Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Correspondence
Mark Harris
mharris{at}bmb.leeds.ac.uk
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The genome of HCV is a 9·5 kb positive-sense RNA molecule that is translated via a cap-independent mechanism to generate a 3000 residue polyprotein, subsequently cleaved by a combination of host cell and viral proteases into structural and non-structural proteins. The NS5A protein is one of six non-structural proteins of HCV that are likely to form an RNA replication complex (Bartenschlager & Lohmann, 2000). Aside from this role, an increasing body of evidence suggests that NS5A may also interfere with host cell functions. Indeed, NS5A has been demonstrated to interact with and inhibit the IFN-induced kinase PKR (Gale et al., 1998
) and has also been shown to interact with the cell cycle regulatory machinery, promoting anchorage-independent growth in NIH3T3 murine fibroblasts and tumour formation in nude mice (Ghosh et al., 1999
); NS5A can repress transcription of the cell cycle repressor gene p21WAF1, while upregulating PCNA expression, effects that may be mediated by reported physical associations of NS5A with p53 (Majumder et al., 2001
) and a novel transcription factor SRCAP (Ghosh et al., 2000
).
Recently, it was shown that NS5A was capable of perturbing mitogenic signalling pathways by interacting with the adaptor protein Grb2 (Tan et al., 1999). This interaction was mapped to a highly conserved polyproline motif near the C terminus of NS5A, which was shown to bind to the SH3 domains of Grb2. SH3 domains are widely distributed among signalling proteins. They share a common structure but individual SH3 domains exhibit different binding specificities mediated both by amino acid sequences within the RT loop of the SH3 domain and sequences flanking the polyproline motifs of the binding protein (Mayer, 2001
). Two classes of polyproline motif are defined by the location of a conserved basic residue: in Class I motifs the basic residue is situated at the N terminus, whereas Class II motifs contain a C-terminal basic residue. The position of the basic residue dictates the binding orientation of the motif. One key group of SH3 domain-containing proteins is the Src family of non-receptor tyrosine kinases (Tatosyan et al., 2000
), nine proteins that share a common domain structure and mode of regulation. Interestingly, these kinases are a common target for virus interference with cell signalling, particularly in the case of viruses that establish chronic infections (Collette & Olive, 1997
). In this regard HCV has not been shown to interact with Src-family kinases and we set out to investigate whether the polyproline motifs in NS5A might mediate such an interaction.
Here we demonstrate that two closely spaced Class II polyproline motifs near the C terminus of NS5A were able to bind to isolated SH3 domains of members of the Src kinase family in vitro. Co-immunoprecipitation analysis coupled with in vitro kinase assays demonstrated that NS5A could interact with, and modulate the activity of, native Src-family kinases in vivo. Importantly, using Huh-7 derived cells harbouring subgenomic HCV replicons, we reveal that expression of NS5A in the context of the other non-structural proteins is able to differentially regulate the activity of endogenous Src-family kinases and perturb Src-family regulated signalling pathways. These data suggest that, in common with other viruses such as human immunodeficiency virus (HIV) and EpsteinBarr virus (EBV) that can establish persistent or chronic infections, HCV is able to interact with and modulate the activity of Src-family kinases.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Tissue culture.
Cos-7 cells were cultured in DMEM supplemented with 10 % fetal calf serum (FCS), 100 IU penicillin ml-1 and 100 µg streptomycin ml-1. Huh-7 cells were cultured as for Cos-7 cells with the addition of 1 % non-essential amino acids. The generation of Huh-7 cells harbouring an HCV replicon has been described elsewhere (Macdonald et al., 2003).
Immunoprecipitation and Western blotting.
