosome that serves as the template of HBV transcription in the nucleus of the infected hepatocytes, enabling maintenance of chronic HBV infection (Allweiss 2017 ).
The Hepadnaviridae form their own taxonomic group as their biological characteristics are not observed in any other viral family. Based on host and phylogenetic differences, the family of Hepadnaviridae contains two genera: the orthohepadnaviruses infecting mammals, and the avihepadnaviruses that infect birds. To date, orthohepadnaviruses have been found in human (HBV), woodchuck (WHV) (Korba 1989), ground squirrel (GSHV) and woolly monkey (WMHBV) (Lanford 1998). Avihepadnaviruses include duck HBV (DHBV) (Mason 1980), heron HBV (HHBV) (Sprengel 1988), snow goose HBV (SGHBV), stork HBV (STHBV) (Pult 2001) and crane HBV (CHBV) (Roggendorf 2007, Funk 2007, Schaefer 2007). Moreover, new hepadnavirus species antigenically related to human HBV and capable of infecting human hepatocytes were also identified in bats (Drexel 2013). The relatedness of these viruses to HBV suggests that bats might constitute ancestral sources of primate hepadnaviruses.
Due to the lack of proofreading activity of the viral polymerase, nucleotide mutations occurs during viral replication. This has led to the emergence of eight HBV genotypes, A-H, which differ in more than 8% of the genome, as well as different subgenotypes, which differ by at least 4% (Fung and Lok 2004, Guirgis 2010). The HBV genotypes have different geographic distribution (Liaw 2010), with predominance of genotype A in northwestern Europe, North and South America, genotype B and C in Asia and genotype D in eastern Europe and in the Mediterranean basin. The less diffuse remaining genotypes are mostly found in West and South Africa (genotype E), in Central and South America (genotypes F and H), while genotype G has been detected in France and in the US (Pujol 2009).
Different types of viral particles can be visualised in the infectious serum by electron microscopy: the infectious virions and the subviral particles. The infectious virus particles are the so-called Dane particles (Dane 1970), have a spherical, double-shelled structure of 42–44 nm containing a single copy of the viral DNA genome, covalently linked to the terminal protein of the virus. A hallmark of HBV infection is the presence of two additional types of non-infectious subviral particles, the spheres and the filaments, which are composed of hepatitis B surface proteins and host-derived lipids, but do not contain the capsid and the HBV genome (Glebe 2007). The spherical structures measure around 22 nm in diameter, while the filaments are of similar width, but of variable lengths (Figure 1).
The viral membrane contains three viral surface proteins and is acquired by the virus during budding into the endoplasmic reticulum (ER), whereas HBV egress appears to occur via multivesicular bodies (MVBs) (Hoffmann 2013). The surface proteins are named L (large or preS1), M (middle or preS2) and S (small). The surface proteins are produced in quantities largely exceeding the amount needed for the assembly of HBV virions and because of their self-assembly abilities, they are secreted abundantly as empty subviral particles (SVPs). As with nearly all enveloped viruses, HBV particles and SVPs also contain proteins of host origin (Glebe 2007, Urban 2010).
The HBV genome consists of a partially double-stranded relaxed circular DNA of approximately 3200 nucleotides in length, varying slightly from genotype to genotype, that in concert with the core protein (HBcAg) forms the nucleocapsids (Nassal 2015). Within the Dane particle the negative strand of the viral DNA is present in full-length, carrying the complete genetic information. In contrast, the positive strand spans only approximately two-thirds of the genome in length, whilst its 3’ end is variable in size (Summers 1988, Lutwick 1977). The viral polymerase is covalently bound to the negative strand by a phosphotyrosine bond. At the 5’ end of the positive strand a short RNA oligomer originating from the pre-genomic (pg) RNA residually remains bound covalently after the viral DNA synthesis. The negative strand also contains a small redundancy of 8–9 nucleotides in length on both the 5’ end and the 3’ end, named the R region. These redundant structures are essential for viral replication (Seeger 1986, Nassal 2015).
