Antiviral Research 182 (2020) 104925

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Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral

Invited Review

Hepatitis B virus biology and life cycle

Senko Tsukuda a, b, Koichi Watashi a, c, d, e, *
a
Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
b
Nuffield Department of Medicine, University of Oxford, Oxford, UK
c
Department of Applied Biological Science, Tokyo University of Science, Noda, Japan
d
Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
e
MIRAI, JST, Saitama, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: Hepatitis B virus (HBV) specifically infects hepatocytes and causes severe liver diseases. The HBV life cycle is
HBV unique in that the genomic DNA (relaxed-circular partially double-stranded DNA: rcDNA) is converted to a
Life cycle molecular template DNA (covalently closed circular DNA: cccDNA) to amplify a viral RNA intermediate, which is
Entry
then reverse-transcribed back to viral DNA. The highly stable characteristics of cccDNA result in chronic
Replication
cccDNA
infection and a poor rate of cure. This complex life cycle of HBV offers a variety of targets to develop antiviral
NTCP agents. We provide here an update on the current knowledge of HBV biology and its life cycle, which may help to
identify new antiviral targets.

(naked nucleocapsids) (Hu and Liu, 2017).

1. Introduction
Hepatitis B virus (HBV) is an enveloped DNA virus, classified in the
Hepadnaviridae (Rajoriya et al., 2017). HBV was first discovered in an
Australian aborigine by the detection of its antigen, currently known as
surface antigen and originally called “Australia antigen” as reported by
Blumberg and colleagues in 1965 (Blumberg et al., 1965). The presence
of this antigen in hepatitis patients was independently reported by
Prince and by Okochi and Murakami in 1968 (Okochi and Murakami,
1968; Prince, 1968), and the virus particles were visualized by an
electron microscope in 1970 by Dane and colleagues (Dane et al., 1970).
At least three types of HBV particles are observed in the serum of
infected patients: spherical structures of 42 nm in diameter, those with a
diameter of 22 nm, and filament structures of variable length that are 22
nm in a diameter (Fig. 1). The 42 nm particles, also called Dane particles,
are infectious virions consisting of a lipid membrane with three viral
surface antigens (HBs), large (L-HBs), middle (M-HBs), and small
(S-HBs), that surround a nucleocapsid composed of hepatitis B core
protein (HBc), viral polymerase (Pol), and viral genome DNA. The 22 nm
particles, which are much more abundant in patient serum, include
subviral particles (SVPs) that lack the nucleocapsid and are thus
non-infectious. Also, other non-infectious particles are currently known
to be produced by infection, including enveloped particles that lack a
viral genome, those containing viral RNA, and envelope-less particles
The HBV genome DNA is a relaxed-circular DNA (rcDNA) of
approximately 3.2 kb in length with a complete minus strand and an
incomplete plus strand (Fig. 2). The viral genome encodes four over­
lapping open reading frames (ORFs), C, P, S, and X, from which func­
tional viral proteins are produced: HBc and its relatives such as E antigen
(HBe) and 22-kDa precore protein (p22cr) from C; Pol from P; three
types of surface antigens, L-HBs, M-HBs, and S-HBs, from S; and HBV X
protein (HBx) from X. rcDNA is converted into covalently closed circular
DNA (cccDNA) in infected cells (see below), and cccDNA produces HBV
RNAs of different lengths (mainly 3.5 Kb, 2.4 Kb, 2.1 Kb, and 0.7 Kb)
transcribed from different promoters in the HBV genome. A 3.5 Kb RNA
produces the protein product from C and P; a 2.4 Kb RNA is translated
into L-HBs and a 2.1 Kb RNA synthesizes the other two surface antigens,
M-HBs and S-HBs; and a 0.7 Kb RNA produces HBx. The produced HBc
protein product initially associates to yield a dimer through its N-ter­
minal domain, and then self-assembles into an icosahedral capsid con­
sisting of 90 or 120 dimers, which incorporates the 3.5 Kb viral
pregenomic RNA (pgRNA) associated with Pol (see below in detail). HBe
is produced by the translation of 3.5 Kb preC mRNA, having an extended
5’ upstream region of the C gene and subsequent cleavage of the protein
product at its C-terminus. Pol is the largest HBV protein, consisting of
four domains with three discrete enzymatic functions: 1) the terminal
protein (TP) domain, which is essential for binding to pgRNA and serves
as a protein primer to initiate minus strand DNA synthesis; 2) the spacer

