A new clue for the pathogenesis of hepatitis C virus infection: Activation of the MAPK/ERK signaling initiated by envelope protein 2

There are highly complicated signal systems in response to a variety of environmental stimuli in organisms. Recently, intensive studies have focused on the relationship between human diseases and alterations of cellular signal transduction. A number of human diseases, such as angiocardiopathy, diabetes and cancer, have been identified to be correlative with disruption of signaling. It was estimated that approximately 3% of world抯 population was infected with hepatitis C virus (HCV), and 70%…     

丙型肝炎病毒E2蛋白对HepG2细胞MAPK／ERK的激活

人CD81是丙型肝炎病毒（hepatitis Cvirus,HCV）的细胞表面特异性受体,HCV包膜蛋白－2（E2）可与其结合。细胞个信号调节激酶（extracellular signal-regulated protein kinase,MAPK/ERK1,2）信号途径主要介导细胞增殖及分化。为探讨HCV E2蛋白与人CD81结合对MAPK／ERK活性变化的影响,以HCV E2蛋白刺激HepG2细胞,采用免疫印迹、免疫组化及免疫荧光等方法动态观察细胞内MAPK／ERK的激活情况,并以流式细胞术检测细胞表面CD81的表达。结果表明：HepG2细胞高表达人CD81;HCV E2蛋白可激活细胞内MAPK／ERK;MAPK／ERK的磷酸化反应与HCV E2蛋白浓度、作用时间呈依赖关系;HCV E2－CD81相互作用引发的细胞异常信号转导可能与HCV致病性相关。     

Adaptation of Hepatitis C Virus to Mouse CD81 Permits Infection of Mouse Cells in the Absence of Human Entry Factors

Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR-BI), CD81, claudin-1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species-specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100-fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2-specific antibodies indicative of major conformational changes of virus-resident E1/E2-complexes. Neutralization with CD81, SR-BI- and claudin-1-specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR-BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species-specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV.     

Down-regulation of HIV-1 infection by inhibition of the MAPK signaling pathway

The human immunodeficiency virus type 1 (HIV-1) can interact with and exploit the host cellular machinery to replicate and propagate itself. Numerous studies have shown that the Mitogen-activated protein kinase (MAPK) signal pathway can positively regulate the replication of HIV-1, but exactly how each MAPK pathway affects HIV-1 infection and replication is not understood. In this study, we used the Extracellular signal-regulated kinase (ERK) pathway inhibitor, PD98059, the Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, and the p38 pathway inhibitor, SB203580, to investigate the roles of these pathways in HIV-1 replication. We found that application of PD98059 results in a strong VSV-G pseudotyped HIV-1_NL4-3 luciferase reporter virus and HIV-1_NL4-3 virus inhibition activity. In addition, SB203580 and SP600125 also elicited marked VSV-G pseudotyped HIV-1_NL4-3 luciferase reporter virus inhibition activity but no HIV-1_NL4-3 virus inhibition activity. We also found that SB203580 and SP600125 can enhance the HIV-1 inhibition activity of PD98059 when cells were treated with all three MAPK pathway inhibitors in combination. Finally, we show that HIV-1 virus inhibition activity of the MAPK pathway inhibitors was the result of the negative regulation of HIV-1 LTR promoter activity.     

Ligand-independent activation of the androgen receptor by insulin-like growth factor-i and the role of the MAPK pathway in skeletal muscle cells

In this study, the roles of the p38 MAPK, ERK1/2 and JNK signaling pathway in IGF-I-induced AR induction and activation were examined. C2C12 cells were treated with IGF-I in the absence or presence of various inhibitors of p38 MAPK (SB203580), ERK1/2 (PD98059), and JNK (SP600125). Inhibition of the MAPK pathway with SB203580, PD98059, or SP600125 significantly decreased IGF-I-induced AR phosphorylation and total AR protein expression. IGF-I-induced nuclear fraction of total AR and phosphorylated AR were significantly inhibited by SB203580, PD98059, or SP600125. Furthermore, IGF-I-induced AR mRNA and skeletal α-actin mRNA were blocked by those inhibitors in dose-dependent manner. Confocal images showed that IGF-I-induced AR nuclear translocation from cytosol was significantly blocked by SB203580, PD98059, or SP600125, suggesting that the MAPK pathway regulates IGF-I-induced AR nuclear localization in skeletal muscle cells. The present results suggest that the MAPK pathways are required for the ligand-independent activation of AR by IGF-I in C2C12 skeletal muscle cells.     

