Binding of Hepatitis C Virus Envelope Protein E2 to CD81 Up-regulates Matrix Metalloproteinase-2 in Human Hepatic Stellate Cells

Abstract

The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage. The hepatitis C virus (HCV) envelope E2 glycoprotein is a key molecule regulating the interaction of HCV with cell surface proteins. E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell types including hepatocytes and B lymphocytes. Regardless, information on the biological functions originating from this interaction are largely unknown. Since human hepatic stellate cells (HSC) express high levels of CD81 at the cell surface, we investigated the E2/CD81 interaction in human HSC and the possible effects arising from this interaction. Matrix metalloproteinase-2 (MMP-2; gelatinase A), a major enzyme involved in the degradation of normal hepatic extracellular matrix, was up-regulated following the interaction between E2 and CD81. In particular, by employing zymography and Western blot, we observed that E2 binding to CD81 induces a time-dependent increase in the synthesis and activity of MMP-2. This effect was abolished by preincubating HSC with an anti-CD81 neutralizing antibody. Similar effects were detected in NIH3T3 mouse fibroblasts transfected with human CD81 with identical time course features. In addition, E2/CD81 interaction in human HSC induced the up-regulation of MMP-2 by increasing activator protein-2/DNA binding activity via ERK/MAPK phosphorylation. Finally, suppression of CD81 by RNA interference in human HSC abolished the described effects of E2 on these cells, indicating that CD81 is essential for the activation of the signaling pathway leading to the up-regulation of MMP-2. These results suggest that HSC may represent a potential target for HCV. The interaction of HCV envelope with CD81 on the surface of human HSC induces an increased expression of MMP-2. Increased degradation of the normal hepatic extracellular matrix in areas where HCV is concentrated may favor inflammatory infiltration and further parenchymal damage. Hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated kinase; HSC, hepatic stellate cell(s); MMPs, matrix metalloproteinases; PI, phosphatidylinositol; AP-2, activator protein-2; SFIF, serum-free/insulin-free; PMSF, phenylmethylsulfonyl fluoride; TIMP, tissue inhibitor of metalloproteinase; mAb, monoclonal antibody; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; siRNA, small interfering RNA; MT1, membrane type 1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.1The abbreviations used are: HCV, hepatitis C virus; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated kinase; HSC, hepatic stellate cell(s); MMPs, matrix metalloproteinases; PI, phosphatidylinositol; AP-2, activator protein-2; SFIF, serum-free/insulin-free; PMSF, phenylmethylsulfonyl fluoride; TIMP, tissue inhibitor of metalloproteinase; mAb, monoclonal antibody; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; BSA, bovine serum albumin; siRNA, small interfering RNA; MT1, membrane type 1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. is the most common cause of chronic liver disease, leading to hepatic fibrosis and ultimately to cirrhosis (1Lauer G.M. Walker B.D. N. Engl. J. Med. 2001; 345: 41-52Crossref PubMed Scopus (2464) Google Scholar). The HCV major envelope E2 glycoprotein exposed on the surface of virions is likely to be involved in the interactions with the host and has been identified as responsible for binding of HCV to target cells (2Pileri P. Uematsu Y. Campagnoli S. Galli G. Falugi F. Petracca R. Weiner A.J. Houghton M. Rosa D. Grandi G. Abrignani S. Science. 1998; 282: 938-941Crossref PubMed Scopus (1776) Google Scholar). Truncated, secreted versions of E2 glycoprotein have been utilized as soluble mimics of viral particles in order to study virus-cell interactions (3Rosa D. Campagnoli S. Moretto C. Guenzi E. Cousens L. Chin M. Dong C. Weiner A.J. Lau J.Y. Choo Q.L. Chien D. Pileri P. Houghton M. Abrignani S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1759-1763Crossref PubMed Scopus (321) Google Scholar, 4Heile J.M. Fong Y.L. Rosa D. Berge R.K. Saletti G. Campagnoli S. Bensi G. Capo S. Coates S. Crawford K. Dong C. Wininger M. Bake R.G. Cousens L. Chien D. Ng P. Archangel P. Grandi G. Houghton M. Abrignani S. J. Virol. 