* Corresponding author. Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.
E-mail address: kwatashi@nih.go.jp (K. Watashi).

https://doi.org/10.1016/j.antiviral.2020.104925
Received 4 May 2020; Received in revised form 24 July 2020; Accepted 26 August 2020
Available online 28 August 2020
0166-3542/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Tsukuda and K. Watashi Antiviral Research 182 (2020) 104925

Abbreviations NTCP sodium taurocholate cotransporting peptide

p22cr 22-kDa precore protein
APOBEC3 apolipoprotein B editing complex 3 PAPD poly(A) RNA polymerase associated domain containing
CBP CREB-binding protein proteins
cccDNA covalently closed circular DNA PCAF CBP-associated factor
C/EBP CCAAT-enhancer-binding protein pgRNA pregenomic RNA
CREB cAMP response element binding protein Pol polymerase
DHBV duck hepatitis B virus Pol α DNA polymerase α
DR1 direct repeat 1 Pol κ DNA polymerase κ
eIF eukaryotic translation initiation factor PPARα peroxisome proliferator-activated receptor alpha
ESCRT endosomal sorting complex required for transport Prdx1 peroxiredoxin 1
FEN1 flap structure-specific endonuclease 1 PRMT protein methyl transferase
FXR farnesoid X receptor PROX1 prosperous-related homeobox protein 1
HBc hepatitis B virus core antigen RBM24 RNA-binding motif protein 24
HBe hepatitis B virus E antigen RBP24 RNA-binding motif protein 24
HBs hepatitis B virus surface antigen rcDNA relaxed-circular DNA
HBV hepatitis B virus RNaseH ribonuclease H
HDAC histone deacetylase RT reverse transcriptase
HNF hepatocyte nuclear factor RXR retinoid X receptor
HP1 heterochromatin protein 1 Set1A histone methyltransferase 1A
Hsc heat stress cognate SETDB1 SET domain bifurcated histone lysine methyltransferase 1
Hsp heat shock protein S-HBs small surface antigen
HSPGs heparan sulfate proteoglycans SIRT sirtuin
IFN interferon SKIV2L superkiller viralicidic activity 2-like
ISG20 IFN-stimulated exoribonuclease gene of 20 kDa Smc structural maintenance of chromosomes
L-HBs large surface antigen SP1 specificity protein 1
LIG DNA ligase SVPs subviral particles
LSD1 histone lysine demethylase 1 TDP2 tyrosyl-DNA phosphodiesterase 2
LRH-1 liver receptor homolog-1 TNF tumor necrosis factor
LTβ lymphotoxin β TOP topoisomerase
m6A N6-methyladenosine TP terminal protein
M-HBs middle surface antigen UBE2L3 ubiquitin conjugating enzyme E2 L3
MSL2 male-specific lethal 2 ZAP zinc finger antiviral protein
MVB multivesicular body ZHX2 zinc finger and homeoboxes 2
NF1 nuclear factor 1