Mitogen-activated protein kinase signalling pathways triggered by the hepatitis C virus envelope protein E2: implications for the prevention of infection

OBJECTIVE: Hepatitis C virus (HCV) is a major pathogenic factor of liver diseases. During HCV infection, interaction of the envelope protein E2 of the virion, with target cells, is a crucial process for viral penetration into the cell and its propagation. We speculate that such interaction may trigger early signalling events required for HCV infection. MATERIALS AND METHODS: Human liver cell line L-02 was treated with HCV E2. The kinase phosphorylation levels of mitogen-activated protein kinase (MAPK) signalling pathways in the treated cells were analyzed by Western blotting. The proliferation of the E2-treated cells was evaluated by MTT assay. RESULTS: HCV E2 was shown to be an efficient activator for MAPK pathways. Levels of phosphorylation of upstream kinases Raf-1 and MEK1/2 were seen to be elevated following E2 treatment and similarly, phosphorylation levels of downstream kinases MAPK/ERK and p38 MAPK also increased in response to E2 treatment, and specificity of kinase activation by E2 was confirmed. E2-induced MAPK/ERK activation was inhibited by the MEK1/2 inhibitor U0126 in a concentration-dependent manner. Blockage of relevant cellular receptors reduced activation of Raf-1, MEK1/2, MAPK/ERK and p38 MAPK by E2, indicating efflux of the E2 signal from extracellular to the intracellular spaces. Thus, kinase cascades of MAPK pathways were continuously affected by E2 presence. Moreover, enhancement of cell proliferation by E2 appeared to be associated with the dynamic phosphorylation of MAPK/ERK and p38 MAPK. CONCLUSION: These results suggest that MAPK signalling pathways triggered by E2 may be a potential target for prevention of HCV infection.     

Binding of hepatitis C virus envelope protein E2 to CD81 up-regulates matrix metalloproteinase-2 in human hepatic stellate cells

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.     

IL-2 activation of NK cells: involvement of MKK1/2/ERK but not p38 kinase pathway

IL-2 stimulates extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) in various immune cell populations. The functional roles that these kinases play are still unclear. In this study, we examined whether MAPK kinase (MKK)/ERK and p38 MAPK pathways are necessary for IL-2 to activate NK cells. Using freshly isolated human NK cells, we established that an intact MKK/ERK pathway is necessary for IL-2 to activate NK cells to express at least four known biological responses: LAK generation, IFN-gamma secretion, and CD25 and CD69 expression. IL-2 induced ERK activation within 5 min. Treatment of NK cells with a specific inhibitor of MKK1/2, PD98059, during the IL-2 stimulation blocked in a dose-dependent manner each of four sequelae, with inhibition of lymphokine-activated killing induction being least sensitive to MKK/ERK pathway blockade. Activation of p38 MAPK by IL-2 was not detected in NK cells. In contrast to what was observed by others in T lymphocytes, SB203850, a specific inhibitor of p38 MAPK, did not inhibit IL-2-activated NK functions. This data indicate that p38 MAPK activation was not required for IL-2 to activate NK cells for the four functions examined. These results reveal selective signaling differences between NK cells and T lymphocytes; in NK cells, the MKK/ERK pathway and not p38 MAPK plays a critical positive regulatory role during activation by IL-2.     

Evaluation of hepatitis C virus glycoprotein E2 for vaccine design: an endoplasmic reticulum-retained recombinant protein is superior to secreted recombinant protein and DNA-based vaccine candidates

Hepatitis C virus (HCV) is the leading causative agent of blood-borne chronic hepatitis and is the target of intensive vaccine research. The virus genome encodes a number of structural and nonstructural antigens which could be used in a subunit vaccine. The HCV envelope glycoprotein E2 has recently been shown to bind CD81 on human cells and therefore is a prime candidate for inclusion in any such vaccine. The experiments presented here assessed the optimal form of HCV E2 antigen from the perspective of antibody generation. The quality of recombinant E2 protein was evaluated by both the capacity to bind its putative receptor CD81 on human cells and the ability to elicit antibodies that inhibited this binding (NOB antibodies). We show that truncated E2 proteins expressed in mammalian cells bind with high efficiency to human cells and elicit NOB antibodies in guinea pigs only when purified from the core-glycosylated intracellular fraction, whereas the complex-glycosylated secreted fraction does not bind and elicits no NOB antibodies. We also show that carbohydrate moieties are not necessary for E2 binding to human cells and that only the monomeric nonaggregated fraction can bind to CD81. Moreover, comparing recombinant intracellular E2 protein to several E2-encoding DNA vaccines in mice, we found that protein immunization is superior to DNA in both the quantity and quality of the antibody response elicited. Together, our data suggest that to elicit antibodies aimed at blocking HCV binding to CD81 on human cells, the antigen of choice is a mammalian cell-expressed, monomeric E2 protein purified from the intracellular fraction.     

