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10.1093/biohorizons/hzn020
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Review
The role of hepatitis C virus in the pathogenesis
of hepatocellular carcinoma
Gui Tran*
Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK.
* Corresponding author: 19 Cambridge Street, Derby DE23 8HG, UK. Tel: þ44 7793531066. Email address: [email protected]
Supervisor: Professor Mark Harris, University of Leeds, Leeds, UK.
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Hepatitis C virus (HCV) is a worldwide pandemic, chronically affecting over 170 million people worldwide. It is a major risk factor for the
development of hepatocellular carcinoma (HCC) and there is increasing experimental evidence to suggest that the virus plays a direct
role in neoplastic transformation. The purpose of this letter is to review the literature regarding two individual proteins of HCV, namely
NS5A and core, and their role in the pathogenesis of HCC through perturbations of cellular pathways, in addition to their immunopathological effects of chronic inflammation. A systematic search of MEDLINE in addition to manual searches of citations in key papers was
employed to identify relevant studies. There is overwhelming evidence suggesting the direct and indirect roles HCV plays in the pathogenesis of HCC. Recent progress in our understanding of the pathophysiology of HCV coupled with advances in in vitro models will
ensure that positive strides are made in the treatment and management of this potentially fatal virus.
Key words: Hepatitis C, hepatocellular carcinoma, core, NS5A.
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Introduction
Hepatitis C virus (HCV) was identified in 19891 and is now
considered to be endemic worldwide, globally affecting at
least 170 million people or 3% of the population.2 HCV is
primarily spread parenterally and its mode of transmission
includes mother to baby, blood –blood transmission and
sexual transmission. The clinical picture of HCV infection
is often an acute stage, followed by chronic infection in
80% of people initially infected. Those in the acute stage
may display mild flu-like symptoms, but are often asymptomatic. Those with chronic HCV infection may progress to
cirrhosis of the liver and finally hepatocellular carcinoma
(HCC). Current estimates suggest 2– 5% of patients with
liver cirrhosis due to HCV develop HCC annually.3
Molecular biology
HCV is an enveloped virus with a positive-sense RNA
genome of 9.6 kb. It is a member of the Flaviviridae
family, of the genus Hepacivirus.4 The viral genome
encodes for a single polyprotein that is subsequently
cleaved into 10 mature proteins, with the structural proteins
located near the 50 end of the polyprotein and the nonstructural proteins located near the 30 end (Figure 1).
The envelope glycoproteins E1 and E2 are integral for cell
recognition and binding. E1 is important in viral fusion with
the target cell membrane. The core protein undergoes two
cleavages to yield the mature protein. It forms the nucleocapsid, interacts with the glycoproteins E1 and E2 to form the
envelope and can also affect the translational ability of the
internal ribosome entry site. p7 is thought to be instrumental
in the production of infectious viruses and may act as a viroporin, which is able to alter the permeability of the
membrane.
NS2 and NS3 autocatalytically cleaves to release another
serine proteinase situated at the N region of the NS3. NS3
then proceeds to cleave the rest of the non-structural proteins
when complexed with its co-factor NS4A to release NS4B,
NS5A and NS5B.5 The C terminal end of NS3 also serves
as an RNA helicase to unwind double-stranded RNA
during replication.
NS4B is involved in the replication complex of HCV by
forming a membranous web which harbours the structural
and non-structural proteins and provides the structural
.........................................................................................................................................................................................................................................