domain, whose function has not been well defined; 3) the reverse
transcriptase (RT) domain, which has DNA elongation activity for both
reverse transcription and DNA-dependent DNA polymerization; and 4)
the ribonuclease H (RNaseH) domain, which digests pgRNA after reverse
transcription. The three HBs proteins share the common C-terminal S
region. In addition, M-HBs and L-HBs carry an extended region, called
the preS2 region, at the N-terminus of the S region. L-HBs further in­
cludes an extension (preS1 region), which is essential for receptor
binding during virus entry, at the N-terminus of the preS2 and S regions.
HBx is a multifunctional protein that promotes viral production at
multiple steps, including viral transcription and replication, and also
plays a role in the development of HBV-related hepatocellular
carcinoma.
HBV proliferates in host cells by taking advantage of these viral
proteins in concert with host-derived factors. As an introduction to HBV
biology, in this article we summarize and update what is currently
known about the mechanism of the HBV life cycle (Fig. 3).
2. HBV entry
HBV entry into host hepatocytes follows multiple steps. Initially, the
virus attaches to the host cell surface through binding to factors
including heparan sulfate proteoglycans (HSPGs) such as glypican 5 in a
non-specific and low-affinity manner, and then interacts with its re­
ceptor(s) more specifically and with high affinity (Leistner et al., 2008;
Schulze et al., 2007; Verrier et al., 2016). Sodium taurocholate
cotransporting peptide (NTCP/SLC10A1), which is specifically
expressed in the liver and originally functions to uptake bile salts into
hepatocytes, was identified as an HBV and HDV entry receptor in 2012.
NTCP was identified as a factor that bound to aa 2–48 of the preS1 re­
gion, which had been already known to be essential for receptor binding
(Barrera et al., 2005; Glebe et al., 2005; Gripon et al., 2005; Ni et al.,
2014; Yan et al., 2012). Virus-receptor interactions are believed to
trigger virus internalization into cells in an endocytosis-dependent
manner (Hao et al., 2011; Huang et al., 2012; Macovei et al., 2010,
2013; Yamada et al., 2012). A recent report found that a receptor
tyrosine kinase, epidermal growth factor receptor (EGFR), triggers the
internalization of HBV/HDV through its direct interaction with NTCP
(Iwamoto et al., 2019, 2020). It has also been reported recently that
NTCP can be oligomerized, and the oligomerization status modulates the
ability of NTCP to mediate viral internalization (Fukano et al., 2018).
Internalization in vesicles is believed to induce fusion between the viral
envelope and the cell-derived vesicular membrane, although its mech­
anism remains largely unknown. The incoming nucleocapsid in the
cytoplasm is directed to the nucleus along with the microtubules (Rabe
et al., 2006) and is imported into the nucleus through the nuclear pore
complex in an importin-dependent manner (Kann et al., 1999; Rabe
et al., 2003).
3. cccDNA formation/maintenance
In the nucleus, HBV genomic DNA is modified by cellular factors. The
Pol-linked terminal redundant sequence in the 5′ -end of the minus
strand DNA and the RNA oligonucleotide attached at the 5′ end of the

