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The role of hepatitis C virus in the pathogenesis of hepatocellular carcinoma

The role of hepatitis C virus in the pathogenesis of hepatocellular carcinoma Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn020 ......................................................................................................................................................................................................................................... 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: guitran@gmail.com Supervisor: Professor Mark Harris, University of Leeds, Leeds, UK. ........................................................................................................................................................................................................................................ 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 immunopatho- logical 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 patho- genesis 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. ........................................................................................................................................................................................................................................ Introduction 1 0 Hepatitis C virus (HCV) was identified in 1989 and is now located near the 5 end of the polyprotein and the non- considered to be endemic worldwide, globally affecting at structural proteins located near the 3 end (Figure 1). least 170 million people or 3% of the population. HCV is The envelope glycoproteins E1 and E2 are integral for cell primarily spread parenterally and its mode of transmission recognition and binding. E1 is important in viral fusion with includes mother to baby, blood–blood transmission and the target cell membrane. The core protein undergoes two sexual transmission. The clinical picture of HCV infection cleavages to yield the mature protein. It forms the nucleocap- is often an acute stage, followed by chronic infection in sid, interacts with the glycoproteins E1 and E2 to form the 80% of people initially infected. Those in the acute stage envelope and can also affect the translational ability of the may display mild flu-like symptoms, but are often asympto- internal ribosome entry site. p7 is thought to be instrumental matic. Those with chronic HCV infection may progress to in the production of infectious viruses and may act as a vir- cirrhosis of the liver and finally hepatocellular carcinoma oporin, which is able to alter the permeability of the (HCC). Current estimates suggest 2–5% of patients with membrane. liver cirrhosis due to HCV develop HCC annually. 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, Molecular biology NS5A and NS5B. The C terminal end of NS3 also serves as an RNA helicase to unwind double-stranded RNA HCV is an enveloped virus with a positive-sense RNA during replication. genome of 9.6 kb. It is a member of the Flaviviridae NS4B is involved in the replication complex of HCV by family, of the genus Hepacivirus. The viral genome forming a membranous web which harbours the structural encodes for a single polyprotein that is subsequently and non-structural proteins and provides the structural cleaved into 10 mature proteins, with the structural proteins ......................................................................................................................................................................................................................................... 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 medium, provided the original work is properly cited. 167 Review Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Figure 1. The structure of the HCV genome. support for the HCV replication complex. The function of replicons, with Huh7 being the most common. The subge- NS5A is thought to be involved in interactions with the cell nomic replicons lack structural protein-encoding genes, so pathways and is a component in the replication complex, these proteins cannot be studied. Although, genomic repli- although its real function is unknown, and NS5B acts as the cons have been created, viral assembly and release have not RNA-dependent RNA polymerase. All these non-structural been demonstrated. proteins form a membranous complex through which RNA is replicated along the perinuclear membrane of the endoplas- HCV, steatosis and fibrosis mic reticulum. Space constraints preclude a detailed analysis of the molecular biology of HCV and more detailed reviews Studies involving the use of transgenic mice have demon- can be found elsewhere in the literature. strated 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 steato- 3, 12 sis. Cell culture systems and replication Fibrosis results from the deposition of extra-cellular models for HCV matrix (ECM) material around the liver parenchyma. Until only recently, there has been a lack of a robust and Stellate cells play an important role in liver fibrogenesis, reliable replication model enabling the HCV life cycle and depositing large amounts of ECM as a result of inflammation its pathogenic role to be easily characterized. A detrimental or hepatocyte damage. Other cells of the immune system consequence of this is that research into HCV has been diffi- such as Kuppffer cells can generate reactive oxygen species cult. There are many cell culture models which support the in (ROS), which stimulate stellate cell activity. ROS are pro- vitro replication of HCV but an in-depth description of these duced naturally as a result of aerobic respiration. ROS, as lies beyond the scope of this project. Therefore, in order to well as having oncogenic properties through DNA damage, provide a framework whereby the limitations of the studies induces the production of TGFb, which has been shown to in the reviewed literature can be realized, a very brief have potent fibrogenic effects. outline of experimental models will be described. More Certain genotypes of HCV, in particular genotype 3, have 6, 7 detailed reviews may be found elsewhere in the literature. been shown to cause an accumulation of lipids within hep- Animal models used for the study of HCV have been atocytes, a pathology known as steatosis. Steatosis may limited by the narrow host range of the virus. Of these, chim- then go on to cause the progression of