The HBV genome displays four major open reading frames (ORFs) that are organised in a unique and highly condensed way (Block 2007). As shown in Figure 2, all ORFs are in an identical orientation, partially overlap and are encoded by the negative strand. On the genome, 6 start codons, four promoters and two transcription-enhancing elements have been identified. The four major ORFs are: I) the preS/S, encoding the three viral surface proteins; II) the precore/core, encoding both the core protein, essential for the formation of the nucleocapsid, and the non-structural pre-core protein, also known as the secreted e-antigen (HBeAg); III) the pol ORF of the viral polymerase, which possesses reverse transcriptase, DNA polymerase and RNase H activities, and the terminal protein; and IV) the X ORF, coding for the small regulatory X protein, which is essential to establish productive viral infection (Zoulim 1994, Lucifora 2011). Characteristic of the 4 major HBV ORFs is that they utilise a single common polyadenylation signal motif (Nassal 2015). In addition, splicing of HBV RNA has been observed both in experimental in vitro systems and in liver of chronic HBV patients. Although the biological relevance of this alternative splicing regulation remains elusive, recent studies indicated that the expression of HBV splicing and splicing generated proteins (HBSP) may contribute to hinder the recruitment of innate immune cells through downregulation of chemokine expression in hepatocytes (Duriez 2017). Moreover, splicing efficiency appears to be cell-type dependent, thus hinting at a possible contribution of HBSP as restriction factors of HBV productive infection (Ito 2019).
The three surface proteins (L, M, and S) are encoded from one open reading frame (PreS/S) which contains three start codons (one for the large, one for the middle and one for the small protein) but promotes the transcription of 2 mRNAs of 2.4 and 2.1 Kb, named preS and S RNAs (Urban 2014). Notably, the preS/S ORF entirely overlaps with the polymerase open reading frame (Nassal 2015). The three HBV envelope proteins share the C-terminal domain of the S-protein, while the M- and L-protein display progressive N-terminal extensions of 55 and, genotype-dependent, 107 or 118 amino acids (preS2 and preS1). The small envelope protein contains the hepatitis B surface antigen (HBsAg). In virions the stoichiometric ratio of L, M and S is about 1:1:4, while the more abundantly secreted non-infectious subviral particles (SVPs) contain only traces of L-protein (Urban 2014). The envelope proteins are co-translationally inserted into the ER membrane, where they aggregate, bud into the ER lumen, and are secreted by the cell, either as 22 nm subviral envelope particles (SVPs) or as 42 nm infectious virions (Dane particles), after having enveloped the DNA-containing nucleocapsids. The surface proteins of mammalian Hepadnaviridae have been shown to be N- and O-glycosylated (Schmitt 2004). These glycosylations have been shown to be responsible for proper secretion of progeny viral particles. During synthesis, the preS1 domain of L is myristoylated and translocated through the ER. This modification and the integrity of the first 77 amino acids of preS1 have been shown to be essential for infectivity (Glebe 2005, Schulze 2010). In particular the region located within the amino acids 21-47 of the large surface protein contains the binding site of the cellular receptor, the sodium-taurocholate co-transporting polypeptide (NTCP). Both spherical and filamentous SVPs are secreted into the blood of infected individuals in a large excess relative to the infectious particles. The biological function of the excess of SVPs in patients is not clear. It was suggested that SVPs might absorb the neutralising antibodies produced by the host and hence increase the ability of the infectious particles to reach the hepatocytes. Of note, most of the anti-HBs antibodies that are developed by vaccination recognize a region located within the first loop (amino acid 124-137) and the second loop (amino acids 139-147) of HBsAg. Different lines of evidence indicate that the the high amounts of SVPs and circulating viral antigens contribute to create a state of immune tolerance on both innate and adaptive immunity against HBV (Dandri 2012, Dembek 2018).
In the cytoplasm, the core protein dimerises and self-assembles to form an icosahedral nucleocapsid. The full-length core protein is 183 amino acids in length and consists of an assembly domain and a nucleic acid-binding domain, which plays an active role in binding and packaging of the pregenomic RNA together with the viral polymerase, and thus enables the RT polymerase/RNA complex to initiate reverse transcription within the newly forming nucleocapsids ( Kann 1999, Daub 2002, Nassal 2015). The core protein can be phosphorylated by several kinases. This step along with the presence of the viral polymerase is important for the specific packaging of the pgRNA (Kann 1999, Porterfield 2010).
The viral polymerase is the single enzyme encoded by the HBV genome and is an RNA-dependent DNA polymerase with RNase H activity. The HBV polymerase consists of three functional domains and a so-called spacer region; the terminal protein (TP) is located at its N-terminal domain, and serves as a primer for reverse transcription of the pgRNA into a negative-strand DNA (Zoulim 1994). The spacer domain separates the terminal protein from the polymerase domains (Nassal 2015).
Despite the occurrence of nucleotide mutations due to the lack of proofreading capacity of the HBV polymerase, the peculiar genomic organisation of HBV, where most of the genes overlap, imposes stronger constraints on the amino acid sequence, which significantly reduces the occurrence of mutations in the absence of strong selective pressures. Nevertheless, it has been shown that antiviral therapy with nucleoside analogues can promote the selection of nucleotide mutations within conserved domains of the reverse transcriptase, which leads to mutations on the amino acid sequence of the envelope proteins. Changes on the HBsAg structure may lead to reduced binding of anti-HBs antibodies, and hence, they may favour the selection of antibody escape mutants (Harrison 2006).