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S. Tsukuda and K. Watashi
plus strand DNA (see below for rcDNA structure and synthesis) are
removed from the rcDNA, and the gaps in both strands are filled and
ligated to generate cccDNA (Gerlich and Robinson, 1980; Guo et al.,
2007; Molnar-Kimber et al., 1983; Wang and Seeger, 1992). Several
factors have been reported to be involved in the process of cccDNA
formation. A DNA repair enzyme, tyrosyl-DNA phosphodiesterase 2
(TDP2), has been shown to cleave the tyrosyl-DNA phosphodiester bond
between Pol and rcDNA in an in vitro assay, although its relevance in
cccDNA formation in the cellular context remains controversial (Cui
et al., 2015; Koniger et al., 2014). Flap structure-specific endonuclease 1
(FEN1), which is implicated in cellular DNA replication and repair, is
another factor reported to cleave the flap structure at the 5′ -end of the
minus strand (Kitamura et al., 2018). After the removal of the Pol and
RNA primers, DNA polymerases and ligases, such as DNA polymerase κ
and α (Pol κ and Pol α), DNA ligase 1 and 3 (LIG1 and LIG3), and
topoisomerase I and II (TOP1 and TOP2), have been reported to function
for filling the gaps in rcDNA (Long et al., 2017; Qi et al., 2016; Sheraz
et al., 2019; Tang et al., 2019). Although it remains unknown how and
where cccDNA is maintained in the nucleus, cccDNA resides episomally
but is inherently stable, which thus functions as a template for viral
replication over the long term. A recent analysis showed a cccDNA
half-life of about 40 days in NTCP-overexpressing HepG2 cells (Ko et al.,
2018). But that in a clinical setting is likely to be far longer. A report
estimated more than 9 months for a cccDNA half-life in hepatitis B pa­
tients (Boyd et al., 2016). Immune responses and cytokine stimulation
are major factors that affect cccDNA maintenance (Hu et al., 2019). The
mechanisms for the regulation of cccDNA maintenance/stability remain
largely unclarified, but a cytidine deaminase, apolipoprotein B editing
complex 3 (APOBEC3), is an example of a protein that modulates
cccDNA stability. Upon stimulation by cytokines such as interferon α
and γ (IFNα, IFNγ), tumor necrosis factor α (TNFα) as well as lympho­
toxin β (LTβ), APOBEC3A and/or APOBEC3B are induced and are re­
ported to destabilize cccDNA (Lucifora et al., 2014; Xia et al., 2016). A
ubiquitin conjugating enzyme E2 L3 (UBE2L3) and male-specific lethal 2
Fig. 1. Schematic representation of HBV particles. Infectious HBV virion (Dane particle) (upper) and non-infectious HBV particles, including enveloped capsids
containing immature DNA/RNA, subviral particles (sphere and filament), and naked nucleocapsids (lower).
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Antiviral Research 182 (2020) 104925
(MSL2) modulate cccDNA stability through degradation of APOBEC3A
and APOBEC3B, respectively (Gao et al., 2017; Zhou et al., 2019). The
ability of APOBEC3G to induce cccDNA hypermutation has also been
reported in an overexpression experiment (Kitamura et al., 2013). In
addition to cccDNA formation and maintenance, a part of the incoming
HBV DNA is integrated into the host genome. A recent cell culture study
showed that the integration occurs as fast as within a week after infec­
tion (Tu et al., 2018). The integrated HBV DNA is
replication-incompetent but can act as a template for the production of
HBs, which is thought to be related to HBV-specific immune tolerance
and the development of HBV-related pathogenesis.
4. HBV transcription
Using cccDNA as a template, four different lengths of RNAs (3.5, 2.4,
2.1, and 0.7 kb) are transcribed. Transcription of viral RNAs is regulated
by four distinct promoters for preS1, preS2, core, and X, and two en­