#
2008 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License
(http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any
167
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Figure 1. The structure of the HCV genome.
support for the HCV replication complex. The function of
NS5A is thought to be involved in interactions with the cell
pathways and is a component in the replication complex,
although its real function is unknown, and NS5B acts as the
RNA-dependent RNA polymerase. All these non-structural
proteins form a membranous complex through which RNA
is replicated along the perinuclear membrane of the endoplasmic reticulum. Space constraints preclude a detailed analysis
of the molecular biology of HCV and more detailed reviews
can be found elsewhere in the literature.5
Cell culture systems and replication
models for HCV
Until only recently, there has been a lack of a robust and
reliable replication model enabling the HCV life cycle and
its pathogenic role to be easily characterized. A detrimental
consequence of this is that research into HCV has been difficult. There are many cell culture models which support the in
vitro replication of HCV but an in-depth description of these
lies beyond the scope of this project. Therefore, in order to
provide a framework whereby the limitations of the studies
in the reviewed literature can be realized, a very brief
outline of experimental models will be described. More
detailed reviews may be found elsewhere in the literature.6, 7
Animal models used for the study of HCV have been
limited by the narrow host range of the virus. Of these, chimpanzees are considered the only robust animal model. As
with all animal models, infection is usually variable and
transient, and susceptibility is low.8 In vitro models such
as Huh7 and human hepatoblastoma cells (HepG2) tend to
produce a low efficiency of HCV replication.9 Recently, the
use of selected subgenomic HCV RNAs, known as replicons,
has yielded promising results, with continuous autonomously replicating HCV RNAs in Huh7 cells, easily detected
using northern and western blotting.10
However, in order to enhance replication, adaptive
mutations in these replicons have occurred, which are
seldom seen in nature. In addition, these mutations have
led to a reduced infectivity in chimpanzees.11 Also, few cell
lines are able to support the efficient replication of the
replicons, with Huh7 being the most common. The subgenomic replicons lack structural protein-encoding genes, so
these proteins cannot be studied. Although, genomic replicons have been created, viral assembly and release have not
been demonstrated.
HCV, steatosis and fibrosis
Studies involving the use of transgenic mice have demonstrated that HCV and its proteins have the ability to induce
fibrosis, either through direct interference with cell activation
pathways or by indirect means, via the induction of steatosis.3, 12
Fibrosis results from the deposition of extra-cellular
matrix (ECM) material around the liver parenchyma.
Stellate cells play an important role in liver fibrogenesis,
depositing large amounts of ECM as a result of inflammation
or hepatocyte damage. Other cells of the immune system
such as Kuppffer cells can generate reactive oxygen species
(ROS), which stimulate stellate cell activity. ROS are produced naturally as a result of aerobic respiration. ROS, as
well as having oncogenic properties through DNA damage,
induces the production of TGFb, which has been shown to
have potent fibrogenic effects.13
Certain genotypes of HCV, in particular genotype 3, have
been shown to cause an accumulation of lipids within hepatocytes, a pathology known as steatosis. Steatosis may
then go on to cause the progression of fibrosis. Research
has shown that HCV genotype 3 is directly involved in
causing steatosis, supported by the fact that treatment of genotype 3 causes disappearance of steatosis.14
Core and NS5A have been hypothesized to be the HCV
proteins involved in the formation of steatosis and may
also be directly involved in liver fibrosis. Core has been
demonstrated to decrease the activity of the microsomal triglyceride transfer protein (MTP). This protein is involved
in the transfer of lipids within cells and its inhibition
would reduce the formation of VLDLs as well as the
secretions of ApoB and triglycerides (TG). This would then
lead to accumulation and build-up of TGs in hepatocytes.15
The process by which this occurs is unknown, but one
hypothesis indicates that HCV core could bind to lipids
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which would then prevent its interaction of MTP with its
target lipids. HCV core has also been demonstrated to bind
to apo AII (a component of HDL), and this binding may
help in the formation of its capsid.16 This binding prevents
core from reducing VLDL levels but it causes secretions of
the naked core into the blood stream, which may then play
a role in immunological evasion.
Core may induce liver damage as it interacts with retinoid
X receptor alpha, which is highly expressed in the liver and is
normally involved in gene regulation and lipid metabolism.