Cells were transfected with pSG5 vectors expressing Src-family kinases alone or in conjunction with pSG5.NS5A vectors using Lipofectin (Invitrogen). After 24 h cells were lysed in modified RIPA buffer (150 mM NaCl, 50 mM Tris/HCl pH 8·0, 1 % NP-40, 0·1 % SDS, 5 mM EDTA, 1 mM Na3VO4, 1 mM NaF, 500 nM cantharidin, 500 nM calyculin and 10 % glycerol) and equal amounts of protein (0·5 mg) precipitated with anti Src-family kinase monoclonal antibodies and protein-G beads (Santa Cruz). Immunoprecipitates were washed three times in lysis buffer and resolved by SDS-PAGE. Western blotting was performed with a sheep polyclonal anti-NS5A antiserum or commercially available antibodies to Src-family kinases, phosphotyrosine (PY69), tyrosine-phosphorylated STAT3 (all Santa Cruz) or STAT3 (Upstate) using standard techniques. Immunoblots were visualized by using an enhanced chemiluminescence system.
Kinase assays.
To assay for Src-family kinase activity cells were lysed as above, and protein was precipitated by using appropriate monoclonal antibodies and protein-G beads. After three washes with lysis buffer and two with kinase buffer (20 mM HEPES/KOH pH 7·4, 10 mM MgCl2, 1 mM NaF, 1 mM Na3VO4, 500 nM cantharidin), immunoprecipitates were incubated with kinase buffer containing 2 µg acid-denatured enolase (Sigma) and 5 µCi [-32P]ATP for 15 min at 30 °C. The reactions were terminated by the addition of Laemmli loading buffer, boiled for 5 min at 90 °C and resolved by SDS-PAGE.
Expression and purification of GST fusion proteins.
Vectors for bacterial expression of the GSTSH3 fusion proteins were obtained from Kalle Saksela (University of Tampere, Finland) and used as described (Hiipakka et al., 1999). GSTGrb2 was obtained from John Ladbury (UCL, UK). Expression and purification of the GST fusion proteins were carried out by standard methods as described (Smith & Johnson, 1988
).
In vitro GSTSH3 binding assay.
GSTSH3 fusion proteins were bound to GA-beads at 4 °C for 1 h. Equal quantities of lysates from cells transiently transfected with the appropriate pSG5 vectors, or [35S]methionine-labelled in vitro transcription/translation reactions (Promega TNT) programmed with pSG5.NS5A(BVDV) were incubated with beads in cell lysis buffer. After 3 h of incubation, beads were washed extensively in lysis buffer. Proteins were analysed by SDS-PAGE followed by autoradiography or Western blotting using an NS5A-specific antibody. GST alone was used as a negative control.
Luciferase assay.
Cells were seeded into six-well dishes 1 day prior to transfection and subsequently transfected using Lipofectin (Invitrogen) as previously described (Macdonald et al., 2003). For Fyn co-transfections, the final concentration of DNA was kept constant with the addition of empty expression vector as appropriate. After transfection cells were incubated in reduced serum growth media (0·5 % FCS) at 37 °C overnight prior to analysis. Where appropriate cells were treated with 10 µM PD98059 or 0·5 µM PP2 (both from Calbiochem) 1 h prior to stimulation with growth media containing 10 % serum. Quantification of relative light units (RLU) was done as previously described (Macdonald et al., 2003
).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
NS5A interacts with and modulates Src-family kinase activity in vivo
These data clearly showed that NS5A was able to interact with the SH3 domains of Src-family kinases. In order to determine whether NS5A was also able to interact with the full-length, native kinases we co-transfected Cos-7 cells with pSG5 vectors expressing both Src-family kinases and either NS5A(wt) or defined proline motif mutants (PA2.2 or PA2.1/2.2). As the interaction with Hck, Lck or Fyn SH3 domains was abolished by the PA2.2 mutation we used the NS5A(PA2.2) mutant as a control in co-transfections with either Hck, Lck or Fyn. Src-family kinases were subsequently immunoprecipitated and their activity analysed by in vitro kinase assay, measuring both autophosphorylation and phosphorylation of the exogenous substrate, enolase. Immunoprecipitates were also analysed by Western blotting for NS5A to determine whether the two proteins could form a stable complex in vivo. The top two Western blots in each panel of Fig. 3 confirm that transfected cells expressed the appropriate Src-family kinase and NS5A. Panels (a) and (b) clearly show that the kinase activity of both Hck and Lck was inhibited by NS5A(wt) but unaffected by NS5A(PA2.2); the corresponding Western blots show that NS5A(wt) was able to bind to both Hck and Lck, but that NS5A(PA2.2) failed to bind to either kinase (although a very low level of binding was observed for Hck). In contrast, panel (c) shows that the activity of Fyn was stimulated by NS5A(wt) but unaffected by NS5A(PA2.2); again this modulation of kinase activity corresponded with the ability of NS5A(wt) to co-precipitate with Fyn. In agreement with the inability of the Src SH3 domain to bind to NS5A (Fig. 1
), NS5A did not co-precipitate with Src, and the presence of either NS5A(wt) or NS5A(PA2.2) had no effect on Src activity (panel d).