hancers (Enhancer I and Enhancer II), which is mediated by host RNA
polymerase II machinery-dependent transcription (Rall et al., 1983).
The transcription is regulated at multiple levels as shown below (Fig. 4).
4.1. Epigenetic modification
cccDNA exists as a minichromosome that associates with viral pro­
teins and host factors (Fig. 4). As cccDNA is assembled with histones, the
post-translational modification status of histones governs the tran­
scriptional activity of cccDNA (Pollicino et al., 2006). Genome-wide
ChIP-seq analysis has revealed the post-translational modification of
cccDNA-associated histones that show a high level of trimethylation or
acetylation of lysine of histone 3 (H3K4me3, H3K27ac, and H3K122ac),
active markers of transcription, enriched at specific sites within the HBV
genome, and very low levels of transcriptional repression markers
(H3K27ac and H3K9me2) even at silent HBV promoters (Tropberger
et al., 2015). There are also accumulating reports of histone
S. Tsukuda and K. Watashi
modification enzymes and other factors directly or indirectly recruited
onto the cccDNA minichromosome to regulate viral transcription. Such
histone modification enzymes includes histone acetyltransferases
[CREB-binding protein (CBP)/p300, CBP-associated factor
(PCAF)/CGN5, HAT1], histone deacetylases [histone deacetylase 1 and
4 (HDAC1, HDAC4), sirtuin 1 and 3 (SIRT1, SIRT3)], methyltransferases
[SET domain bifurcated histone lysine methyltransferase 1 (SETDB1),
protein methyl transferase 1 and 5 (PRMT1, PRMT5), histone methyl­
transferase 1A (Set1A)], and demethylases [histone lysine demethylase
1 (LSD1)] (Alarcon et al., 2016; Belloni et al., 2009; Ren et al., 2018;
Riviere et al., 2015; Xing et al., 2019; Yang et al., 2019; Zhang et al.,
2017). More precise mechanisms for cccDNA-associated histone modi­
fication and its regulation of viral transcription are mentioned elsewhere
(Hong et al., 2017). Importantly, these cccDNA-associated histone
modifications have been revealed to be targeted by the antiviral activity
of interferon (IFN): IFNα induces hypoacetylation of cccDNA-bound
histones and the recruitment of transcriptional corepressors that result
in transcriptional silencing both in cell culture and in chimeric mouse
models (Belloni et al., 2012). It has been also reported that IFNα alters
the acetylation status of H3K9 and H3K27 to suppress transcription of
duck hepatitis B virus (DHBV) cccDNA (Liu et al., 2013).
4.2. Transcription factors
In addition to epigenetic control, the recruitment of cellular tran­
scription factors to the viral promoter/enhancer regions in cccDNA
governs the activity of transcription. The viral promoter/enhancer re­
gions contain the binding sites for numerous transcription factors,
including the elements for the liver-specific nuclear receptors. The
transcription factors involved in HBV transcription have been studied
for more than two decades (Fig. 4). These include liver-enriched hepa­
tocyte nuclear factor 3 and 4 (HNF3, HNF4), retinoid X receptor alpha
(RXRα), peroxisome proliferator-activated receptor alpha (PPARα), and
farnesoid X receptor (FXR) as nuclear receptors (Chen et al., 1994;
Guidotti et al., 1999; Huan et al., 1995; Li et al., 1995; Ori and Shaul,
1995; Reese et al., 2013; Tang and McLachlan, 2001, 2002; Wang et al.,
1998). Other transcription factors that have been reported to activate
transcription to augment pgRNA expression include
CCAAT-enhancer-binding protein (C/EBP), nuclear factor 1 (NF1),
specificity protein 1 (SP1), cAMP response element binding protein
(CREB), and liver receptor homolog-1 (LRH-1), and those that report­
edly suppress pgRNA transcription include HNF6, prosperous-related
homeobox protein 1 (PROX1), p53, and zinc finger and homeoboxes 2