fibrosis. Research panzees are considered the only robust animal model. As has shown that HCV genotype 3 is directly involved in with all animal models, infection is usually variable and causing steatosis, supported by the fact that treatment of gen- 8 14 transient, and susceptibility is low. In vitro models such otype 3 causes disappearance of steatosis. as Huh7 and human hepatoblastoma cells (HepG2) tend to Core and NS5A have been hypothesized to be the HCV produce a low efficiency of HCV replication. Recently, the proteins involved in the formation of steatosis and may use of selected subgenomic HCV RNAs, known as replicons, also be directly involved in liver fibrosis. Core has been has yielded promising results, with continuous autono- demonstrated to decrease the activity of the microsomal tri- mously replicating HCV RNAs in Huh7 cells, easily detected glyceride transfer protein (MTP). This protein is involved using northern and western blotting. in the transfer of lipids within cells and its inhibition However, in order to enhance replication, adaptive would reduce the formation of VLDLs as well as the mutations in these replicons have occurred, which are secretions of ApoB and triglycerides (TG). This would then seldom seen in nature. In addition, these mutations have lead to accumulation and build-up of TGs in hepatocytes. led to a reduced infectivity in chimpanzees. Also, few cell The process by which this occurs is unknown, but one lines are able to support the efficient replication of the hypothesis indicates that HCV core could bind to lipids ......................................................................................................................................................................................................................................... 168 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Review ......................................................................................................................................................................................................................................... which would then prevent its interaction of MTP with its RNA-activated protein kinase, PKR, which is involved in target lipids. HCV core has also been demonstrated to bind many cellular processes such as apoptosis and cell to apo AII (a component of HDL), and this binding may growth. PKR is activated by IFN and phosphorylates the help in the formation of its capsid. This binding prevents eukaryotic initiation factor, eIF2a, limiting protein synthesis core from reducing VLDL levels but it causes secretions of and so inhibiting cell and viral growth. PKR has also been the naked core into the blood stream, which may then play shown to be involved in mediating several forms of a role in immunological evasion. stress-induced apoptosis. PKR also phosphorylates and Core may induce liver damage as it interacts with retinoid deactivates IkB (the cognate NF-kB inhibitor). As well as X receptor alpha, which is highly expressed in the liver and is mediating inflammation, NF-kB protects against apoptosis. normally involved in gene regulation and lipid metabolism. Core may mediate G2/M phase arrest via a p53- This interaction upregulates genes involved in lipid metab- independent manner. It has been shown that core induces olism, although the mechanisms of how this occurs are the phosphorylation of PKR at threonine 446, which unknown. HCV core has also been shown to induce oxi- would alter PKR activity on different substrates as well as dative stress in murine models through lipid peroxidation, prevent apoptosis. In a previous study, mutations of PKR that is to say, the degradation of lipids by oxidation. This at residue 446 in mice have been shown to cause is a result of its ability to induce the generation of ROS in tumours. However, the results and conclusion of the the absence of any inflammation which is also implicated study were limited with regard to the method employed in ROS production. The importance of the absence of and what is currently known about PKR pathways. Alisi inflammation suggests that it is core itself which is respon- et al. used hepatoma cells expressing core protein only sible for ROS generation. but the disadvantage of this method is that core protein is Perhaps, the chronic inflammatory process of HCV infec- not found independently in the cell during natural infections. tion would lead to increased mutagenesis in the regenerating Indeed, NS5A may interact with PKR and so may interfere hepatocytes, leading to a multi-step process of mutations with core. It would be interesting to see whether core and finally presenting as HCC. However, in auto-immune hepa- NS5A compete or complement each other with regard to titis, the occurrence of HCC is extremely rare, raising PKR, if either of them influences PKR at all. Also, it has doubts as to whether inflammation alone is able to lead to been suggested that core interferes with the PKR pathway such a high incidence of HCC in patients infected with to impair apoptosis, but no known pathways have yet been HCV. This coupled with experimental evidence demonstrat- identified by which this may occur. Therefore, the con- ing the role of HCV in the development of HCC has impli- clusions drawn are as yet limited. cated the direct role of HCV in HCC. 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 HCV and hepatocellular carcinoma a multi-step process that requires perturbations in negative The pathogenesis of HCC from HCV infection is yet to be and positive regulators of cell growth, and given the promi- fully understood, with various viral protein–host cell inter- nent role that p53/p73 has in cell proliferation, these path- actions hypothesized to play a direct role in the development ways are thought to play influential roles in the of HCC. Perturbations in the cell cycle, combined with upre- pathogenesis of HCC. gulation of oncogenes and loss of tumour-suppressor gene The core protein is able to bind to p53, a key component functions, may combine to lead to HCC development; in cell cycle arrest and apoptosis. By allowing HCV to modu- HCV proteins have been shown to interact with these cellular late the cell cycle, it can either prevent apoptosis and allow pathways. The natural history of HCV infection is pro- the replication of its progeny or induce apoptosis to enable gression to fibrosis and