This interaction upregulates genes involved in lipid metabolism, although the mechanisms of how this occurs are
unknown.17 HCV core has also been shown to induce oxidative stress in murine models through lipid peroxidation,
that is to say, the degradation of lipids by oxidation.18 This
is a result of its ability to induce the generation of ROS in
the absence of any inflammation which is also implicated
in ROS production. The importance of the absence of
inflammation suggests that it is core itself which is responsible for ROS generation.
Perhaps, the chronic inflammatory process of HCV infection would lead to increased mutagenesis in the regenerating
hepatocytes, leading to a multi-step process of mutations
finally presenting as HCC. However, in auto-immune hepatitis, the occurrence of HCC is extremely rare, raising
doubts as to whether inflammation alone is able to lead to
such a high incidence of HCC in patients infected with
HCV. This coupled with experimental evidence demonstrating the role of HCV in the development of HCC19 has implicated the direct role of HCV in HCC.
HCV and hepatocellular carcinoma
The pathogenesis of HCC from HCV infection is yet to be
fully understood, with various viral protein–host cell interactions hypothesized to play a direct role in the development
of HCC. Perturbations in the cell cycle, combined with upregulation of oncogenes and loss of tumour-suppressor gene
functions, may combine to lead to HCC development;
HCV proteins have been shown to interact with these cellular
pathways. The natural history of HCV infection is progression to fibrosis and cirrhosis, leading to HCC in a significant proportion of the infected population. These viral
protein–host cell interactions may play a role separate
from cirrhosis in the development of HCC but they also
play an integral role in the cause of chronic infection,
leading to fibrosis and cirrhosis.
HCV core
HCV core has been shown to perturb and alter cell cycle
growth at various stages, resulting in deregulation of
mitosis (Figure 2). One study demonstrated that core is
involved in interaction with the double-stranded
RNA-activated protein kinase, PKR, which is involved in
many cellular processes such as apoptosis and cell
growth.20 PKR is activated by IFN and phosphorylates the
eukaryotic initiation factor, eIF2a, limiting protein synthesis
and so inhibiting cell and viral growth. PKR has also been
shown to be involved in mediating several forms of
stress-induced apoptosis.21 PKR also phosphorylates and
deactivates IkB (the cognate NF-kB inhibitor). As well as
mediating inflammation, NF-kB protects against apoptosis.
Core may mediate G2/M phase arrest via a p53independent manner.20 It has been shown that core induces
the phosphorylation of PKR at threonine 446, which
would alter PKR activity on different substrates as well as
prevent apoptosis. In a previous study, mutations of PKR
at residue 446 in mice have been shown to cause
tumours.22 However, the results and conclusion of the
study were limited with regard to the method employed
and what is currently known about PKR pathways. Alisi
et al. 20 used hepatoma cells expressing core protein only
but the disadvantage of this method is that core protein is
not found independently in the cell during natural infections.
Indeed, NS5A may interact with PKR23 and so may interfere
with core. It would be interesting to see whether core and
NS5A compete or complement each other with regard to
PKR, if either of them influences PKR at all. Also, it has
been suggested that core interferes with the PKR pathway
to impair apoptosis, but no known pathways have yet been
identified by which this may occur. Therefore, the conclusions drawn are as yet limited.
The functional consequences of the interaction of HCV
with p53 and p73 (a related tumour-suppressor protein)
has yet to be fully elucidated, but considering that cancer is
a multi-step process that requires perturbations in negative
and positive regulators of cell growth, and given the prominent role that p53/p73 has in cell proliferation, these pathways are thought to play influential roles in the
pathogenesis of HCC.