|
Taken together, these data demonstrate that NS5A is able to modulate the activity of Src-family kinases in vivo; this effect correlates with the ability of NS5A to bind to Src-family kinases in vivo. The correlation between the binding of NS5A to native Src-family kinases, and the binding to isolated SH3 domains, points to a critical role for polyproline:SH3 domain binding in mediating these interactions.
HCV replicon cells exhibit differential activity of endogenous Fyn and Lyn in response to mitogenic stimulation
To further address the physiological significance of the NS5A:Src-family kinase interaction, we employed the Huh-7 pFK-I389neo/NS3-3'/5.1 cell clone (Krieger et al., 2001) as a model system. These cells carry a selectable self-replicating culture-adapted subgenomic HCV RNA and express the NS5A protein in the context of the non-structural polyprotein. Preliminary experiments had shown that Huh7 cells express both Fyn and Lyn, in contrast, we were unable to detect significant levels of either Hck or Lck in these cells (data not shown); thus we focussed our analysis on Fyn and Lyn. Cells expressing the HCV replicon (FK5.1) or a control Huh-7 cell line stably expressing neomycin phosphotransferase (Huh-7neo) were grown in reduced serum growth medium to suppress mitogenic signalling pathways, and then stimulated with mitogens (EGF, insulin and PMA). Lysates were immunoprecipitated with antibodies to either Fyn or Lyn and subjected to either in vitro kinase assays (Fig. 4b, e
) or Western blotting for Fyn (Fig. 4a
), Lyn (Fig. 4d
) or NS5A (Fig. 4c, f
). The addition of mitogens stimulated the autophosphorylation and activation of both kinases. In FK5.1 cells, EGF- or insulin-stimulated Fyn activity was significantly higher than in control Huh-7neo cells, consistent with the transient transfection data in Fig. 3(c)
. In contrast EGF- or insulin-stimulated Lyn activity was reduced in FK5.1 cells, again consistent with the data in Fig. 3(e)
. However, for both Fyn and Lyn, PMA treatment appeared to partially override any replicon specific modulation of kinase activity (lanes 7 and 8). Importantly, Fig. 4(c, f)
also shows that NS5A forms a stable complex with both Fyn and Lyn in FK5.1 cells, confirming the data in Fig. 3(c, e)
. These data demonstrate that in a physiologically relevant context NS5A both interacts with and differentially modulates Src-family kinases.