cirrhosis, leading to HCC in a signifi- viral spread. The disruption of checkpoints in various cant proportion of the infected population. These viral stages of the cell cycle is one way that tumourigenesis can protein–host cell interactions may play a role separate occur. Core may enhance p53 function by increasing the affi- from cirrhosis in the development of HCC but they also nity of p53 to its DNA-binding site or by increasing its tran- play an integral role in the cause of chronic infection, scriptional activity without increasing p53 expression leading to fibrosis and cirrhosis. itself. However, although a small proportion of core is found in the nucleus, core is predominantly cytoplasmic and p53 is located within the nucleus, which may suggest HCV core that p53 enhancement may not be fully explained by these HCV core has been shown to perturb and alter cell cycle mechanisms. A separate study has suggested that core inhi- growth at various stages, resulting in deregulation of bits p53, but this was performed using core protein only mitosis (Figure 2). One study demonstrated that core is and did not employ an appropriate control, as the endogen- involved in interaction with the double-stranded ous p53 levels were not measured. ......................................................................................................................................................................................................................................... 169 Review Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... 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 full-length HCV replicon in Huh7 and Huh7.5 cells, respect- WAF1/CIP1 target gene of p53, p21 . This protein normally ively. Microarray analysis also revealed 372 genes (out of translocates to the nucleus and inhibits cyclin-dependent 12 500 analysed) to be transcriptionally affected by core. kinase (CDK) complexes, resulting in cells failing to exit In particular, most oncogenes were upregulated in contrast 26 24 the G1 phase of their life cycle. Otsuka et al. demon- to most tumour-suppressor genes which were downregu- WAF1/CIP1 strated that p21 activity is increased by p53 acti- lated. One of the genes found to be upregulated, wnt-1, is 27 WAF1/CIP1 4 vation but Ray et al. concluded that p21 implicated in the oncogenic process, especially in HCC. activity is repressed by a p53-independent pathway. These Although this study demonstrated these abilities of core, the differences may be reconciled by another study whereby mechanism by how theyoccur isyet to be identified. Indeed, the WAF1/CIP1 p21 activation depends on the form taken by complex nature of cell-signalling pathways coupled with core, i.e. either the innate or mature form. Full-length the multi-step process of hepatocarcinogenesis implies that a core (191 amino acids long) found in the cytoplasm increases combination of sequential gene up/downregulation is required WAF1/CIP1 the expression of p21 by activating p53, whereas for HCC to occur. Therefore, the fact that these genes are WAF1/CIP1 mature core found in the nucleus decreases p21 . affected does not necessarily imply how, if at all, they are Subcellular localization of the mature form is regulated by involved in tumourigenesis. This study, however, does the innate form. Therefore, the core protein function with provide firm evidence of the ability of core to alter cellular WAF1/CIP1 regard to p21 activation is dependent on its localiz- pathways. ation within the cell and this must be considered when inves- Core is involved in a multitude of host cell pathways, and tigating core—something not considered in previous studies. space constraints prevent a detailed discussion of each A recent study by Fukutomi et al. has been able to pathway. For other postulated interactions of core with demonstrate the ability of core to increase cell proliferation, host cell pathways and their relevant references, please using both transient transfection of the core protein and a refer to Table 1. ......................................................................................................................................................................................................................................... 170 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Review ......................................................................................................................................................................................................................................... Table 1. Postulated interactions of core with host cellular pathways Host cellular protein with which core Host cellular protein function Reference interacts ........................................................................................................................................................................................................................................ PKR Apoptosis and cell growth 20 p53 and p73 Tumour-suppressor protein 24 WAF1/CIP1 p21 Prevents exit of G1 phase of cell cycle 28 TNFa 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-x 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. ......................................................................................................................................................................................................................................... 171 Review Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Table 2. Postulated interactions of NS5A with host cellular pathways incongruent results between the two studies and demon- strates the importance of effective in vitro cell culture Host cellular protein with Host cellular protein Reference systems in correctly reflecting natural infection: by expressing which NS5A may interact function ................................................................................................................ a single HCV protein, an incorrect interpretation on the PKR Apoptosis and cell growth 23, 32 importance of NS5A and its ability to bind to PKR may Growth factor receptor-bound Involvement in cell 55 have been realized. protein 2 (Grb2) growth, differentiation and A separate in vitro study using Huh7 cell lines harbouring HCV replicons seems to demonstrate NS5A does inhibit transformation PKR pathway, supporting the initial data. However, Interleukin 8 Polymorph chemotaxis 