The core protein is able to bind to p53, a key component
in cell cycle arrest and apoptosis. By allowing HCV to modulate the cell cycle, it can either prevent apoptosis and allow
the replication of its progeny or induce apoptosis to enable
viral spread. The disruption of checkpoints in various
stages of the cell cycle is one way that tumourigenesis can
occur. Core may enhance p53 function by increasing the affinity of p53 to its DNA-binding site or by increasing its transcriptional activity without increasing p53 expression
itself.24 However, although a small proportion of core is
found in the nucleus, core is predominantly cytoplasmic5
and p53 is located within the nucleus, which may suggest
that p53 enhancement may not be fully explained by these
mechanisms. A separate study has suggested that core inhibits p53, but this was performed using core protein only
and did not employ an appropriate control, as the endogenous p53 levels were not measured.25
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Figure 2. The complex nature of HCV core interactions with host cellular processes is demonstrated here. Red arrows indicate inhibition, black arrows
indicate activation and purple arrows show activation or repression. Each group of pathways is colour-coded to allow easier visualization of interactions.
Core has also been shown to interact with the activity of a
target gene of p53, p21WAF1/CIP1. This protein normally
translocates to the nucleus and inhibits cyclin-dependent
kinase (CDK) complexes, resulting in cells failing to exit
the G1 phase of their life cycle.26 Otsuka et al. 24 demonstrated that p21WAF1/CIP1 activity is increased by p53 activation but Ray et al. 27 concluded that p21WAF1/CIP1
activity is repressed by a p53-independent pathway. These
differences may be reconciled by another study whereby
p21WAF1/CIP1 activation depends on the form taken by
core, i.e. either the innate or mature form.28 Full-length
core (191 amino acids long) found in the cytoplasm increases
the expression of p21WAF1/CIP1 by activating p53, whereas
mature core found in the nucleus decreases p21WAF1/CIP1.
Subcellular localization of the mature form is regulated by
the innate form. Therefore, the core protein function with
regard to p21WAF1/CIP1 activation is dependent on its localization within the cell and this must be considered when investigating core—something not considered in previous studies.
A recent study by Fukutomi et al. 29 has been able to
demonstrate the ability of core to increase cell proliferation,
using both transient transfection of the core protein and a
full-length HCV replicon in Huh7 and Huh7.5 cells, respectively. Microarray analysis also revealed 372 genes (out of
12 500 analysed) to be transcriptionally affected by core.
In particular, most oncogenes were upregulated in contrast
to most tumour-suppressor genes which were downregulated. One of the genes found to be upregulated, wnt-1, is
implicated in the oncogenic process, especially in HCC.4
Although this study demonstrated these abilities of core, the
mechanism by how they occur is yet to be identified. Indeed, the
complex nature of cell-signalling pathways coupled with
the multi-step process of hepatocarcinogenesis implies that a
combination of sequential gene up/downregulation is required
for HCC to occur. Therefore, the fact that these genes are
affected does not necessarily imply how, if at all, they are
involved in tumourigenesis. This study, however, does
provide firm evidence of the ability of core to alter cellular
pathways.
Core is involved in a multitude of host cell pathways, and
space constraints prevent a detailed discussion of each
pathway. For other postulated interactions of core with
host cell pathways and their relevant references, please
refer to Table 1.
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Table 1. Postulated interactions of core with host cellular pathways
Host cellular protein with which core
interacts
Host cellular protein function
Reference
PKR
Apoptosis and cell growth
20
p53 and p73
Tumour-suppressor protein
24
........................................................................................................................................................................................................................................
p21
WAF1/CIP1
TNFa
Prevents exit of G1 phase of cell cycle
28
Apoptosis
43, 44
NFkB
Anti-apoptotic, chemoattractant for immune cells
44, 45
LZIP
Tumour suppressor
46
hnRNP K
Stimulating the promoter of the oncogene c-myc; inhibiting the thymidine kinase promoter, which
47
is important for G1/S transition
14-3-31
Ras/Raf/MAPK pathway
48
Bcl-xL
Anti-apoptotic
49
Bax
Pro-apoptotic
50
TGF-b
Cell cycle arrest in the G1 phase; fibrogenesis; limiting the antiviral immune response
51
Cyclin E
Cell proliferation
52
MAPK pathway
Cell proliferation
53
Lymphotoxin b receptor
Cell differentiation
54
Fas pathway
Apoptosis
60
Figure 3. The complexities of NS5A interactions with relevant host cellular pathways. Black arrows indicate activation, red arrows show repression and
purple arrows show either activation or repression.