(ZHX2) (Cai et al., 2003; Hao et al., 2015; Kim et al., 2008; Lopez-­
Cabrera et al., 1991; Ori et al., 1994; Qin et al., 2009; Uchida et al.,
1996; Xu et al., 2018; Yuh and Ting, 1991; Zhang and McLachlan, 1994;
Zhang et al., 1993).
Fig. 2. Schematic representation of the structure of HBV genomic DNA, RNAs, and proteins. Left, HBV genomic rcDNA and the encoded ORFs (C, P, S, X). Center,
HBV RNAs (gray lines) produced by cccDNA transcription and the proteins (boxes) produced from the RNAs. The RNA lengths, as well as the names of the RNAs, are
shown on the left. Right, HBV proteins and the domain structures. Amino acid numbers and the lengths are shown above the box and on the right, respectively.
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Antiviral Research 182 (2020) 104925
4.3. Role of HBx in HBV transcription
HBx has been demonstrated to be essential for HBV replication after
infection (Lucifora et al., 2011). HBx is a multifunctional protein with
many studies showing a wide variety of functions, although the rele­
vance of such functions in physiological settings needs to be further
clarified. HBx has been reported to associate with the cccDNA mini­
chromosome in close parallel to the kinetics of cccDNA-bound H3
acetylation (Belloni et al., 2009). HBx modulates the recruitment of
chromatin-modifying enzymes (p300, HDAC, SIRT1) and controls the
epigenetic status of cccDNA-associated histones for active transcription
(Belloni et al., 2009). HBx has also been reported to affect not only
acetylation but also methylation and phosphorylation of
cccDNA-associated histones in HepG2 cells (Luo et al., 2013). In the
absence of HBx, cccDNA is transcriptionally silenced by a decrease in H3
acetylation and H3K4me3 and an increase in H3K9me2/3 that results in
the recruitment of heterochromatin protein 1 (HP1) and chromatin
condensation, while HBx expression relieves this transcriptional
silencing by recovering the increased H3K4me3 and dissociating HP1
recruitment on cccDNA (Riviere et al., 2015). This evidence supports the
activity of HBx on the epigenetic profile of cccDNA-associated histones
to regulate HBV transcription.
In addition, a recent finding on HBx action on Smc5/6 has opened a
new aspect of its function (Fig. 4). The structural maintenance of chro­
mosomes (Smc) family are ATPases that generally regulate higher-order
chromosome organization (Palecek, 2018). Recently, it has been
revealed that the Smc5/6 complex associates with an episomal HBV
DNA reporter and suppresses its transcriptional activity (Decorsiere
et al., 2016). In the presence of HBx, DDB1-containing E3 ubiquitin
ligase is recruited to Smc5/6 and induces complex degradation to relieve
Smc5/6-mediated transcriptional silencing. Genetic knockdown of
Smc5/6 causes the replication of HBx-deficient HBV, suggesting an
essential role for Smc5/6 antagonism in HBx’s function to support viral
replication (Murphy et al., 2016). This function requires a CCCH motif in
the HBx sequence that is highly conserved among strains (Ramakrishnan
et al., 2019). A study using clinical samples has shown that the
anti-Smc5/6 function can be retained in HBx variants found in HCC
patients (Riviere et al., 2019). Interestingly, nitazoxanide has been
found to inhibit HBx-DDB1 binding and restore the expression of Smc5
to suppress HBV replication in HBV-infected cells, proposing that this
mechanism can serve as a therapeutic target (Sekiba et al., 2019).
5. HBV RNAs stability
Recent accumulating evidence has focused on the HBV RNA stability
as a major step that limits the viral replication level. HBV pgRNA con­
tains a stem loop, called epsilon, at both the 3’and 5′ termini. Epsilon is
S. Tsukuda and K. Watashi Antiviral Research 182 (2020) 104925