Besides the production of large amounts of empty SVPs, HBV produces and secretes a non-particulate form of the nucleoprotein, the precore protein, or HBeAg, which is not required for viral infection or replication, but appears to act as a decoy for the immune system, and hence, has tolerogenic functions in promoting viral persistence in the neonates of viremic mothers (Chen 2005, Visvanathan 2006). The precore and core proteins are translated from two distinct RNA species that have different 5’ initiation sites: the precore RNA and the pgRNA. Indeed, the precore transcript, which also contains the full core gene, encodes a signal sequence that directs the precore protein to the lumen of the endoplasmic reticulum, where it is post-translationally processed. Here, the precore protein undergoes N- and C-terminal cleavage to produce the mature HBeAg form (p17), which is then secreted as a monomeric protein. Interestingly, 20 to 30% of the mature protein is retained in the cytoplasm, where it may antagonise TLR signalling pathways and so contribute to the suppression of the host innate immune responses (Lang 2011). As an important marker for active viral replication, the HBeAg is widely used in molecular diagnostics (Chen 2005, Hadziyannis 2006).
The X protein is a multifunctional regulatory protein with transactivating and pro-apoptotic potential, which can modify several cellular pathways (Bouchard 2004) and act as a carcinogenic cofactor (Kim 1991, Slagle 1996, Bouchard 2004). Numerous DNA transfection experiments have shown that over-expression of the X protein (HBx) causes transactivation of a wide range of viral elements and cellular promoters (Bouchard 2004). In vitro studies have shown that HBx can affect various cytoplasmic signal transduction pathways by activating the Src kinase, Ras/Raf/MAP kinase, members of the protein kinase C, as well as Jak1/STAT (Bouchard 2001, Bouchard 2004). Furthermore, in vitro binding studies show that HBx can regulate the proteasome function, and thus affecting the degradation of cellular and viral proteins (Zhang 2004), as well as mitochondrial function, by altering its transmembrane potential, and that HBx can modulate calcium homeostasis (Bouchard 2001, Yang 2011). In addition, several independent studies obtained using the woodchuck model (Zoulim 1994), human liver chimeric mice (Tsuge 2010) and HepaRG™ cells (Lucifora 2011), have shown that HBx is required to initiate HBV replication and to maintain virion productivity. Notably, these studies indicated that despite the establishment of comparable cccDNA amounts, transcription of HBV RNAs was dramatically impaired in cells inoculated with HBV X-minus mutants, indicating that HBx is essential to promote cccDNA-driven viral transcription. These findings are also in agreement with data showing that HBx is recruited to the cccDNA minichromosome, where it was shown to participate in epigenetic control of cccDNA-driven HBV transcription (Belloni 2009, Levrero 2009). Of note, HBx was shown to mediate the degradation of the ‘structural maintenance of chromosomes’ (Smc) complex Smc5/6 (Decorsiere 2016, Murphy 2016). The study of Decorsiere et al. shows that HBx uses the damaged DNA binding protein 1 (DDB1) as an adaptor protein to interact with an E3 ubiquitin ligase enzyme named CRL4, which is a component of the ubiquitin–proteasome system. Several viruses are known to exploit the ubiquitin–proteasome system to ensure productive infection. Being involved in chromosome organisation and DNA repair, the smc5/6 complex probably binds to the cccDNA acting as a host factor suppressing viral transcription. Thus, ubiquitination and degradation of the Smc5/6 complex by the cell’s proteasome machinery, which was demonstrated to occur both in HBV infected human hepatocytes in vitro and in humanised mice in vivo, represents a new mechanism by which HBx can contribute to initiate and maintain active cccDNA transcription .
Most HBV-related HCC show the integration of HBV DNA sequences including the X gene (Brechot 2004, Pollicino 2011, Lupberger 2007). Although HBV integrated forms are frequently rearranged and hence not compatible with the expression of functional proteins, HBx sequences deleted in the C-terminal portion have been frequently detected in tumoural cells (Iavarone 2003). Intriguingly, such HBx deletion variants were shown to retain their ability to support cccDNA transcription when expressed in vitro (Riviere 2019), thus suggesting that not only HBx wildtype, but also such HBx variant forms may participate in cell transformation.
In virus-associated cancers, viral proteins have been shown to participate in epigenetic alterations by disturbing the host DNA methylation system. Interestingly, HBx appears to act as an epigenetic modifying factor in the human liver, which can modulate the transcription of DNA methyltransferases required for normal levels of genomic methylation and maintenance of hypomethylation of tumour suppressor genes (TSGs) (Park 2007). HBx-promoted hypermethylation of TSGs suggests a novel mechanism by which this promiscuous transactivating protein may accelerate hepatocarcinogenesis.