|
To test this hypothesis Huh-7neo and FK5.1 cells were transfected with a -casein promoter luciferase reporter construct (responsive to STAT3) (Berchtold et al., 1997
) and treated with inhibitors of Src-family kinases (PP2) or the RasERK pathway (PD98059). As shown in Fig. 5
(a), in comparison to Huh-7neo, FK5.1 cells exhibited a fivefold elevation in STAT3 activity (bars 1 and 2), in both cell lines this activity was inhibited by PP2 (bars 3 and 4) but unaffected by PD98059 (bars 5 and 6), suggesting that STAT3 activity in Huh7 cells is regulated by Src kinases but not by the RasERK pathway. Further proof of this was shown by co-transfection with pSG5-Fyn, which resulted in a significant increase in STAT3 activity. In control Huh-7neo cells the addition of exogenous Fyn resulted in a sevenfold increase in STAT3 activity (compare bars 1 and 7); however, in FK5.1 cells, this increase was only twofold (compare bars 2 and 8), consistent with the higher basal STAT3 activity observed in FK5.1 cells. Again, the increase in STAT3 activation following Fyn transfection was partially inhibited by PP2 (bars 9 and 10), but unaffected by PD98059 (data not shown). These data were supported by Western blot analysis of STAT3 tyrosine phosphorylation (Fig. 5b
), showing that levels of STAT3 phosphorylation correlate very closely with transcriptional activity. Overall expression (Fig. 5c
) and serine phosphorylation of STAT3 (data not shown) were unaltered. To confirm that the activity of Fyn also correlated with STAT3 activity we analysed the autophosphorylation of Fyn as previously shown in this paper (Figs 3 and 4
) and by others (e.g. Briggs et al., 2000
) this correlates with kinase activity. Fyn was therefore immunoprecipitated and analysed by Western blot using a phosphotyrosine-specific antibody (PY69). This analysis confirmed that levels of Fyn autophosphorylation (Fig. 5d
) correlated with both the activity and tyrosine phosphorylation of STAT3. These data are thus consistent with the hypothesis that NS5A binds to and activates Fyn, thereby stimulating Fyn-mediated tyrosine phosphorylation of STAT3.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our data confirm that a class II polyproline motif near the C terminus (PP2.2) mediates Grb2 binding and show that this motif also interacts with the SH3 domains of Hck, Lck, Fyn and Lyn. Intriguingly, the Lyn SH3 domain was also able to bind to a second Class II polyproline motif at the C terminus of NS5A (PP2.1). The lack of conservation of the PP2.1 motif (it is only completely conserved in isolates of genotype 1) suggests that it may not be functionally significant; conversely the absolute conservation of the PP2.2 motif points to a role at some stage in virus replication.
Our data demonstrate that NS5A is not only able to interact with native Src-family kinases in cell culture, but also to differentially modulate their activity in the case of Hck, Lck and Lyn activity was inhibited, whereas the activity of Fyn was stimulated. This situation is reminiscent of two other viral proteins that interact with Src-family kinases: the HIV-1 Nef protein and the murine polyomavirus middle-T antigen. Nef binds to the SH3 domains of Hck, Lck, Fyn, Lyn and Src; it activates Hck, suppresses Fyn and has little effect on the other three kinases (Briggs et al., 2000). Middle-T binds to the catalytic domains of Src, Yes and Fyn; binding to Src and Yes activates kinase activity whereas middle-T has no effect on Fyn (Ichaso et al., 2001
). Regulation of Src-family kinase activity is mediated by a number of factors including intramolecular interactions involving the SH2/SH3 domains. It is conceivable that there are subtle differences in the regulation of individual Src-family kinases that are exploited by NS5A to effect differential modulation; alternatively, there may be other intermolecular interactions between NS5A and Src-family kinases that contribute to the observed effects in this regard it is pertinent that Hck exhibited some binding to the NS5A(PA2.2) mutant, suggesting that other determinants may contribute to binding.
A key question that remains to be answered is: what are the physiological consequences of NS5ASrc-family interactions for virus replication and pathogenesis? Although providing evidence for this is hampered by the absence of a robust in vitro replication system, as a first stage in answering this question we analysed both kinase activity and signalling pathways regulated by Src-family kinases in Huh-7 cells harbouring a subgenomic replicon. Activity of both Fyn and Lyn was dysregulated in these cells in comparison to control cells, consistent with data from transient transfections.