56 mutations within the NS5A-coding region in order to and degranulation; enhance replication of HCV in vitro may not necessarily inhibiting IFNa have reflected the true course of natural HCV infection. PI3K Anti-apoptotic 57 Indeed, these mutations altered the subcellular localization GSK-3b Proto-oncogene 57 of the protein, which may impact on its interaction with P53 Apoptosis and suppress 34 PKR. Molecular epidemiological studies have further indi- oncogenesis cated the interaction of NS5A with PKR, where mutations WAF1/CIP1 p21 Prevents exit of G1 phase 37 within the ISDR correlated to response to IFNa treatment. of cell cycle This controversy surrounding the link between PKR and TNFa Apoptotic protein 58 NS5A has demonstrated perfectly how, up until recently, a Human vesicle-associated Vesicle transport 59 lack of both robust cell culture models and efficient HCV replication systems have impeded research in this subject, membrane protein-associated leading to conflicting results. protein (hVAPA) The direct binding and cytoplasmic co-localization of p53 Bad Proto-apoptotic protein 57 as well as interaction of NS5A with the p53 co-activator Bax Apoptotic protein 50 hTAF 32, a key component of TFIID has also been II shown. Cytoplasmic sequestration of p53 could lead to the transcriptional repression of p53 target genes in a dose- dependent manner, and NS5A binding to hTAF 32 through II NS5A an FXXff (where X is any residue and f a hydrophobic The role of NS5A in cell transformation, differentiation and residue) motif may also play a vital role in abrogating the func- oncogenesis has been intensely studied and many cell- tion of p53. NS5A may bind to the N-terminus of p53, which signalling pathways have been implicated. Indeed, the is a common activation site for other proteins such MDM2. importance of the role of NS5A in HCC can be highlighted This finding has been supported by another study, where p53 WAF1 from a study where nude mice expressing NS5A all devel- binding led to a reduction in p21 and consequently oped tumours. NS5A has been shown to interact with a increased cell growth. However, in this latter study, it was multitude of proteins, but space constraints preclude the dis- suggested that binding to p53 is mediated by the N-terminal cussion of all of these. For an overview of NS5A interactions 150 residues of NS5A, whereas Lan et al. suggest it is mentioned in what follows, please refer to Figure 3 and mediated by the C-terminal 285 residues of NS5A. One Table 2. possibility is that NS5A may bind to p53 via both the N and NS5A may cause the activation of NF-kB via a PKR C terminus. However, the mechanism by which NS5A pathway, leading to inflammation. It has been shown using causes p53 cytoplasmic sequestration remains unknown. All yeasts and mammalian cell systems solely expressing NS5A these studies used the ectopic expression of NS5A which that NS5A has a direct role in its ability to inhibit PKR. may not reflect natural infection, both in terms of NS5A It has been suggested that a region within NS5A, known as expression levels and the potential cell interactions during the interferon sensitivity-determining region (ISDR), binds virus morphogenesis. to PKR preventing it from dimerizing. This then disrupts In contrast to these two studies demonstrating the ability WAF1 37 PKR function and prevents phosphorylation of eIF2. of NS5A to reduce p21 levels, Arima et al. showed WAF1 However, in a study involving cell lines expressing all the that p21 may be increased as a result of NS5A interact- structural and non-structural HCV proteins, it was demon- ing with Cdk1/2 cyclin complexes. It has been suggested that strated that NS5A had no significant effect on PKR. The these conflicting results may have arisen due to the use of dif- co-expression of other proteins may have localized NS5A fering non-hepatic cell lines. Another explanation may be to organelle membranes, thus reducing its potential to bind that NS5A may act on different parts of the cell pathway WAF1 to PKR, as PKR is normally found throughout the cyto- that influence p21 , although the reasons as to why plasm. This provides an adequate explanation of the remain unknown. ......................................................................................................................................................................................................................................... 172 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Review ......................................................................................................................................................................................................................................... Conclusion Acknowledgements Since the discovery of HCV nearly 20 years ago, great strides I would like to thank Professor Mark Harris for his invalu- have been made in understanding the epidemiology, molecu- able advice and guidance, and Dr John Heritage and lar biology and pathological nature of the virus. There is now Professor Richard Killington for their support. 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 Funding immunopathological effects, generate ROS and cause steato- sis, fibrosis and cirrhosis. It is also able to exert direct onco- This work was carried out whilst in receipt of scholarships genic effects on the host cell through perturbations in cell from the Yorkshire Cancer Research Campaign and the signalling, secretory pathways and the cell cycle. However, McCleod Award, both of which are gratefully many of the mechanisms of selected repression or induction acknowledged. 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, References recently, a subgenomic replicon was derived from a viral 1. 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Submitted on 29 September 2007; accepted on 5 March 2008; advance access publication 22 April 2008 ......................................................................................................................................................................................................................................... http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bioscience Horizons Oxford University Press

The role of hepatitis C virus in the pathogenesis of hepatocellular carcinoma

Tran, Gui
Bioscience Horizons , Volume 1 (2) – Jun 22, 2008