The generation of various HBV-transfected human hepatoma cell lines and the use of related HBV viruses – including the duck hepatitis B virus (DHBV) and the woodchuck hepatitis virus (WHV) – have significantly contributed to elucidate many steps of the hepadnavirus replication cycle (Dandri 2013). Nevertheless, the limited availability of robust in vitro infection systems and accessible animal models of HBV infection has significantly hindered the identification of cellular factors mediating the early steps of HBV infection in human hepatocytes.
The first step in HBV infection involves a non-cell-type specific primary attachment to the cell-associated heparan sulfate proteoglycans (Schulze 2007). This first reversible attachment step is then followed by an irreversible binding of the virus to a specific hepatocyte-specific receptor (Urban 2014). Using mutational analysis, important determinants for infectivity were identified within the HBV envelope proteins. These include 75 amino acids of the preS1 domain of the HBV L-protein, its myristoylation and the integrity of a region in the antigenic loop of the S domain (Gripon 2005, Engelke 2006, Meier 2013). Of note, HBV and HDV infection can be blocked by a small myristoylated lipopeptide (Myrcludex-B) containing the same aminoacid sequence of the preS1 domain of the HBV-L protein (Petersen 2008, Lütgehetmann 2012). Although cell polarisation, in addition to the differentiation status of the hepatocytes, was shown to play an essential role in the infection process (Schulze 2011), the identity of the receptor has remained a mystery for many years. By using a method called zero-length photo cross-linking and tandem affinity purification, the preS1 peptide was seen to specifically interact with a sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter localised to the basolateral membrane of highly differentiated primary hepatocytes (Yan 2012). NTCP mediates the transport of conjugated bile acids and some drugs from portal blood to the liver. Based on the discovery that NTCP functions as viral entry receptor by interacting with the large surface protein of HBV, cell lines susceptible to HBV infection could be established demonstrating that both HBV and HDV infection can be established in human hepatoma cell lines (Yan 2012, Nkongolo 2013). Although large amounts of input viruses (MOI >1000) are generally used to achieve efficient HBV infection in these culture systems, the availability of in vitro assays permitting investigation of the early steps of infection as well as rapid screening of new anti-HBV agents has opened new opportunities in HBV research. Such in vitro studies showed for instance that HBV entry is inhibited by cyclosporins and oxysterols, which are known to bind to NTCP, in hNTCP-transfected hepatoma cells (Nkongolo 2013, Watashi 2013). In addition, b inding of HBV or of Myrcludex-B to the cellular receptor NTCP was shown to limit its function, thus altering the hepatocellular uptake of bile salts and the expression profile of genes of the bile acid metabolism (Oehler 2014).
Despite the importance of having discovered the functional cellular receptor mediating HBV entry, additional hepatocyte-specific and species-specific factors appear to be involved in the HBV infection process, as infection rates and virion productivity are generally low in NTCP expressing human cell lines. Intriguingly, establishment of transient HDV infection could be described in murine cells engineered to express the human NTCP, whereas HBV infection establishment failed in mouse hepatocytes expressing the human NTCP (Li 2014, He 2015). Since HBV and HDV utilise the same envelope proteins for cell entry, additional downstream species-specific factors appear responsible for these discrepancies. As a consequence, no transgenic mice permissive for HBV infection are currently available.
Upon binding to the cell membrane, two possible entry pathways have been proposed. Experimental evidence suggests that HBV can be either involved in an endocytosis process, followed by the release of the nucleocapsid from endocytic vesicles, or HBV may enter the hepatocytes after fusion of the viral envelope at the plasma membrane. As soon as the viral nucleocapsids are released into the cytoplasm, the relaxed circular partially double-stranded DNA (rcDNA) with its covalently linked polymerase needs to enter the cell nucleus in order to convert the rcDNA genome into a covalently closed circular form (cccDNA) (Nassal 2015). Previous studies indicated that the viral capsids are transported via microtubules to the nuclear periphery (Rabe 2006). The accumulation of the capsids at the nuclear envelope would then facilitate interactions with nuclear transport receptors and adaptor proteins of the nuclear pore complex (Kann 1999). Although immature capsids may remain trapped within the nuclear baskets by the pore complexes, the mature capsids eventually disintegrate, permitting the release of both core capsid subunits and of the viral DNA polymerase complexes, which diffuse into the nucleoplasm (Schmitz 2010).