Lastly, our data demonstrate that NS5A-mediated activation of both endogenous and exogenous Fyn in replicon cells results in the activation of the transcription factor STAT3. This observation sheds light on the mechanism of STAT3 activation by NS5A reported by other groups (Gong et al., 2001), as it has been shown that STAT3 is a substrate for multiple Src-family kinases, including Fyn. Increasingly hepatocellular carcinoma (HCC) is associated with HCV infection (Zoulim et al., 2003
) and in this regard it is of interest that constitutive STAT3 activation is associated with many tumours including HCC. Usually these tumours exhibit dysregulation of STAT3 activity (Yoshikawa et al., 2001
), and we speculate therefore that indirect activation of STAT3 by NS5A might contribute to the development of HCC in HCV-infected individuals.
Clearly, given that NS5A both interacts with and modulates the activity of Fyn, the two proteins must co-localize within replicon cells. Although we do not have any direct evidence for co-localization, it is pertinent that many studies have demonstrated that NS5A associates predominantly with the cytosolic face of the ER membrane (Brass et al., 2002). Fyn has also been reported to associate with, and phosphorylate, the ER membrane-located IP3-receptor (Jayaraman et al., 1996
), providing indirect evidence for co-localization of NS5A and Fyn.
In conclusion, Src-family kinases regulate a multitude of signalling pathways, including the arrangement of the actin cytoskeleton, the response to growth factor stimulation and, importantly, the response to cytokine stimulation. Although we have identified a specific effect of NS5A on a signalling pathway controlled by Src-family kinases, the differential modulation of individual Src-family kinases by NS5A is likely to have complex effects on the final response of infected cells to a variety of signalling events. Thus it may be possible that by varying the activity of each individual kinase, the role of NS5A is to subvert and re-route the response of the cell to best suit HCV replication. Further speculation must await the results of further experimentation in this area.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berchtold, S., Moriggl, R., Gouilleux, F., Silvennoinen, O., Beisenherz, C., Pfitzner, E., Wissler, M., Stocklin, E. & Groner, B. (1997). Cytokine receptor-independent, constitutively active variants of STAT5. J Biol Chem 272, 3023730243.
Brass, V., Bieck, E., Montserret, R., Wolk, B., Hellings, J. A., Blum, H. E., Penin, F. & Moradpour, D. (2002). An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J Biol Chem 277, 81308139.
Briggs, S. D., Lerner, E. C. & Smithgall, T. E. (2000). Affinity of Src family kinase SH3 domains for HIV Nef in vitro does not predict kinase activation by Nef in vivo. Biochemistry 39, 489495.[CrossRef][Medline]
Collette, Y. & Olive, D. (1997). Non-receptor protein tyrosine kinases as immune targets of viruses. Immunol Today 18, 393400.[CrossRef][Medline]
Gale, M. J., Korth, M. J. & Katze, M. G. (1998). Repression of the PKR protein kinase by the hepatitis C virus NS5A protein: a potential mechanism of interferon resistance. Clin Diagn Virol 10, 157162.[CrossRef][Medline]
Ghosh, A. K., Steele, R., Meyer, K., Ray, R. & Ray, R. B. (1999). Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. J Gen Virol 80, 11791183.[Abstract]
Ghosh, A. K., Majumder, M., Steele, R., Yaciuk, P., Chrivia, J., Ray, R. & Ray, R. B. (2000). Hepatitis C virus NS5A protein modulates transcription through a novel cellular transcription factor SRCAP. J Biol Chem 275, 71847188.
Gong, G. Z., Waris, G., Tanveer, R. & Siddiqui, A. (2001). Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-B. Proc Natl Acad Sci U S A 98, 95999604.
Green, S., Issemann, I. & Sheer, E. (1988). A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Res 16, 369.[Medline]
Higuchi, R. (1992). Using PCR to engineer DNA. In PCR Technology. Principles and Applications. Edited by H. A. Erlich. New York: Freeman.
