Book on hepatitis from page 68 to 74

Book on hepatitis from page 68 to 74

68  Hepatology 2012
Figure 2. Genome organization and transcripts of the human hepatitis B virus. The
outer thin lines represent the viral transcripts that initiate at different sites, under the
control of distinct promoters, but are all terminated after a common polyadenylation site.
The RNA signal on the terminally redundant pgRNA is indicated as a hairpin. The thick
lines represent the rcDNA form of the genome as present in infectious virions. The 5’ end
of the minus-strand DNA is covalently linked to the terminal protein of the polymerase.
The 5´ end of the incomplete plus-strand DNA is constituted by an RNA oligo derived from
the 5’ end of pgRNA. DR1 and DR2 indicate the direct repeats. The inner arrows indicate
the open reading frames (adapted from Nassal 2008).
HBV structural and non-structural proteins
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 (Glebe 2007). Notably, the preS/S ORF entirely
overlaps with the polymerase open reading frame (Lee 2004). 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 (Bruss 2007). The envelope
proteins are cotranslationally 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
(Schildgen 2004, 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
HBV Virology  69
to be essential for infectivity (Glebe 2005, Nassal 2008) (Schulze 2010). Both
spherical and filamentous SVPs are secreted into the blood of infected individuals in
a 10
3
-10
6
-fold 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 neutralizing antibodies produced by the host and hence increase the ability of the
infectious particles to reach the hepatocytes. It has also been suggested that SVPs
contribute to create a state of immune tolerance, which is a precondition for highly
productive persistent infection.
In the cytoplasm, the core protein dimerises and self-assembles to form an
icosahedral nucleocapsid. The full-lengthcore protein is 183 amino acids in length
and consistsof an assembly domain and a nucleic acid-bindingdomain, 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 1994, Kann
2007, Kann 1999, Daub 2002). 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, Nassal
2008). The spacer domain separates the terminal protein from the polymerase
domains (Beck 2007)
Despite the occurrence of nucleotide mutations due to the lack of proofreading
capacity of the HBV polymerase, the peculiar genomic organization 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 analogs can promote the selection of nucleotide mutations within
conserved domains of the reverse transcriptase, which lead to mutations also 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).
HBV also produces distinct non-structural proteins whose exact functions are not
fully elucidated. 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 promotingviral persistence in the neonates of viremic mothers (Chen
2005, Visvanathan 2006). Theprecore and core proteins are translated from 2
distinct RNAspecies that have different 5' initiation sites: the precore RNA and the
pgRNA. Indeed, the precoretranscript, which also contains the full core gene,
encodes a signal sequence that directs theprecore protein to the lumen of the
endoplasmic reticulum, where it is post-translationally processed. Here, the precore
protein undergoesN- 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
70  Hepatology 2012
signaling 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, Dandri 1996, Slagle 1996).
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). The evidence that HBx responsive enhancers/
promoters do not share any common DNA sequence and that HBx does not bind
double-stranded DNA suggested that HBx may exert its transactivating activity
through protein-protein interactions. 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.
Furthermore, in vitro binding studies show that HBx can regulate the proteasome
function, and thus, may control the degradation of cellular and viral proteins (Zhang
2004). It has also been reported that HBx can affect mitochondria function, by
altering its transmembrane potential, as well as that HBx can modulate calcium
homeostasis (Bouchard 2001, Nassal 2008, Yang 2011).
Although the exact role of HBx in the context of HBV infection has not been
clarified, several lines of evidence obtained first using the woodchuck model
(Zoulim 1994) and more recently using uPA/SCID mice (Tsuge 2010) and
HepaRG
TM
cells (Lucifora 2011), have convincingly 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, indicating that HBx is essential for viral transcription. These findings are
also in agreement with data showing that HBx is recruited to the cccDNA
minichromosome, where it appears to be involved in epigenetic control of HBV
replication (Belloni 2009, Levrero 2009). In addition, HBx has been shown to
enhance encapsidation of the pgRNA by increasing phosphorylation of the core
protein (Melegari 2005), indicating that HBx may support virion productivity in
various steps of the HBV life cycle.