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Table 2. Postulated interactions of NS5A with host cellular pathways
Host cellular protein with
which NS5A may interact
Host cellular protein
function
Reference
PKR
Apoptosis and cell growth
23, 32
Growth factor receptor-bound
Involvement in cell
protein 2 (Grb2)
growth, differentiation and
Interleukin 8
Polymorph chemotaxis
................................................................................................................
55
transformation
56
and degranulation;
inhibiting IFNa
PI3K
Anti-apoptotic
57
GSK-3b
Proto-oncogene
57
P53
Apoptosis and suppress
34
oncogenesis
p21WAF1/CIP1
Prevents exit of G1 phase
37
of cell cycle
TNFa
Apoptotic protein
58
Human vesicle-associated
Vesicle transport
59
Bad
Proto-apoptotic protein
57
Bax
Apoptotic protein
50
membrane protein-associated
protein (hVAPA)
NS5A
The role of NS5A in cell transformation, differentiation and
oncogenesis has been intensely studied and many cellsignalling pathways have been implicated. Indeed, the
importance of the role of NS5A in HCC can be highlighted
from a study where nude mice expressing NS5A all developed tumours.30 NS5A has been shown to interact with a
multitude of proteins, but space constraints preclude the discussion of all of these. For an overview of NS5A interactions
mentioned in what follows, please refer to Figure 3 and
Table 2.
NS5A may cause the activation of NF-kB via a PKR
pathway, leading to inflammation. It has been shown using
yeasts and mammalian cell systems solely expressing NS5A
that NS5A has a direct role in its ability to inhibit PKR.23
It has been suggested that a region within NS5A, known as
the interferon sensitivity-determining region (ISDR), binds
to PKR preventing it from dimerizing. This then disrupts
PKR function and prevents phosphorylation of eIF2.
However, in a study involving cell lines expressing all the
structural and non-structural HCV proteins, it was demonstrated that NS5A had no significant effect on PKR. The
co-expression of other proteins may have localized NS5A
to organelle membranes, thus reducing its potential to bind
to PKR, as PKR is normally found throughout the cytoplasm.31 This provides an adequate explanation of the
incongruent results between the two studies and demonstrates the importance of effective in vitro cell culture
systems in correctly reflecting natural infection: by expressing
a single HCV protein,23 an incorrect interpretation on the
importance of NS5A and its ability to bind to PKR may
have been realized.
A separate in vitro study using Huh7 cell lines harbouring
HCV replicons32 seems to demonstrate NS5A does inhibit
PKR pathway, supporting the initial data.23 However,
mutations within the NS5A-coding region in order to
enhance replication of HCV in vitro may not necessarily
have reflected the true course of natural HCV infection.
Indeed, these mutations altered the subcellular localization
of the protein, which may impact on its interaction with
PKR. Molecular epidemiological studies have further indicated the interaction of NS5A with PKR, where mutations
within the ISDR correlated to response to IFNa treatment.33
This controversy surrounding the link between PKR and
NS5A has demonstrated perfectly how, up until recently, a
lack of both robust cell culture models and efficient HCV
replication systems have impeded research in this subject,
leading to conflicting results.