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Volume 1 † Number 2 † June 2008 10.1093/biohorizons/hzn020 ......................................................................................................................................................................................................................................... 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: guitran@gmail.com Supervisor: Professor Mark Harris, University of Leeds, Leeds, UK. ........................................................................................................................................................................................................................................ 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 immunopatho- logical 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 patho- genesis 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. ........................................................................................................................................................................................................................................ Introduction 1 0 Hepatitis C virus (HCV) was identified in 1989 and is now located near the 5 end of the polyprotein and the non- considered to be endemic worldwide, globally affecting at structural proteins located near the 3 end (Figure 1). least 170 million people or 3% of the population. HCV is The envelope glycoproteins E1 and E2 are integral for cell primarily spread parenterally and its mode of transmission recognition and binding. E1 is important in viral fusion with includes mother to baby, blood–blood transmission and the target cell membrane. The core protein undergoes two sexual transmission. The clinical picture of HCV infection cleavages to yield the mature protein. It forms the nucleocap- is often an acute stage, followed by chronic infection in sid, interacts with the glycoproteins E1 and E2 to form the 80% of people initially infected. Those in the acute stage envelope and can also affect the translational ability of the may display mild flu-like symptoms, but are often asympto- internal ribosome entry site. p7 is thought to be instrumental matic. Those with chronic HCV infection may progress to in the production of infectious viruses and may act as a vir- cirrhosis of the liver and finally hepatocellular carcinoma oporin, which is able to alter the permeability of the (HCC). Current estimates suggest 2–5% of patients with membrane. liver cirrhosis due to HCV develop HCC annually. 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, Molecular biology NS5A and NS5B. The C terminal end of NS3 also serves as an RNA helicase to unwind double-stranded RNA HCV is an enveloped virus with a positive-sense RNA during replication. genome of 9.6 kb. It is a member of the Flaviviridae NS4B is involved in the replication complex of HCV by family, of the genus Hepacivirus. The viral genome forming a membranous web which harbours the structural encodes for a single polyprotein that is subsequently and non-structural proteins and provides the structural cleaved into 10 mature proteins, with the structural proteins ......................................................................................................................................................................................................................................... 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 medium, provided the original work is properly cited. 167 Review Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Figure 1. The structure of the HCV genome. support for the HCV replication complex. The function of replicons, with Huh7 being the most common. The subge- NS5A is thought to be involved in interactions with the cell nomic replicons lack structural protein-encoding genes, so pathways and is a component in the replication complex, these proteins cannot be studied. Although, genomic repli- although its real function is unknown, and NS5B acts as the cons have been created, viral assembly and release have not RNA-dependent RNA polymerase. All these non-structural been demonstrated. proteins form a membranous complex through which RNA is replicated along the perinuclear membrane of the endoplas- HCV, steatosis and fibrosis mic reticulum. Space constraints preclude a detailed analysis of the molecular biology of HCV and more detailed reviews Studies involving the use of transgenic mice have demon- can be found elsewhere in the literature. strated 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 steato- 3, 12 sis. Cell culture systems and replication Fibrosis results from the deposition of extra-cellular models for HCV matrix (ECM) material around the liver parenchyma. Until only recently, there has been a lack of a robust and Stellate cells play an important role in liver fibrogenesis, reliable replication model enabling the HCV life cycle and depositing large amounts of ECM as a result of inflammation its pathogenic role to be easily characterized. A detrimental or hepatocyte damage. Other cells of the immune system consequence of this is that research into HCV has been diffi- such as Kuppffer cells can generate reactive oxygen species cult. There are many cell culture models which support the in (ROS), which stimulate stellate cell activity. ROS are pro- vitro replication of HCV but an in-depth description of these duced naturally as a result of aerobic respiration. ROS, as lies beyond the scope of this project. Therefore, in order to well as having oncogenic properties through DNA damage, provide a framework whereby the limitations of the studies induces the production of TGFb, which has been shown to in the reviewed literature can be realized, a very brief have potent fibrogenic effects. outline of experimental models will be described. More Certain genotypes of HCV, in particular genotype 3, have 6, 7 detailed reviews may be found elsewhere in the literature. been shown to cause an accumulation of lipids within hep- Animal models used for the study of HCV have been atocytes, a pathology known as steatosis. Steatosis may limited by the narrow host range of the virus. Of these, chim- then go on to cause the progression of fibrosis. Research panzees are considered the only robust animal model. As has shown that HCV genotype 3 is directly involved in with all animal models, infection is usually variable