2000; 74: 6885-6892Crossref PubMed Scopus (66) Google Scholar). In particular, E2 glycoprotein has been reported to bind to the major loop of human transmembrane molecule CD81, a member of tetraspanin protein superfamily, expressed on various cell types including hepatocytes and B lymphocytes, and, accordingly, CD81 has been proposed as a putative receptor for HCV (2Pileri P. Uematsu Y. Campagnoli S. Galli G. Falugi F. Petracca R. Weiner A.J. Houghton M. Rosa D. Grandi G. Abrignani S. Science. 1998; 282: 938-941Crossref PubMed Scopus (1776) Google Scholar). The most striking feature of CD81, as well as other tetraspanin molecules, is a propensity to associate with a wide variety of membrane proteins, acting as a molecular facilitator for downstream intracellular signaling (5Maecker H.T. Todd S.C. Levy S. FASEB J. 1997; 11: 428-442Crossref PubMed Scopus (801) Google Scholar). The wide range of complexes into which CD81 assembles suggests that the association of CD81 with different partners may have important and diverse effects on different cell types (6Levy S. Todd S.C. Maecker H.T. Annu. Rev. Immunol. 1998; 16: 89-109Crossref PubMed Scopus (435) Google Scholar). Accordingly, binding of E2 glycoprotein to CD81 may be relevant for explaining the development and the persistence of HCV infection in the liver as well as in extrahepatic tissues (7Flint M. McKeating J.A. Rev. Med. Virol. 2000; 10: 101-117Crossref PubMed Scopus (71) Google Scholar). study in on of normal human liver that CD81 is expressed at the cell surface of as well as HCV E2 binds different cell types in liver tissue with different and this binding is by of liver tissue with anti-CD81 R. Falugi F. Galli G. N. Rosa D. Campagnoli S. E. Houghton M. Abrignani S. Grandi G. J. Virol. 2000; 74: PubMed Scopus Google Scholar). In addition, we have reported that human HSC, and key of hepatic express high levels of CD81 protein at the cell surface A. S. C. P. A. P. M. J. PubMed Scopus Google Scholar). essential in the of hepatic is the degradation of normal liver extracellular matrix by increased expression of as matrix These are involved in the of liver as well as chronic hepatic and J. 2000; PubMed Google and may to the inflammatory and to D. J. Immunol. 1996; Google Scholar, K. Y. S. M. A. 1997; PubMed Scopus Google including viral Y. G. J. C. J. Virol. 2000; 74: PubMed Scopus Google Scholar). The activity of MMPs, secreted by tissue cells as is by and tissue J. PubMed Scopus Google Scholar). MMP-2 as gelatinase the most relevant involved in of normal liver hepatic types and and 1997; PubMed Scopus Google Scholar). of MMP-2 may be by including the inflammatory of the are to the of MMP-2 by a membrane type matrix which MMP-2 M. J. Sci. 2001; Google Scholar). is that activation of MMP-2 a key in the following chronic tissue damage. of the normal in areas of of inflammatory cells these activation of MMP-2 be as a relevant to tissue In the we investigated the interaction of E2/CD81 in human HSC and the expression of MMP-2 following this interaction as a possible host to viral results that human HSC represent a potential target for HCV the liver and that binding of E2 to CD81 on HSC surface to an increased expression of by ERK/MAPK activation by HCV E2 protein used this study was a by the protein was from a cell transfected with L. PubMed Scopus Google in which the E2 from was to the tissue activator cell and by the protein was by and described of the HCV envelope E2 were J.M. Fong Y.L. Rosa D. Berge R.K. Saletti G. Campagnoli S. Bensi G. Capo S. Coates S. Crawford K. Dong C. Wininger M. Bake R.G. Cousens L. Chien D. Ng P. Archangel P. Grandi G. Houghton M. Abrignani S. J. Virol. 