Although the mechanism of cccDNA formation is still not defined, the establishment of productive HBV infection requires the removal of the covalently attached viral polymerase and completion of the positive-strand by the cellular replicative machinery to form the supercoiled cccDNA molecule, which is then incorporated into the host chromatin and serves as the template of viral transcription and replication (Nassal 2015, Newbold 1995). Because of similarities between rcDNA and cellular topoisomerase-DNA adducts that are repaired by tyrosyl-DNA-phosphodiesterase (TDP) 1 or TDP2, recent studies have provided evidence that HBV indeed uses these cellular enzymes to release the P protein from the rcDNA and thus initiates cccDNA biogenesis (Königer 2014). Unlike the provirus DNA of retroviruses, the cccDNA does not need to be incorporated into the host genome. Nevertheless, integration of HBV DNA sequences does occur, particularly in the course of hepatocyte turnover and in the presence of DNA damage (Petersen 1998, Summers 2004, Mason 2005, Allweiss 2018).
Disguised as a stable minichromosome (Bock 1994, Bock 2001, Levrero 2009, Tropberger 2015), the cccDNA uses the cellular transcriptional machinery to produce all viral RNAs necessary for protein production and viral replication, which takes place in the cytoplasm after reverse transcription of an over-length pregenomic RNA (pgRNA) (Figure 3).
Experimental DHBV infection studies indicate that the cccDNA can be formed not only from incoming virions, but also from newly synthesised nucleocapsids, which instead of being enveloped and secreted into the blood, are transported into the nucleus to ensure accumulation, and later maintenance, of the cccDNA pool (Zoulim 2005b, Nassal 2015). According to this scenario, multiple rounds of infection are not needed to establish a cccDNA pool in infected duck hepatocytes. Moreover, expression of the DHBV viral large surface (LS) protein was shown to induce a negative-feedback mechanism, whereby the accumulation of the LS protein would be fundamental to shut off the cccDNA amplification pathway and redirect the newly synthesised rcDNA-containing nucleocapsids to envelopment and extracellular secretion (Kock 2010). Although this peculiar nuclear re-entry mechanism has been clearly demonstrated for the duck HBV (Summers 1991, Wu 1990) and a high copy number of cccDNA molecules is generally detected in chronically infected ducks and woodchucks (up to 50 copies/cell) (Zhang 2003, Moraleda 1997, Dandri 2000), lower cccDNA intrahepatic loads are generally determined in human liver biopsies obtained from chronically HBV-infected patients (median 0.1 to 5 cccDNA copy/cell) (Werle-Lapostolle 2004, Wong 2004, Laras 2006, Volz 2007, Wursthorn 2006, Lutgehetmann 2008) and in chronically HBV-infected human-liver chimeric mice (Petersen 2008, Lutgehetmann 2011, Allweiss 2018 ), suggesting that different viral and host mechanisms may control cccDNA dynamics and cccDNA pool size in human infected hepatocytes . One elegant study showed that HBV converts the rcDNA into cccDNA less efficiently than DHBV in the same human cell background (Kock 2010).
Although the formation of the cccDNA minichromosome is essential to establish productive infection, studies performed in humanised mice indicated that several weeks are necessary for HBV to spread among human hepatocytes in vivo, even in the absence of adaptive immune responses, whereas the increase of the amount of cccDNA molecules per cell seems to remain low (Volz 2013, Allweiss 2018).
HBV polymerase inhibitors do not directly affect cccDNA activity and various in vitro and in vivo studies support the notion that the cccDNA minichromosome is very stable in quiescent hepatocytes, while cell division severely impact its stability ( Dandri 2000 , Allweiss 2018 ). Thus, the significant decrease in cccDNA levels (approximately 1 log10 reduction) generally determined after one year of therapy with polymerase inhibitors (Werle-Lapostolle 2004) is supposed to derive from the lack of sufficient recycling of viral nucleocapsids to the nucleus, due to the strong inhibition of viral DNA synthesis in the cytoplasm, and less incoming viruses from the blood. Nevertheless, cccDNA depletion is expected to require many years of nucleos(t)ide drug administration. Thus, despite the absence of detectable viraemia, the persistence of the cccDNA minichromosome within the infected liver is responsible for the failure of viral clearance and the relapse of viral activity after cessation of antiviral therapy with polymerase inhibitors in chronically infected individuals. Furthermore, if viral suppression is not complete, the selection of resistant variants escaping antiviral therapy is likely to occur ( Zoulim 2009). Resistant HBV genomes can be archived in infected hepatocytes when nucleocapsids produced in the cytoplasm by reverse transcription and containing resistant mutants are transported into the nucleus and added to the cccDNA pool. Under antiviral pressure, these variants will coexist with wild-type cccDNA molecules and function as templates for the production and possibly further selection of replication-competent resistant mutants, which will spread to other hepatocytes and, eventually may even replace the wild-type cccDNA molecules in the liver (Zoulim 2006, Zoulim 2009, Allweiss 2017).