Fig. 3. Schematic overview of the HBV life cycle. See the main text for a precise description of the HBV life cycle.

Fig. 4. Schematic representation of the regulation of HBV transcription. HBV transcription is regulated at the epigenetic (left), transcription factor (center), and the
restriction (right) levels. Left, histone modification enzymes that positively (upper) and negatively (lower) regulate transcription are shown. Center, transcription
factors positively (upper) and negatively (lower) regulate the transcription of HBV core promoter/enhancer. Right, SMC5/6 suppresses the transcription, and HBx
counteracts the SMC5/6 restriction by the recruitment of the DDB1/Cul4 ubiquitin ligase complex for protein degradation.

5
S. Tsukuda and K. Watashi
essential for the RNA packaging into capsids (see below), and recent
reports have shown a role for these elements in modulating the stability
of HBV RNAs. Zinc finger antiviral protein (ZAP) interacts with HBV
RNAs through a region containing the epsilon and promotes its decay
mainly in the nucleus, and this mechanism is potentiated upon IFN
treatment (Mao et al., 2013). An RNA helicase, superkiller viralicidic
activity 2-like (SKIV2L: a homolog of the Saccharomyces cerevisiae Ski2),
interacts with HBV RNAs, especially X-mRNA (0.7 Kb RNA), and me­
diates their degradation through a non-stop-mediated RNA decay
mechanism (Aly et al., 2016). A ribonuclease, IFN-stimulated exoribo­
nuclease gene of 20 kDa (ISG20), promotes HBV RNA degradation by
binding to the lower stem portion of the epsilon (Leong et al., 2016; Liu
et al., 2017). The growing list of other cellular factors that bind to HBV
RNAs and promote their degradation includes a cytidine deaminase, AID
(Liang et al., 2015), a splicing factor and a U2 small nuclear ribonu­
cleoprotein auxiliary factor, PUF60 (Sun et al., 2017), RNA-binding
motif protein 24 (RBP24) (Yao et al., 2018), and peroxiredoxin 1
(Prdx1) (Deng et al., 2019). Interestingly, using a dihydroquinolizinone
derivative, RG7834, which is a new drug candidate that suppresses HBV
replication and gene expression, the non-canonical poly(A) RNA poly­
merase associated domain containing proteins 5 and 7 (PAPD5 and
PAPD7) has been revealed to function in stabilizing HBV RNA, and
RG7834 has been proved to target these proteins (Mueller et al., 2018,
2019). It has also been recently reported that covalent modifications of
viral RNA can regulate the translation and/or stability of RNA in many
viruses including HIV-1, influenza virus, enterovirus, and respiratory
syncytial virus, which is known as epitranscriptomic regulation (Tsai
and Cullen, 2020). In HBV, it has been reported that the
post-transcriptional modification, N6-methyladenosine (m6A) of HBV
Fig. 5. Schematic representation of reverse transcription, RNA degradation, and DNA synthesis in nucleocapsids. Pol (shown by TP, RT, RH) is recruited to the 5′ -
terminal epsilon and mediates the priming reaction. After translocation to the 3′ -terminal DR1, Pol mediates the DNA elongation to synthesize the full-length (− )DNA
and simultaneously digests the template RNA. The remaining 5′ -terminal RNA fragment containing DR1 is translocated to DR2 on the other terminus and starts the
synthesis of (+)DNA.
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RNA at its 3′ epsilon stem loop structure, destabilizes HBV RNAs,
although the m6A at the 5′ epsilon stem loop of pgRNA has another
function that facilitates the reverse transcription of pgRNA (Imam et al.,
2018). m6A-modified HBV RNA at A1907 is recognized by YTH-domain
family 2 (YTHDF2) and an IFN-induced RNase, ISG20, which then
processes HBV RNA for degradation (Imam et al., 2020). Thus, HBV RNA
stability has been revealed to be strictly regulated by multiple cellular
factors.
6. Encapsidation and DNA synthesis
RNA encapsidation requires HBc, Pol, and viral RNA. Synthesized
HBc monomers initially associate to yield a dimer, and then 90 or 120
dimers subsequently self-assemble to constitute an icosahedral capsid
(Bottcher et al., 1997; Conway et al., 1997). Concomitantly with the
encapsidation, Pol at its TP domain interacts with the epsilon stem loop
of the pgRNA at the 5’ terminus to form a ribonucleoprotein complex,
which is then incorporated into the capsid (Bartenschlager and Schaller,
1992; Pollack and Ganem, 1994). This process is facilitated by host
chaperones, heat shock protein 90 (Hsp90), Hsp40, and heat stress
cognate 70 (Hsc70), through interactions with Pol and its conforma­
tional optimization(Beck and Nassal, 2003; Hu et al., 1997, 2004; Hu