2000; 74: 6885-6892Crossref PubMed Scopus (66) Google Scholar). The was from with and for the to E2 to target cells as described J.M. Fong Y.L. Rosa D. Berge R.K. Saletti G. Campagnoli S. Bensi G. Capo S. Coates S. Crawford K. Dong C. Wininger M. Bake R.G. Cousens L. Chien D. Ng P. Archangel P. Grandi G. Houghton M. Abrignani S. J. Virol. 2000; 74: 6885-6892Crossref PubMed Scopus (66) Google Scholar). The CD81, and were from and from The was from The was from The was from was from in was the from was from and were on of human liver as described in M. S. R. C. A. A. G. R.G. P. J. 1996; Google Scholar). were with the anti-CD81 and, with the antibody. the of the were in and with and mouse was used as and of HSC were from of normal human liver for as reported A. M. S. C. Galli G. D. C. PubMed Google Scholar). a with HSC were from other liver cells by of was as described A. M. S. C. Galli G. D. C. PubMed Google Scholar). were on in with by and bovine serum described in this study were on cells between and cell these of cells and of In particular, of cell surface at these of in HSC with a cell identical to the detected in liver tissue at the between and the of the liver D. J. PubMed Scopus Google Scholar). In HSC were for in the of the different cell cells were and with serum in E2 protein was to cells for on were and with for further on cells were in and for on were in and a with were used as CD81 cells were with anti-CD81 with the mouse as and with an and as described A. S. C. P. A. P. M. J. PubMed Scopus Google Scholar). and cells were in with serum on for cells were for with in PBS, were for with were with E2 in and, with mAb, and was with the were with was by a of with a and were and by the anti-CD81 was as a of and a mouse were as a for E2 and CD81, E2 of E2 to HSC was by a HSC were with increasing of anti-CD81 and as and E2 for at cells, were with and cells were in and with in were with to cells were in and the of was by a at The of E2 protein binding to CD81 on human HSC was further by the of E2 from the binding to CD81 in the of the CD81 these HSC were with increasing of E2 protein to with anti-CD81 for at cells were and in and the of was as described from the cell were for degradation activity by The and was at a with the in the was by with were in a and at for The were by the with The of MMP-2 was with Western for Western were for and for were with and were by at The was to In the the MMP-2 enzyme was by of of with of The was with and the protein was with were by and to were with in for at and at with in The was used at were for with a at a in and with In cells were with a of the protein synthesis inhibitor a mouse was used to of CD81 cells were in essential bovine and at in a and cells at were used in into cells was by the with the to the with the of of of The transfected cell were by the cells in with bovine serum and a high of human CD81 was by and used for the and HSC were in CHAPS, PMSF, and were protein with anti-CD81 protein with mouse as a complexes were in and time in were on the of and as a was in these as an in inhibitor of type A. G. J. PubMed Scopus Google Scholar). were for at and with were with and the was by on to was a activity is expressed as a the cell on were in for to with were with PBS, in PMSF, and and by at for of was by to and for at with in and The membrane was at with an of at and by for with a of the antibody. The was and the HSC were in and in for to to E2 protein at different were with PBS, by and were in of PMSF, on for and at for were in of on for and at for The was in of B PMSF, and for at with were at for and were for at of C PMSF, were by at for at were a protein of were with of in of a of and as well as and The was on for by the of of binding was for The was to on a in The were and was at with an were by were with a of binding CD81 by RNA human CD81 was and is for human CD81 on was and was to of the was used as a The were by and, for were in the for at by at the was HSC were in the as of the cell following the for cell of cell with of the of was to a and with an were transfected the and were into in the CD81 protein expression levels at the cell surface were on a the The of was an antibody. for were to the of are expressed as was by of and, the was by and of CD81 in and with a study the binding of E2 in normal human liver R. Falugi F. Galli G. N. Rosa D. Campagnoli S. E. Houghton M. Abrignani S. Grandi G. J. Virol. 