During chronic HBV infection immune-mediated cell injury and compensatory hepatocyte proliferation appear to favour cccDNA decline and selection of cccDNA-free cells (Mason 2005, Allweiss 2018). Notably, studies with the duck model showed that antiviral therapy with polymerase inhibitors induced a greater cccDNA reduction in animals displaying higher hepatocyte proliferation rates (Addison 2002). Furthermore, the identification of uninfected cccDNA negative cell clones containing traces of infection in the form of viral integration indicates that cccDNA clearance without cell destruction can occur in chronically infected woodchucks (Mason 2005). Thus, killing of hepatocytes may be instrumental not only to eliminate HBV infected cells but also to induce hepatocyte proliferation, an event that was shown to promote a clear reduction of cccDNA amounts per cell, and even its loss ( Allweiss 2018). On the other hand, studies have shown that very low levels of cccDNA can persist indefinitely, possibly explaining lifelong immune responses to HBV despite clinical resolution of HBV infection (Rehermann 1996).
As mentioned previously, the cccDNA acts chemically and structurally as an episomal DNA with a plasmid-like structure, which is organised as a minichromosome by histone and non-histone proteins (Bock 1994, Bock 2001, Newbold 1995). Hence its function is regulated, similarly to the cellular chromatin, by the activity of various nuclear transcription factors, including transcriptional coactivators, repressors and chromatin-modifying enzymes (Levrero 2009, Belloni 2012, Tropberger 2015). Congruent with the fact that HBV infects hepatocytes, the cccDNA presents a broad panel of binding sites for liver-specific transcription factors (Levrero 2009, Quasdorff 2008).
The different HBV transcripts are transported into the cytoplasm, where they are respectively translated or used as the template for progeny genome production. Thus, the transcription of the pgRNA is the critical step for genome amplification and determines the rate of HBV replication. Of note, antiviral cytokines such as IFN α were shown to have the capacity to repress cccDNA transcription (Belloni 2012), as well as to promote its partial degradation (Lucifora 2014). Such findings point out the important role that immune modulating factors may play in reducing cccDNA loads and activity. Thus, identification of the factors affecting stability and transcriptional activity of the cccDNA in the course of infection and under antiviral therapy may assist in the design of new therapeutic strategies aimed at silencing and eventually depleting the cccDNA reservoir (Nassal 2015).
The next crucial step in HBV replication is the specific packaging of pgRNA plus the reverse transcriptase into new capsids. The pgRNA bears a secondary structure – named the ε structure - that is present at both the 5’ and the 3’ ends. The ε hairpin loop at the 5’ end is recognised by the viral polymerase and acts as the initial packaging signal (Bartenschlager 1992). Binding of polymerase to the RNA stem-loop structure ε initiates packaging of one pgRNA molecule and its reverse transcription. The first product is single-stranded (ss) DNA of minus polarity; due to its unique protein priming mechanism, its 5’ end remains covalently linked to the polymerase. The pgRNA is concomitantly degraded, except for its 5’ terminal (approximately 15 to 18 nucleotides which serve as primer for plus-strand DNA synthesis), resulting in rcDNA. The heterogeneous lengths of the plus-strand DNAs generated by capsid-assisted reverse transcription may result from a non-identical supply of dNTPs inside individual nucleocapsids at the moment of their enclosure by the dNTP impermeable envelope. This predicts that intracellular cores produced in the absence of envelopment should contain further extended positive DNAs. Alternatively, space restrictions in the capsid lumen could prevent plus-strand DNA completion; in this view, further plus-strand elongation after infection of a new cell might destabilise the nucleocapsid and thus be involved in genome uncoating (Nassal 2015).
The final replication step, the assembly and release of HBV Dane particles, is also not fully understood. The envelopment of the DNA-containing nucleocapsids requires a balanced coexpression of the S and L proteins in order to recruit the nucleocapsid to the budding site. Moreover, the release of infectious viral particles was shown to occur via multivesicular bodies (MVBs), whereas the release of subviral particles (SVPs) proceeds via the general secretory pathway (Hoffmann 2013). Although the role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well-characterised, studies indicate that the lack of expression of the envelope proteins increase cccDNA levels, while co-expression of the envelope proteins not only favours the secretion of viral particles, but also limits the completion of the plus-strand (Lentz 2011).