and causing steatosis, supported by the fact that treatment of gen- 8 14 transient, and susceptibility is low. In vitro models such otype 3 causes disappearance of steatosis. as Huh7 and human hepatoblastoma cells (HepG2) tend to Core and NS5A have been hypothesized to be the HCV produce a low efficiency of HCV replication. Recently, the proteins involved in the formation of steatosis and may use of selected subgenomic HCV RNAs, known as replicons, also be directly involved in liver fibrosis. Core has been has yielded promising results, with continuous autono- demonstrated to decrease the activity of the microsomal tri- mously replicating HCV RNAs in Huh7 cells, easily detected glyceride transfer protein (MTP). This protein is involved using northern and western blotting. in the transfer of lipids within cells and its inhibition However, in order to enhance replication, adaptive would reduce the formation of VLDLs as well as the mutations in these replicons have occurred, which are secretions of ApoB and triglycerides (TG). This would then seldom seen in nature. In addition, these mutations have lead to accumulation and build-up of TGs in hepatocytes. led to a reduced infectivity in chimpanzees. Also, few cell The process by which this occurs is unknown, but one lines are able to support the efficient replication of the hypothesis indicates that HCV core could bind to lipids ......................................................................................................................................................................................................................................... 168 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Review ......................................................................................................................................................................................................................................... which would then prevent its interaction of MTP with its RNA-activated protein kinase, PKR, which is involved in target lipids. HCV core has also been demonstrated to bind many cellular processes such as apoptosis and cell to apo AII (a component of HDL), and this binding may growth. PKR is activated by IFN and phosphorylates the help in the formation of its capsid. This binding prevents eukaryotic initiation factor, eIF2a, limiting protein synthesis core from reducing VLDL levels but it causes secretions of and so inhibiting cell and viral growth. PKR has also been the naked core into the blood stream, which may then play shown to be involved in mediating several forms of a role in immunological evasion. stress-induced apoptosis. PKR also phosphorylates and Core may induce liver damage as it interacts with retinoid deactivates IkB (the cognate NF-kB inhibitor). As well as X receptor alpha, which is highly expressed in the liver and is mediating inflammation, NF-kB protects against apoptosis. normally involved in gene regulation and lipid metabolism. Core may mediate G2/M phase arrest via a p53- This interaction upregulates genes involved in lipid metab- independent manner. It has been shown that core induces olism, although the mechanisms of how this occurs are the phosphorylation of PKR at threonine 446, which unknown. HCV core has also been shown to induce oxi- would alter PKR activity on different substrates as well as dative stress in murine models through lipid peroxidation, prevent apoptosis. In a previous study, mutations of PKR that is to say, the degradation of lipids by oxidation. This at residue 446 in mice have been shown to cause is a result of its ability to induce the generation of ROS in tumours. However, the results and conclusion of the the absence of any inflammation which is also implicated study were limited with regard to the method employed in ROS production. The importance of the absence of and what is currently known about PKR pathways. Alisi inflammation suggests that it is core itself which is respon- et al. used hepatoma cells expressing core protein only sible for ROS generation. but the disadvantage of this method is that core protein is Perhaps, the chronic inflammatory process of HCV infec- not found independently in the cell during natural infections. tion would lead to increased mutagenesis in the regenerating Indeed, NS5A may interact with PKR and so may interfere hepatocytes, leading to a multi-step process of mutations with core. It would be interesting to see whether core and finally presenting as HCC. However, in auto-immune hepa- NS5A compete or complement each other with regard to titis, the occurrence of HCC is extremely rare, raising PKR, if either of them influences PKR at all. Also, it has doubts as to whether inflammation alone is able to lead to been suggested that core interferes with the PKR pathway such a high incidence of HCC in patients infected with to impair apoptosis, but no known pathways have yet been HCV. This coupled with experimental evidence demonstrat- identified by which this may occur. Therefore, the con- ing the role of HCV in the development of HCC has impli- clusions drawn are as yet limited. cated the direct role of HCV in HCC. 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 HCV and hepatocellular carcinoma a multi-step process that requires perturbations in negative The pathogenesis of HCC from HCV infection is yet to be and positive regulators of cell growth, and given the promi- fully understood, with various viral protein–host cell inter- nent role that p53/p73 has in cell proliferation, these path- actions hypothesized to play a direct role in the development ways are thought to play influential roles in the of HCC. Perturbations in the cell cycle, combined with upre- pathogenesis of HCC. gulation of oncogenes and loss of tumour-suppressor gene The core protein is able to bind to p53, a key component functions, may combine to lead to HCC development; in cell cycle arrest and apoptosis. By allowing HCV to modu- HCV proteins have been shown to interact with these cellular late the cell cycle, it can either prevent apoptosis and allow pathways. The natural history of HCV infection is pro- the replication of its progeny or induce apoptosis to enable gression to fibrosis and cirrhosis, leading to HCC in a signifi- viral spread. The disruption of checkpoints in various cant proportion of the infected population. These viral stages of the cell cycle is