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 tumoral cells (Iavarone 2003). In virus-associated
cancers, viral proteins have been shown to participate in epigenetic alterations by
disturbing the host DNA methylation system. Interestingly, a study suggested that
the HBV regulatory X protein is a potent 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
tumor suppressor genes (TSGs) (Park 2007). HBx-promoted hypermethylation of
TSGs suggests a novel mechanism by which this promiscuous transactivating
protein may accelerate hepatocarcinogenesis (Kekule 1993, Dandri 1996).
HBV Virology  71
The HBV replication cycle
During the last 30 years, the generation of various HBV–transfected human
hepatoma cell lines and the use of related HBV viruses, like the duck hepatitis B
virus (DHBV) and the woodchuck hepatitis virus (WHV) have  significantly
contributed to elucidate many steps of the hepadnavirus replication cycle (Schultz
2004, Roggendorf 1995, Roggendorf 2007). Nevertheless, the lack of efficient in
vitro infection systems and of easily accessible animal models has significantly
hindered the identification of mechanisms and cellular factors mediating viral entry
and uncoating in human hepatocytes. Although primary hepatocytes remain
permissive in vitro for only a short time after plating, the availability of primary
hepatocytes from tree-shrews (Tupaia belangeri) for infection studies with HBV
and the closely-related woolly monkey hepatitis B virus (WM-HBV) (Kock 2001),
and the discovery of a human hepatoma cell line (HepaRG) able to gain
susceptibility for HBV infection upon induction of differentiation in vitro (Gripon
2002), have lately expanded our possibilities to functionally dissect the HBV entry
process (Glebe 2007, Schulze 2010).
The first step in HBV infection appears to involve 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, but still unknown hepatocyte-specific receptor
(Urban 2010, Glebe 2007). This step probably requires activation of the virus,
resulting in exposure of the myristoylated N-terminus of the L-protein. Important
determinants for infectivity within the HBV envelope proteins were identified using
mutational analyses. 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). Potential HBV receptor candidates have
been described in the past, but none of them has been confirmed in a functional
assay (Glebe 2007). Recent studies indicated that cell polarization, in addition to the
differentiation status of the hepatocytes, plays an important role in the infection
process (Schulze 2011).
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 viral 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 2008).
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 2007). 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 polymerase-viral DNA complexes, which diffuse into the
nucleoplasm (Schmitz 2010).
72  Hepatology 2012
Within the infected nuclei 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 2008, Newbold 1995). For
the formation of the cccDNA, the terminal protein and one of the redundant
terminal repeats present on the rcDNA need to be removed. So far it is assumed that
cellular ligases and probably other enzymes involved in DNA repair mechanisms
become active and convey the relaxed circular form into the cccDNA (Seeger
2000). Unlike the provirus DNA of retroviruses, the cccDNA does not need to be
incorporated into the host genome. Nevertheless, integrations of HBV DNA
sequences do occur, particularly in the course of hepatocyte turnover and in the
presence of DNA damage, as has been shown in cell culture (Dandri 2002) and in
the woodchuck system (Petersen 1998, Summers 2004, Mason 2005).
Disguised as a stable non-integrated minichromosome (Bock 1994, Bock 2001),
the cccDNA utilizes 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).
cccDNA
AAA
AAA
cccDNA
AAA
AAA
AAA
AAA
Figure 3. The HBV lifecycle. Upon hepatocyte infection the nucleocapsid is released into
the cytoplasm and the rcDNA transferred to the cell nucleus where it is converted into the
cccDNA minichromosome. After transcription of the viral RNAs, the pgRNA is
encapsidated and reverse-transcribed by the HBV polymerase. Through Golgi and ER
apparatus the core particles acquire the envelope and are secreted. Via viral entry and
retransporting of the newly synthesized HBV DNA into the cell nucleus, the cccDNA pool
can be amplified.
Experimental DHBV infection studies indicate that the cccDNA can be formed
not only from incoming virions, but also from newly synthesized nucleocapsids,
which instead of being enveloped and secreted into the blood, are rather transported
into the nucleus to ensure accumulation, and later maintenance, of the cccDNA pool
(Zoulim 2005b, Nassal 2008). According to this scenario, multiple rounds of
infection are not needed to establish a cccDNA pool in infected duck hepatocytes.