Notably, in addition to HBV DNA, pregenomic RNA encapsidated and enveloped in virus-like particles is also found in the serum of chronically HBV-infected patients (van Bömmel 2015; Wang 2016) and such release of pgRNA-containing particles seems to accompany that of DNA-containing virions under normal conditions, whereas the amount of pgRNA-containing particles is not diminished after blocking the reverse transcription activity of the HBV polymerase with nucleotide/nucleoside analogues (NUCs) (Wang 2016) . In contrast to NUC therapy, a study in HBV-infected human liver chimeric mice indicated that administration of peg-IFNα decreased the levels of both serum HBV DNA and pgRNA (Giersch 2016). Moreover, this study showed that levels of serum pgRNA correlated with levels of pgRNA and cccDNA determined intrahepatically, thus suggesting that measurements of serum pgRNA may serve as a suitable serological marker to determine the persistence of active cccDNA molecules in the liver of infected patients (Giersch 2016).
Because of the narrow host range of infection, the study of HBV biology has been limited. Consequently researchers have attempted to establish animal models and cell culture systems that are permissive for HBV replication and at least partially reproduce some stages of HBV infection and can be used, e.g., for the preclinical testing of novel antiviral drugs.
Major fundamental progresses in HBV research were based on infection studies performed with HBV-related animal viruses: DHBV, which infects Peking ducks (Mason 1980) and WHV (Summers 1978), which infects the Eastern American woodchuck (Marmota monax).
One of the major advantages of the DHBV model was that DHBV-permissive primary hepatocytes from ducklings or embryos were easily accessible and showed very high infectivity rates in vitro and in vivo with high levels of DHBV replication and antigen expression (Jilbert 1996). However, in contrast to mammalian hepadnaviruses, DHBV infection is cleared within a few days postinfection if the virus is not transmitted vertically and the DHBV genome shares little primary nucleotide sequence homology (40%) with HBV. Furthermore, DHBV infection is usually not associated with liver disease and development of hepatocellular carcinoma (HCC). Nevertheless, the duck model has contributed substantially to elucidate the hepadnaviral replication scheme (Mason 1982, Summers 1988, Delmas 2002) and has been also used for preclinical studies (Zimmerman 2008, Reaiche 2010, Chayama 2011).
WHV is more similar to HBV in terms of genomic organisation than the avian hepadnaviruses. Consequently, in vitro and in vivo studies with woodchuck hepatitis B virus (WHV) have been largely used for the preclinical evaluation of antiviral drugs now in use for treatment of HBV infection (Moraleda 1997, Tennant 1998, Mason 1998, Block 1998, Dandri 2000, Korba 2004, Menne 2005, Fletcher 2015). Moreover, experimental infection of newborn woodchucks almost invariably leads to chronic infection, whereas most animals infected at older ages develop acute hepatitis that results in an efficient immune response leading to viral clearance.
Since acute and chronic WHV infections in woodchucks show serological profiles similar to those of HBV infection in humans, the woodchuck system has provided important information about factors involved in the establishment of virus infection, replication and viral persistence (Lu 2001). Of note, virtually all WHV chronic carrier woodchucks succumb to HCC 2–4 years post infection and regenerative hepatocellular nodules and hepatocellular adenomas are characteristically observed in WHV-infected woodchuck livers (Korba 2004). Proto-oncogene activation by WHV DNA integration has been observed frequently and is thought to play a key role in driving hepatocarcinogenesis in woodchucks, often activating a member of the myc family by various mechanisms (Tennant 2004). Interestingly, WHV viral integration was used as a genetic marker to follow the fate of infected hepatocytes during resolution of transient infection in woodchucks (Summers 2003) and to estimate the amount of cell turnover occurring in the course of chronic infection (Mason 2005). Experimental infection studies in woodchucks also demonstrated that WHV mutants that lacked the X gene were unable or severely impaired to replicate in vivo (Chen 1993, Zoulim 1994, Zhang 2001). The woodchuck model of virally induced HCC has been used to test chemoprevention of HCC using long-term antiviral nucleoside therapy and for the development of new imaging agents for the detection of hepatic neoplasms by ultrasound and magnetic resonance imaging (Tennant 2004). Nevertheless, one main difference between human and rodent hepatitis B resides in the absence of associated cirrhosis in woodchuck and squirrel livers, even after prolonged viral infection (Buendia 1998). It is possible that the rapid onset of hepatocyte proliferation following liver damage in rodents does account for this discrepancy. One general disadvantage for using woodchucks is that they are genetically heterogeneous animals, difficult to breed in captivity and to handle in a laboratory setting. Nevertheless, the woodchuck model has greatly contributed in advancing our understanding of the pathogenesis of HBV infection.