2000; 74: PubMed Scopus Google expression of CD81 was in the liver a was on a expression of CD81 was detected in In human liver with fibrosis and the expression of this tetraspanin was increased and in with and fibrosis HCV E2 CD81 on HSC of HCV E2 glycoprotein to human HSC surface was by The of E2 glycoprotein on HSC surface was high and with CD81 expression by human HSC, as detected by the anti-CD81 The of E2 glycoprotein in human HSC was investigated by and E2 the cell surface of HSC, the of membrane was observed by the anti-CD81 the of E2 binding to CD81 on human HSC, we E2 binding of HSC with increasing of anti-CD81 and HSC on in a as detected by The CD81 was to be in binding anti-CD81 The mouse was used as a The of E2 binding to CD81 on human HSC was further by the of HSC with E2 glycoprotein at increasing The cells were on with the anti-CD81 in C and the of HSC was in a HCV E2/CD81 the of MMP-2 in and in of HSC with HCV E2 glycoprotein induced a time-dependent increase of MMP-2 activity detected in HSC increased activity was observed of In addition, the increased MMP-2 activity induced by E2 glycoprotein in HSC was and at The effects of E2 on the expression of MMP-2 were further Western of HSC and cell a time-dependent increase of MMP-2 expression and MMP-2 expression was as as In order to the increased expression of MMP-2 was to synthesis to of the protein from intracellular the were by preincubating the cells with the protein synthesis inhibitor in induced the of increase in MMP-2 indicating that the up-regulation of MMP-2 E2 is to protein E2 induces synthesis of MMP-2 in human were with E2 glycoprotein for and HSC HSC was for Western were for and for cells were by and to were and with at with a the was with cells were for with of and for and with E2 cells with were used as with to protein are of HCV E2 to CD81 the of MMP-2 in E2 protein is to synthesis and activity of MMP-2 the binding to CD81, HSC were with different anti-CD81 and with E2 in the effect of E2 on MMP-2 activity was abolished by preincubating HSC with the neutralizing anti-CD81 (2Pileri P. Uematsu Y. Campagnoli S. Galli G. Falugi F. Petracca R. Weiner A.J. Houghton M. Rosa D. Grandi G. Abrignani S. Science. 1998; 282: 938-941Crossref PubMed Scopus (1776) Google with anti-CD81 in with the results of the E2 binding with anti-CD81 was HCV MMP-2 on HCV E2 glycoprotein is to bind CD81 in human and and other (2Pileri P. Uematsu Y. Campagnoli S. Galli G. Falugi F. Petracca R. Weiner A.J. Houghton M. Rosa D. Grandi G. Abrignani S. Science. 1998; 282: 938-941Crossref PubMed Scopus (1776) Google Scholar). In order to that the observed effects were on the binding of E2 to human CD81, identical were in NIH3T3 mouse fibroblasts transfected with human CD81. Accordingly, E2 is of binding CD81, of NIH3T3 mouse fibroblasts with E2 cause increase in MMP-2 activity the this effect was detected in human with identical time course observed in human HSC effect was observed in NIH3T3 cells transfected with the HCV E2/CD81 has been reported that a cell surface molecular between CD81 and the type by ERK/MAPK activation A. G. J. PubMed Scopus Google Scholar, F. K. J. 1997; PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). the activity of type following E2/CD81 in of HSC with E2 induced activation of type In order to the that E2 with other and we in the of which has been to be an in inhibitor of type A. G. J. PubMed Scopus Google Scholar, F. K. J. 1997; PubMed Scopus Google Scholar, A. PubMed Scopus Google Scholar). this a of activity was with an anti-CD81 was as a HCV E2/CD81 ERK/MAPK have that of CD81 by ERK/MAPK activity and of the