one way that tumourigenesis can protein–host cell interactions may play a role separate occur. Core may enhance p53 function by increasing the affi- from cirrhosis in the development of HCC but they also nity of p53 to its DNA-binding site or by increasing its tran- play an integral role in the cause of chronic infection, scriptional activity without increasing p53 expression leading to fibrosis and cirrhosis. itself. However, although a small proportion of core is found in the nucleus, core is predominantly cytoplasmic and p53 is located within the nucleus, which may suggest HCV core that p53 enhancement may not be fully explained by these HCV core has been shown to perturb and alter cell cycle mechanisms. A separate study has suggested that core inhi- growth at various stages, resulting in deregulation of bits p53, but this was performed using core protein only mitosis (Figure 2). One study demonstrated that core is and did not employ an appropriate control, as the endogen- involved in interaction with the double-stranded ous p53 levels were not measured. ......................................................................................................................................................................................................................................... 169 Review Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... 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 full-length HCV replicon in Huh7 and Huh7.5 cells, respect- WAF1/CIP1 target gene of p53, p21 . This protein normally ively. Microarray analysis also revealed 372 genes (out of translocates to the nucleus and inhibits cyclin-dependent 12 500 analysed) to be transcriptionally affected by core. kinase (CDK) complexes, resulting in cells failing to exit In particular, most oncogenes were upregulated in contrast 26 24 the G1 phase of their life cycle. Otsuka et al. demon- to most tumour-suppressor genes which were downregu- WAF1/CIP1 strated that p21 activity is increased by p53 acti- lated. One of the genes found to be upregulated, wnt-1, is 27 WAF1/CIP1 4 vation but Ray et al. concluded that p21 implicated in the oncogenic process, especially in HCC. activity is repressed by a p53-independent pathway. These Although this study demonstrated these abilities of core, the differences may be reconciled by another study whereby mechanism by how theyoccur isyet to be identified. Indeed, the WAF1/CIP1 p21 activation depends on the form taken by complex nature of cell-signalling pathways coupled with core, i.e. either the innate or mature form. Full-length the multi-step process of hepatocarcinogenesis implies that a core (191 amino acids long) found in the cytoplasm increases combination of sequential gene up/downregulation is required WAF1/CIP1 the expression of p21 by activating p53, whereas for HCC to occur. Therefore, the fact that these genes are WAF1/CIP1 mature core found in the nucleus decreases p21 . affected does not necessarily imply how, if at all, they are Subcellular localization of the mature form is regulated by involved in tumourigenesis. This study, however, does the innate form. Therefore, the core protein function with provide firm evidence of the ability of core to alter cellular WAF1/CIP1 regard to p21 activation is dependent on its localiz- pathways. ation within the cell and this must be considered when inves- Core is involved in a multitude of host cell pathways, and tigating core—something not considered in previous studies. space constraints prevent a detailed discussion of each A recent study by Fukutomi et al. has been able to pathway. For other postulated interactions of core with demonstrate the ability of core to increase cell proliferation, host cell pathways and their relevant references, please using both transient transfection of the core protein and a refer to Table 1. ......................................................................................................................................................................................................................................... 170 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Review ......................................................................................................................................................................................................................................... Table 1. Postulated interactions of core with host cellular pathways Host cellular protein with which core Host cellular protein function Reference interacts ........................................................................................................................................................................................................................................ PKR Apoptosis and cell growth 20 p53 and p73 Tumour-suppressor protein 24 WAF1/CIP1 p21 Prevents exit of G1 phase of cell cycle 28 TNFa 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-x 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. ......................................................................................................................................................................................................................................... 171 Review Bioscience Horizons † Volume 1 † Number 2 † June 2008 ......................................................................................................................................................................................................................................... Table 2. Postulated interactions of NS5A with host cellular pathways incongruent results between the two studies and demon- strates the importance of effective in vitro cell culture Host cellular protein with Host cellular protein Reference systems in correctly reflecting natural infection: by expressing which NS5A may interact function ................................................................................................................ a single HCV protein, an incorrect interpretation on the PKR Apoptosis and cell growth 23, 32 importance of NS5A and its ability to bind to PKR may Growth factor receptor-bound Involvement in cell 55 have been realized. protein 2 (Grb2) growth, differentiation and A separate in vitro study using Huh7 cell lines harbouring HCV replicons seems to demonstrate NS5A does inhibit transformation PKR pathway, supporting