and Seeger, 1996; Prange et al., 1999; Stahl et al., 2007). RNA-binding
motif protein 24 (RBM24) interacts with both Pol and epsilon RNA to
mediate the encapsidation (Yao et al., 2019). The interaction of nucle­
ophosmin B23 with HBc also increases capsid assembly (Jeong et al.,
2014; Lee et al., 2009). The reported host factors incorporated into the
capsid include eukaryotic translation initiation factor 4E (eIF4E),
DEAD-box RNA helicase DDX3, and APOBEC3G (Kim et al., 2010;
S. Tsukuda and K. Watashi
Nguyen and Hu, 2008; Turelli et al., 2004; Wang et al., 2009). DDX3 and
APOBEC3G have a negative effect on the HBV replication.
After RNA incorporation, viral genome synthesis proceeds through
several steps inside the nucleocapsid (Fig. 5). The epsilon in pgRNA has
an internal bulge possessing the sequence 5′ -UUC-3′ , which functions as
a template for priming. A tyrosine residue at aa 63 in the TP domain of
Pol acts as a protein primer: the first dGTP residue is covalently linked to
a hydroxyl residue in Y63 (priming initiation), and two further dAMPs
are extended to produce 5′ -dGAA-3’ (priming polymerization) (Jones
et al., 2012; Nassal and Rieger, 1996; Wang and Seeger, 1992, 1993;
Wang and Hu, 2002). The produced Pol-dGAA complex is then trans­
located to the complementary direct repeat 1 (DR1) sequence at the 3′
end of pgRNA to allow for minus strand DNA synthesis (Lin et al., 2008;
Wang and Hu, 2002). This strand is extended to the 3′ end of pgRNA,
resulting in a unit length minus strand DNA containing an additional
terminal redundancy. The pgRNA template is simultaneously degraded
during the minus strand DNA synthesis through digestion by the RNase
H domain, and eventually leaves a short RNA fragment containing the
capped 5′ terminal region including 5′ DR1. This RNA fragment is then
translocated to DR2 at the 3′ terminus and is extended to the 5′ terminus
of the minus strand DNA. Having a redundant sequence, extending the 3′
end of the plus strand DNA switches to the 3′ redundant sequence on the
minus strand DNA, enabling further elongation of the plus strand DNA to
eventually produce rcDNA.
7. HBV morphogenesis
The viral genome-containing nucleocapsids are either shuttled to the
nucleus to amplify and maintain the cccDNA pool (recycling) or
assembled with mature envelope proteins to be secreted outside of cells
(virus egress). Capsids containing the replication intermediates, pgRNA,
and those without viral DNA/RNA are also secreted from cells but with
different secretion efficiencies. The secretion process for infectious vi­
rions depends on multivesicular body (MVB)-associated endosomal
sorting complex required for transport (ESCRT) machinery, which in­
volves gamma 2-adaptin, CHMP3/4, Vps4, Nedd4, and α-taxilin (Hoff­
mann et al., 2013; Lambert et al., 2007; Rost et al., 2006, 2008; Stieler
and Prange, 2014; Watanabe et al., 2007). In contrast, the egress of
naked capsids does not require the above ESCRT machinery, but rather
involves other factors such as Alix and HGS (Bardens et al., 2011; Chou
et al., 2015). Subviral particles in the spherical structure that predom­
inantly contain S-HBs are released from the endoplasmic reticulum
through the general secretory pathway (Patient et al., 2007, 2009), and
filamentous subviral particles that also contain L-HBs share the
ESCRT-dependent egress pathway (Jiang et al., 2015). Thus, the virus
egress pathway is closely associated with the cellular membrane sorting
machinery.
8. Conclusions
Numerous studies have been conducted to better comprehend HBV
biology. This has led to the identification of numerous targets that are
currently under clinical and preclinical development (see other articles
in this issue). Further clarification will provide additional anti-HBV drug
candidates with novel modes of action.
Acknowledgments
We appreciate Drs. Julie Lucifora and Marion Delphin at INSERM,
U1052, Université de Lyon for their scientific comments. The authors’
work was supported by the Japan Society for the Promotion of Science
KAKENHI Grants JP17H04085 and JP20H03499; the Japan Science and
Technology Agency (JST) MIRAI program; the Japan Agency for Medi­
cal Research and Development (AMED) Grants JP20fk0310114j0004,
JP20fk0310101j1004, JP20fk0310103j0204, JP20fk0210036j0003,
and JP20jm0210068j0002; the Takeda Science Foundation; the
7
Antiviral Research 182 (2020) 104925
Smoking Research Foundation; and the Mochida Memorial Foundation
for Medical and Pharmaceutical Research. The authors declare that they
have no conflicts of interest with the contents of this article.
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