protein in liver cells A. PubMed Scopus Google Scholar). Accordingly, we investigated the of ERK/MAPK activation following the binding of HCV E2 to CD81 in human with an the of that of these was increased in HSC by E2 glycoprotein In particular, the time course of ERK/MAPK activation that the with ERK/MAPK levels were to induced by a ERK/MAPK activator in with a levels of that the increase in was to an increase of the activity an increase in the of In order to the of ERK/MAPK in the expression of MMP-2 induced by we employing an inhibitor that the ERK/MAPK signaling in of HSC with was to the of induced by E2/CD81 the of E2 to gelatinase activity and increase MMP-2 protein levels was by the essential of ERK/MAPK activity in MMP-2 up-regulation E2 HCV ERK/MAPK for the of MMP-2 by and order to further into the of MMP-2 by the HCV E2/CD81 we the expression and binding activity of AP-2, a by ERK/MAPK and involved in MMP-2 K. J. 10: PubMed Google Scholar, S. J. 1997; PubMed Scopus Google Scholar, Y. J. PubMed Scopus Google Scholar, A. S. 1996; 16: PubMed Scopus Google Scholar, J. F. J. G. J. 1998; PubMed Scopus Google Scholar). the and the expression of binds to the binding of the MMP-2 and expression of MMP-2. in protein levels were increased by E2 The effect of E2 on protein levels at levels to by The of E2 to increase protein levels at was by on ERK/MAPK effect on protein levels In addition, from HSC exposed to E2 for increased binding to an binding This effect was of was observed in the of and binding was by a of binding Finally, the of E2 to increase the binding of HSC to the binding was by of CD81 the of HCV E2 on the CD81 to MMP-2 further the of CD81 in the intracellular signaling leading to the up-regulation of MMP-2 E2 a of CD81 was by the RNA interference HSC were transfected by with a a of CD81 The expression levels of CD81 protein at cell surface were by the of the of CD81. The CD81 protein expression was to of at to in the protein expression levels of the MMP-2 protein and the activity were in HSC with E2 in the synthesis of E2 was abolished in HSC with as well as the MMP-2 expression and the activity indicating that CD81 is essential in these effects in The HCV envelope E2 glycoprotein has been proposed as a key molecule regulating the interaction of HCV with surface of target E2 binds the major extracellular loop of human CD81, a tetraspanin expressed on various cell including hepatocytes and B lymphocytes. Regardless, the interaction between E2 and CD81 is for cell and the biological functions originating from this interaction are J.M. Fong Y.L. Rosa D. Berge R.K. Saletti G. Campagnoli S. Bensi G. Capo S. Coates S. Crawford K. Dong C. Wininger M. Bake R.G. Cousens L. Chien D. Ng P. Archangel P. Grandi G. Houghton M. Abrignani S. J. Virol. 2000; 74: 6885-6892Crossref PubMed Scopus (66) Google Scholar). In addition, has been on the interaction between HCV and information is the possible effects of HCV on liver This is by the expression of CD81 in areas in normal and human liver and in areas of and of chronic liver In particular, expression of this tetraspanin is in human HSC the of A. S. C. P. A. P. M. J. PubMed Scopus Google Scholar). This cell type is to a key in the of chronic liver the of extracellular matrix, the synthesis and of and other soluble involved in the of the inflammatory and the of a tissue to J. 2000; PubMed Scopus Google Scholar). Accordingly, the of the was to the possible effects induced by E2 binding to CD81 expressed on the surface of human that the interaction between the HCV E2 glycoprotein and CD81 expressed on the surface of human HSC to relevant effects to be in the and of this cell type chronic liver and, in the of chronic HCV degradation by MMP-2 is to and other MMPs, the activation of MMP-2 at the cell surface, which to this a key in cell 1996; PubMed Scopus Google Scholar). MMP-2 activity at the cell surface has been to with of the cell membrane to as for surface signaling molecules, and A. R. M. S. 2001; PubMed Scopus Google Scholar). of the is to be to the of a including and J. G. J. 1998; PubMed Scopus Google Scholar, A. E. K. Y. M. J. 1998; PubMed Scopus Google Scholar). from in different cell including HSC N. A. J. 1997; Google suggests that this is HSC express and A. J.A. J. PubMed Scopus Google Scholar, S. 1996; PubMed Scopus Google Scholar, N. A. 