the initial data. However, Interleukin 8 Polymorph chemotaxis 56 mutations within the NS5A-coding region in order to and degranulation; enhance replication of HCV in vitro may not necessarily inhibiting IFNa have reflected the true course of natural HCV infection. PI3K Anti-apoptotic 57 Indeed, these mutations altered the subcellular localization GSK-3b Proto-oncogene 57 of the protein, which may impact on its interaction with P53 Apoptosis and suppress 34 PKR. Molecular epidemiological studies have further indi- oncogenesis cated the interaction of NS5A with PKR, where mutations WAF1/CIP1 p21 Prevents exit of G1 phase 37 within the ISDR correlated to response to IFNa treatment. of cell cycle This controversy surrounding the link between PKR and TNFa Apoptotic protein 58 NS5A has demonstrated perfectly how, up until recently, a Human vesicle-associated Vesicle transport 59 lack of both robust cell culture models and efficient HCV replication systems have impeded research in this subject, membrane protein-associated leading to conflicting results. protein (hVAPA) The direct binding and cytoplasmic co-localization of p53 Bad Proto-apoptotic protein 57 as well as interaction of NS5A with the p53 co-activator Bax Apoptotic protein 50 hTAF 32, a key component of TFIID has also been II shown. Cytoplasmic sequestration of p53 could lead to the transcriptional repression of p53 target genes in a dose- dependent manner, and NS5A binding to hTAF 32 through II NS5A an FXXff (where X is any residue and f a hydrophobic The role of NS5A in cell transformation, differentiation and residue) motif may also play a vital role in abrogating the func- oncogenesis has been intensely studied and many cell- tion of p53. NS5A may bind to the N-terminus of p53, which signalling pathways have been implicated. Indeed, the is a common activation site for other proteins such MDM2. importance of the role of NS5A in HCC can be highlighted This finding has been supported by another study, where p53 WAF1 from a study where nude mice expressing NS5A all devel- binding led to a reduction in p21 and consequently oped tumours. NS5A has been shown to interact with a increased cell growth. However, in this latter study, it was multitude of proteins, but space constraints preclude the dis- suggested that binding to p53 is mediated by the N-terminal cussion of all of these. For an overview of NS5A interactions 150 residues of NS5A, whereas Lan et al. suggest it is mentioned in what follows, please refer to Figure 3 and mediated by the C-terminal 285 residues of NS5A. One Table 2. possibility is that NS5A may bind to p53 via both the N and NS5A may cause the activation of NF-kB via a PKR C terminus. However, the mechanism by which NS5A pathway, leading to inflammation. It has been shown using causes p53 cytoplasmic sequestration remains unknown. All yeasts and mammalian cell systems solely expressing NS5A these studies used the ectopic expression of NS5A which that NS5A has a direct role in its ability to inhibit PKR. may not reflect natural infection, both in terms of NS5A It has been suggested that a region within NS5A, known as expression levels and the potential cell interactions during the interferon sensitivity-determining region (ISDR), binds virus morphogenesis. to PKR preventing it from dimerizing. This then disrupts In contrast to these two studies demonstrating the ability WAF1 37 PKR function and prevents phosphorylation of eIF2. of NS5A to reduce p21 levels, Arima et al. showed WAF1 However, in a study involving cell lines expressing all the that p21 may be increased as a result of NS5A interact- structural and non-structural HCV proteins, it was demon- ing with Cdk1/2 cyclin complexes. It has been suggested that strated that NS5A had no significant effect on PKR. The these conflicting results may have arisen due to the use of dif- co-expression of other proteins may have localized NS5A fering non-hepatic cell lines. Another explanation may be to organelle membranes, thus reducing its potential to bind that NS5A may act on different parts of the cell pathway WAF1 to PKR, as PKR is normally found throughout the cyto- that influence p21 , although the reasons as to why plasm. This provides an adequate explanation of the remain unknown. ......................................................................................................................................................................................................................................... 172 Bioscience Horizons † Volume 1 † Number 2 † June 2008 Review ......................................................................................................................................................................................................................................... Conclusion Acknowledgements Since the discovery of HCV nearly 20 years ago, great strides I would like to thank Professor Mark Harris for his invalu- have been made in understanding the epidemiology, molecu- able advice and guidance, and Dr John Heritage and lar biology and pathological nature of the virus. There is now Professor Richard Killington for their support. 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 Funding immunopathological effects, generate ROS and cause steato- sis, fibrosis and cirrhosis. It is also able to exert direct onco- This work was carried out whilst in receipt of scholarships genic effects on the host cell through perturbations in cell from the Yorkshire Cancer Research Campaign and the signalling, secretory pathways and the cell cycle. However, McCleod Award, both of which are gratefully many of the mechanisms of selected repression or induction acknowledged. 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, References recently, a subgenomic replicon was derived from a viral 1. 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Submitted on 29 September 2007; accepted on 5 March 2008; advance access publication 22 April 2008 .........................................................................................................................................................................................................................................

Journal

Bioscience Horizons – Oxford University Press

Published: Jun 22, 2008

Keywords: Key words Hepatitis C hepatocellular carcinoma core NS5A

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