The direct binding and cytoplasmic co-localization of p53
as well as interaction of NS5A with the p53 co-activator
hTAFII32, a key component of TFIID has also been
shown.34 Cytoplasmic sequestration of p53 could lead to
the transcriptional repression of p53 target genes in a dosedependent manner, and NS5A binding to hTAFII32 through
an FXXff (where X is any residue and f a hydrophobic
residue) motif may also play a vital role in abrogating the function of p53. NS5A may bind to the N-terminus of p53, which
is a common activation site for other proteins such MDM2.35
This finding has been supported by another study, where p53
binding led to a reduction in p21WAF1 and consequently
increased cell growth.36 However, in this latter study, it was
suggested that binding to p53 is mediated by the N-terminal
150 residues of NS5A, whereas Lan et al. 34 suggest it is
mediated by the C-terminal 285 residues of NS5A. One
possibility is that NS5A may bind to p53 via both the N and
C terminus. However, the mechanism by which NS5A
causes p53 cytoplasmic sequestration remains unknown. All
these studies used the ectopic expression of NS5A which
may not reflect natural infection, both in terms of NS5A
expression levels and the potential cell interactions during
virus morphogenesis.
In contrast to these two studies demonstrating the ability
of NS5A to reduce p21WAF1 levels, Arima et al. 37 showed
that p21WAF1 may be increased as a result of NS5A interacting with Cdk1/2 cyclin complexes. It has been suggested that
these conflicting results may have arisen due to the use of differing non-hepatic cell lines.38 Another explanation may be
that NS5A may act on different parts of the cell pathway
that influence p21WAF1, although the reasons as to why
remain unknown.
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Conclusion
Acknowledgements
Since the discovery of HCV nearly 20 years ago, great strides
have been made in understanding the epidemiology, molecular biology and pathological nature of the virus. There is now
an overwhelming weight of evidence suggesting direct and
indirect roles of HCV in the pathogenesis of HCC, stemming
from both in vitro and in vivo studies. HCV is able to induce
immunopathological effects, generate ROS and cause steatosis, fibrosis and cirrhosis. It is also able to exert direct oncogenic effects on the host cell through perturbations in cell
signalling, secretory pathways and the cell cycle. However,
many of the mechanisms of selected repression or induction
of specific genes and pathways remain vague and difficult to
elucidate.
The major obstacle to research in HCV has been a lack of
a robust, reliable and efficient cell culture system. However,
recently, a subgenomic replicon was derived from a viral
isolate termed Japanese fulminant hepatitis 1 (JFH-1), a
variant of HCV genotype 2a obtained from the serum of a
Japanese patient. This replicon did not harbour any adaptive
mutations yet retained its replicative efficiency and
infectivity.39 Recent studies have subsequently led to the
development of a full-length JFH-1 genome40 as well as chimeric full-length HCV genomes41, 42 demonstrating efficient
production of infectious HCV. New in vitro systems involving the use of JFH-1 will enable the study of the complete
HCV life cycle as well as HCV interactions within the
context of the whole genome.
One limitation of previous studies has been the individual
expression (even potential over-expression) of single proteins,
which do not reflect natural HCV infection. This problem has
been further amplified by the variable use of truncated proteins among the different experiments, as well as the use of
differing genotypes and subtypes. This may go part of the
way to explaining the conflicting results of many studies.
However, the past two decades have not been without
progress in our understanding of HCV. Core and NS5A
have all been shown to interact with a multitude of proteins,
impacting on cellular pathways which may contribute to
tumourigenesis.
What does the future hold for HCV? Recent advances in
cell culture models combined with a growing understanding
of the pathological processes involved with HCV infection
indicate a very positive stride towards effective treatment
and management. Indeed, the ultimate objective to our
understanding of HCV is to enhance and progress current
antiviral therapies and potentially develop a vaccine against
this worldwide pandemic. Only by first understanding
HCV and its pathological role in the development of HCC
can effective treatment be developed and delivered. Given
the progress so far and the advent in new in vitro techniques
to accelerate research, there is much hope for this in the not
too distant future.
I would like to thank Professor Mark Harris for his invaluable advice and guidance, and Dr John Heritage and
Professor Richard Killington for their support.
Funding
This work was carried out whilst in receipt of scholarships
from the Yorkshire Cancer Research Campaign and the
McCleod Award, both of which are gratefully
acknowledged.
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Submitted on 29 September 2007; accepted on 5 March 2008; advance access
publication 22 April 2008
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