Although HBV infects humans exclusively, it can be used to infect chimpanzees experimentally and, to a certain extent, tupaia, the Asian tree shrew (Baumert 2005). Chimpanzees were the first animals found to be susceptible to HBV infection (Barker 1973) and played an important role in the development of vaccines and in the evaluation of the efficacy of therapeutic antibodies (Ogata 1999, Dagan 2003). Though chimpanzees are not prone to develop chronic liver disease (Gagneux 2004), they provide an ideal model for the analysis of early immunological events of HBV acute infection and pathogenesis (Guidotti 1999). Infection experiments with chimpanzees showed that the majority of viral DNA is eliminated from the liver by non-cytolytic mechanisms that precede the peak of T cell infiltration (Guidotti 1999). T cell depletion studies in chimpanzees also indicate that the absence of CD8 positive cells greatly delays the onset of viral clearance (Thimme 2003). Chimpanzees have been used for preclinical testing of preventive and therapeutic vaccines (Will 1982, Guidotti 1999, Kim 2008, Murray 2005). Nonetheless, the large size, the strong ethical constraints and the high costs of chimpanzees severely limit their use for research purposes.
The tree shrew species Tupaia belangeri has been analysed for the study of HBV both in vitro and in vivo, taking advantage of the adaptability of these non-rodent mammals to the laboratory environment (Baumert 2005, von Weizsacker 2004). Inoculation of tree shrews with HBV positive human serum was shown to result in viral DNA replication in their livers, HBsAg secretion into the serum, and production of antibodies to HBsAg and HBeAg (Walter 1996). Although experimental infection of tree shrew with HBV infectious serum is not highly efficient, productive HBV infection was successfully passed through five generations of tree shrews and was specifically blocked by immunisation with hepatitis B vaccine (Yan 1996). Whereas experimental infection of tree shrews causes only a mild, transient infection with low viral titres, primary hepatocytes isolated from them turned out to be a valuable alternative source of HBV-permissive cells (von Weizsacker 2004). Interestingly, the woolly monkey hepatitis B virus (WMHV), which was isolated from the endangered new world primate woolly monkey (Lagothrix lagotricha) (Lanford 1998), was shown to infect primary tupaia hepatocytes very efficiently (Kock 2001, Dandri 2005a), thus providing a useful and more accessible alternative system for studying the early steps of hepadnaviral infection in vitro (Schulze 2011, Yan 2012).
Because of the different limitations encountered using chimpanzees and models based on HBV-related viruses, recent developments have focused on using the natural target of HBV infection: the human hepatocyte. However, primary human hepatocytes are not easy to handle, cannot be propagated in vitro and their susceptibility to HBV infection is generally low and highly variable. Furthermore, cultured cells may respond differently to the infection than hepatocytes in the liver. The generation of mice harbouring human chimeric livers offered new possibilities to overcome some of these limitations (Dandri 2001 ).
Two major models are currently available: the urokinase-type plasminogen activator (uPA) transgenic mouse (Rhim 1994) and the knockout fumarylacetoacetate hydrolase (FAH) mouse (Azuma 2007). In both systems, the absence of adaptive immune responses permits the engraftment of transplanted xenogenic hepatocytes, while the presence of transgene-induced hepatocyte damage creates the space and the regenerative stimulus necessary for the transplanted cells to repopulate the mouse liver. Both models permit the establishment of HBV infection, which can then persist for the lifespan of the chimeric mice (Dandri 2001, Bissig 2010). While mouse hepatocytes do not support HBV infection, human chimeric mice can be efficiently infected by injecting infectious serum derived from either patients or chimeric mice. Furthermore, genetically engineered viruses created in cell culture can be used to investigate phenotype and in vivo fitness of distinct HBV genotypes and variants (Tsuge 2005). Within the mouse liver human hepatocytes maintain a functional innate immune system and respond to stimuli induced by exogenously applied human IFNα (Belloni 2012, Allweiss 2014). The lack of an adaptive immune system and the undetectable responsiveness of mouse liver cells to human IFNα make the model ideal to exploit the capacities of HBV to interfere with pathways of the innate antiviral response in human hepatocytes (Lutgehetmann 2011), as well as to assess the efficacy of new therapeutic approaches (Petersen 2008, Volz 2013, Klumpp 2018). Moreover, humanised chimeric mice can be superinfected or simultaneously infected with different human hepatotropic viruses, such as HDV (Lütgehetmann 2012, Giersch 2014, Giersch 2019) and HCV (Hiraga 2009) to investigate the mechanisms of viral interference and response to antiviral treatment in the setting of coinfection. Moreover, different chimeric mouse models with a dual reconstitution of both components of the human immune system and human hepatocytes are emerging and their potential for the study of immune responses in HBV infection or immunotherapeutic strategies is currently explored (Dusséaux 2017 ).
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