Hiipakka, M., Poikonen, K. & Saksela, K. (1999). SH3 domains with high affinity and engineered ligand specificities targeted to HIV-1 Nef. J Mol Biol 293, 10971106.[CrossRef][Medline]
Ichaso, N. & Dilworth, S. M. (2001). Cell transformation by the middle T-antigen of polyoma virus. Oncogene 20, 79087916.[CrossRef][Medline]
Jayaraman, T., Ondrias, K., Ondriasova, E. & Marks, A. R. (1996). Regulation of the inositol 1,4,5-trisphosphate receptor by tyrosine phosphorylation. Science 272, 14921494.[Abstract]
Krieger, N., Lohmann, V. & Bartenschlager, R. (2001). Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75, 46144624.
Lavanchy, D. & 31 other authors (1999). Global surveillance and control of hepatitis C. J Viral Hepat 6, 3547.[CrossRef][Medline]
Macdonald, A., Crowder, K., Street, A., McCormick, C., Saksela, K. & Harris, M. (2003). The hepatitis C virus NS5A protein inhibits activating protein-1 (AP1) function by perturbing Ras-ERK pathway signalling. J Biol Chem 278, 1777517784.
Majumder, M., Ghosh, A. K., Steele, R., Ray, R. & Ray, R. B. (2001). Hepatitis C virus NS5A physically associates with p53 and regulates p21/waf1 gene expression in a p53-dependent manner. J Virol 75, 14011407.
Mayer, B. J. (2001). SH3 domains: complexity in moderation. J Cell Sci 114, 12531263.
Meyers, G., Tautz, N., Becher, P., Thiel, H. J. & Kummerer, B. M. (1996). Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs. J Virol 70, 86068613.[Abstract]
Miller, C. L., Burkhardt, A. L., Lee, J. H., Stealey, B., Longnecker, R., Bolen, J. B. & Kieff, E. (1995). Integral membrane-protein 2 of EpsteinBarr virus regulates reactivation from latency through dominant-negative effects on protein-tyrosine kinases. Immunity 2, 155166.[Medline]
Saksela, K., Cheng, G. & Baltimore, D. (1995). Proline-rich (PxxP) motifs in HIV-1 Nef bind to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+ viruses but not for down-regulation of CD4. EMBO J 14, 484491.[Abstract]
Schreiner, S. J., Schiavone, A. P. & Smithgall, T. E. (2002). Activation of STAT3 by the Src family kinase Hck requires a functional SH3 domain. J Biol Chem 277, 4568045687.
Smith, D. B. & Johnson, K. S. (1988). Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-S-transferase. Gene 67, 3140.[CrossRef][Medline]
Tan, S. L., Nakao, H., He, Y. P., Vijaysri, S., Neddermann, P., Jacobs, B. L., Mayer, B. J. & Katze, M. G. (1999). NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling. Proc Natl Acad Sci U S A 96, 55335538.
Tatosyan, A. G. & Mizenina, O. A. (2000). Kinases of the Src family: structure and functions. Biochemistry (Mosc) 65, 4958.[Medline]
Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. (1997). Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc Natl Acad Sci U S A 94, 87388743.
Yanagi, M., St Claire, M., Shapiro, M., Emerson, S. U., Purcell, R. H. & Bukh, J. (1998). Transcripts of a chimeric cDNA clone of hepatitis C virus genotype 1b are infectious in vivo. Virology 244, 161172.[CrossRef][Medline]
Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. (1999). Hepatitis C virus: an infectious molecular clone of a second major genotype (2a) and lack of viability of intertypic 1a and 2a chimeras. Virology 262, 250263.[CrossRef][Medline]
Yoshikawa, H., Matsubara, K., Qian, G. S., Jackson, P., Groopman, J. D., Manning, J. E., Harris, C. C. & Herman, J. G. (2001). SOCS-1, a negative regulator of the JAK/STAT pathway, is silenced by methylation in human hepatocellular carcinoma and shows growth-suppression activity. Nat Genet 28, 2935.[CrossRef][Medline]
Zoulim, F., Chevallier, M., Maynard, M. & Trepo, C. (2003). Clinical consequences of hepatitis C virus infection. Rev Med Virol 13, 5768.[CrossRef][Medline]
Received 2 October 2003;
accepted 4 November 2003.