HBV Virology  73
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 synthesized rcDNA-containing nucleocapsids to envelopment and
extracellular secretion (Kock 2010). Although this peculiar nuclear reentry
mechanism has been clearly demonstrated for the duck HBV (Summers 1991,
Nassal 2008, Wu 1990) and a high copy number of cccDNA molecules is generally
detected in chronically infected ducks and woodchucks (1 to 50 copies/cell) (Zhang
2003, Dandri 2000), lower cccDNA intrahepatic loads are generally determined in
human liver biopsies obtained from chronically HBV -infected patients (median 0.1
to 1 cccDNA copy/cell) (Werle-Lapostolle 2004, Wong 2004, Laras 2006, Volz
2007, Wursthorn 2006, Lutgehetmann 2008) and in chronically HBV-infected
human-liver chimeric uPA/SCID mice (Petersen 2008, Lutgehetmann 2011a,
Lutgehetmann 2011b, Lutgehetmann 2010), suggesting that different viral and host
mechanisms may control cccDNA dynamics and cccDNA pool size in human
infected hepatocytes (Levrero 2009). A recent study elegantly showed that HBV
converted 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, recent studies performed in uPA/SCID mice indicate that this
step is achieved, initially, only in a minority of human hepatocytes. Indeed, three
weeks post-infection, the intrahepatic cccDNA load is very low (ca. 1 copy/50
human hepatocytes) and only sporadic cells stain HBcAg-positive, while within 8
weeks the majority of human hepatocytes become infected. Thus, several weeks
appear to be necessary for HBV to spread among human hepatocytes in vivo, even
in the absence of adaptive immune responses (Dandri 2011).
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 (Moraleda 1997, Dandri 2000, Dandri 2005,
Lutgehetmann 2010). Thus, the significant decrease in cccDNA levels
(approximately 1 log10 reduction) generally determined after 1 year of therapy with
polymerase inhibitors (Werle-Lapostolle 2004) is imagined 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 viremia,
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 2005a,
Zoulim 2005b, 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
74  Hepatology 2012
other hepatocytes and, eventually may even replace the wild-type cccDNA
molecules in the liver (Zoulim 2006, Zoulim 2009).
During chronic HBV infection immune-mediated cell injury and compensatory
hepatocyte proliferation may favour cccDNA decline and selection of cccDNA-free
cells (Mason 2005, Zhang 2003, Thermet 2008). Notably, studies with the duck
model show that antiviral therapy with polymerase inhibitors induce a greater
cccDNA reduction in animals displaying higher hepatocyte proliferation rates
(Addison 2002). cccDNA decrease was also determined in chronically WHV-infected woodchuck hepatocytes when cell turnover was induced  in vitro  by
addition of cellular growth factors and viral replication was suppressed by adefovir
treatment (Dandri 2000). Furthermore, the identification of uninfected cccDNA-negative cell clones containing “traces” of the infection in form of viral integrations
indicate that cccDNA clearance without cell destruction can occur in chronically
infected woodchucks  (Mason 2005). Thus, in chronic infection, killing of
hepatocytes may be instrumental not only to eliminate infected cells but also to
induce hepatocyte proliferation which, in turn, may favour cccDNA loss (Dandri
2005, Lutgehetmann 2010). 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 (Bock 1994, Bock 2001, Newbold
1995), which is organized as a minichromosome by histone and non-histone
proteins. 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).
Congruent with the fact that HBV infects hepatocytes, nearly all elements regulating
viral transcription have binding sites for liver-specific transcription factors (Levrero
2009, Quasdorff 2008). Nevertheless, although a number of factors regulating viral
transcription are known, the exact molecular mechanisms regulating HBV
transcription are still poorly defined. Both messenger and pregenomic RNAs 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. 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.
The next crucial step in HBV replication is the specific packaging of pgRNA, plus
the reverse transcriptase, into newly forming 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 loops at the 5’ end are first recognized by the viral polymerase and act 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 (~15–18 nucleotides which serve as primer for plus-strand DNA synthesis),
resulting in rcDNA. The heterogeneous lengths of the plus-strand DNAs generated