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In this the observed increase in the expression and activity of MMP-2 following the interaction between the HCV E2 glycoprotein and CD81 in human HSC of The of E2 binding to CD81 responsible for the effects was by binding of E2 to CD81 a time-dependent increase in the of the of with a at of as by zymography and Western on the expression of MMP-2 in of HSC with of In these in the expression of MMP-2 are observed following is that a time-dependent increase was for of and The of MMP-2 was by Western in and cell This effect was to protein synthesis of from intracellular was abolished by preincubating the cells with In addition, the time of this effect suggests that up-regulation of MMP-2 by intracellular by the interaction of E2 with CD81. The results of by preincubating HSC with an anti-CD81 that the binding of E2 to CD81 further that the increased expression of MMP-2 was to the interaction between E2 and CD81. In order to that this effect was on the binding of E2 with human CD81, identical were in NIH3T3 mouse fibroblasts transfected with human CD81. E2 is to bind CD81, of NIH3T3 cells with E2 cause increase in MMP-2 this effect was detected in NIH3T3 cells transfected with human CD81 with identical time course features. This was further by in human HSC in which a of CD81 was by the RNA interference important of is to with and with various other transmembrane into has been that CD81 J. Google and as on the cell surface that an important in regulating Since we that E2 was to type the binding with CD81 in HSC, we that the of CD81 induced by E2 at the membrane activity and synthesis an essential in regulating of signaling at the membrane Annu. Rev. 1998; PubMed Scopus Google Scholar). In particular, membrane of is an important that activation of the ERK/MAPK Accordingly, we have that CD81 synthesis of with the of to the membrane via the binding and this of to the activation of ERK/MAPK A. PubMed Scopus Google Scholar). this we investigated the effect of E2/CD81 binding on the ERK/MAPK signaling pathway in human that the binding of E2 to CD81 ERK/MAPK to in human In addition, the of E2 to MMP-2 enzyme activity and protein levels was by a inhibitor of the signaling that ERK/MAPK activity is essential for MMP-2 expression E2 The human MMP-2 a in which an binding that MMP-2 expression K. J. 10: PubMed Google Scholar, S. J. 1997; PubMed Scopus Google Scholar, Y. J. PubMed Scopus Google Scholar). suggests that the is as an important of MMP-2 in other as cells M. S. J. J. J. 282: PubMed Scopus Google Scholar). activation of the ERK/MAPK has been to binding activity A. S. 1996; 16: PubMed Scopus Google Scholar, J. F. J. G. J. 1998; PubMed Scopus Google Scholar). results that HCV E2 binding to CD81 results in increased protein levels in human This increase is the ERK/MAPK the of E2 to levels is by an inhibitor of the ERK/MAPK In addition, increased levels induced by binding are with an increase in the binding of HSC to an binding The binding activity as well as the increase in MMP-2 protein levels and gelatinase activity are by These suggest that HCV E2 glycoprotein MMP-2 activity by of the MMP-2 and that activation is by the ERK/MAPK signaling as in the proposed in The results of the study the possible interaction between HCV and liver cells leading to and these R. PubMed Scopus Google reported that HCV and the and into HSC by effects in this cell including an increase of and of from the that HCV into HSC and the suggest that may cause an increased MMP-2 synthesis and by with the membrane of of these a of HCV relevant for the of the of and fibrosis in the of chronic HCV M. S. and for and