Involvement of mitogen-activated protein kinase in agonist-induced phosphorylation of the mu-opioid receptor in HEK 293 cells

Agonist exposure of many G protein-coupled receptors stimulates an activation of extracellular signal-regulated protein kinases (ERKs) 1 and 2, members of the mitogen-activated protein kinase (MAPK) family. Here, we show that treatment of human embryonic kidney (HEK) 293 cells stably transfected to express the rat micro-opioid receptor (MOR1) with [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO) stimulated a rapid and transient (3-5-min) activation and nuclear translocation of MAPK. Exposure of these cells to the MAPK kinase 1 inhibitor PD98059 not only prevented MAPK activation but also inhibited homologous desensitization of the mu-opioid receptor. We have therefore determined the effect of PD98059 on agonist-induced mu-receptor phosphorylation. DAMGO stimulated a threefold increase in MOR1 phosphorylation within 20 min that could be reversed by the antagonist naloxone. PD98059 produced a dose-dependent inhibition of agonist-promoted mu-receptor phosphorylation with an IC50 of 20 microM. DAMGO also induced MOR1 internalization that peaked at 30 min. Confocal microscopy revealed that DAMGO-induced MOR1 internalization was also largely inhibited in the presence of PD98059. U0126, another chemically unrelated inhibitor of the MAPK cascade, mimicked the effect of PD98059 on mu-receptor phosphorylation and desensitization. MOR1 itself, however, appears to be a poor substrate for MAPK because mu-receptors immunoprecipitated from stably transfected HEK 293 cells were not phosphorylated by exogenous ERK 2 in vitro. The fact that morphine also triggered MAPK activation but did not induce MOR1 internalization indicates that receptor internalization was not required for MOR1-mediated mitogenic signaling. We conclude that MOR1 stimulates a rapid and intemalization-independent MAPK activation. Activation of the MAPK cascade in turn may not only relay mitogenic signals to the nucleus but also trigger initial events leading to phosphorylation and desensitization of the mu-opioid receptor.     

Hepatitis C virus protects human B lymphocytes from Fas-mediated apoptosis via E2-CD81 engagement

HCV infection is often associated with B-cell regulatory control disturbance and delayed appearance of neutralizing antibodies. CD81 is a cellular receptor for HCV and can bind to HCV envelope protein 2 (E2). CD81 also participates to form a B cell costimulatory complex. To investigate whether HCV influences B cell activation and immune function through E2 -CD81 engagement, here, human Burkitt's lymphoma cell line Raji cells and primary human B lymphocytes (PHB) were treated with HCV E2 protein and cell culture produced HCV particles (HCVcc), and then the related cell phenotypes were assayed. The results showed that both E2 and HCVcc triggered phosphorylation of IκBα, enhanced the expression of anti-apoptosis Bcl-2 family proteins, and protected Raji cells and PHB cells from Fas-mediated death. In addition, both E2 protein and HCVcc increased the expression of costimulatory molecules CD80, CD86 and CD81 itself, and decreased the expression of complement receptor CD21. The effects were dependent on E2-CD81 interaction on the cell surface, since CD81-silenced Raji cells did not respond to both treatments; and an E2 mutant that lose the CD81 binding activity, could not trigger the responses of both Raji cells and PHB cells. The effects were not associated with HCV replication in cells, for HCV pseudoparticle (HCVpp) and HCVcc failed to infect Raji cells. Hence, E2-CD81 engagement may contribute to HCV-associated B cell lymphoproliferative disorders and insufficient neutralizing antibody production.     

E5 Protein of Human Papillomavirus Type 16 Protects Human Foreskin Keratinocytes from UV B-Irradiation-Induced Apoptosis

The human papillomavirus type 16 (HPV16) E5 protein associates with the epidermal growth factor receptor (EGFR) and enhances the activation of the EGFR after stimulation by EGF in human keratinocytes. Phosphatidylinositol 3-kinase (PI3K) and ERK1/2 mitogen-activated protein kinase (ERK1/2 MAPK), two signal molecules downstream of the EGFR, have been recognized as participants in two survival signal pathways in response to stress. The fact that E5 can enhance EGFR activation suggests that E5 might act as a survival factor. To test this hypothesis, the apoptotic response of UV B-irradiated primary keratinocytes infected with either control retrovirus, LXSN, or HPV16 2E5-expressing recombinant retrovirus was quantitated. Under the same conditions, LXSN-infected cells showed extensive apoptosis, while E5-expressing cells demonstrated a significant reduction in UV B-irradiation-induced apoptosis. The E5-mediated protection against apoptosis was blocked by wortmannin and PD98059, specific inhibitors of the PI3K and ERK1/2 MAPK pathways, respectively, suggesting that the PI3K and ERK1/2 MAPK pathways are involved in this process. Western blot analysis showed that Akt (also named protein kinase B), which is a downstream effector of PI3K, and ERK1/2 MAPK were activated by EGF. When cells were stimulated by EGF and irradiated by UV B, the levels of phospho-Akt and phospho-ERK1/2 activated by EGF in E5-expressing cells were about twofold greater than those in LXSN-infected cells. Two other UV-activated stress pathways, p38 and JNK, were activated to the same level during UV B irradiation in both LXSN-infected cells and E5-expressing cells, indicating that E5 protein did not affect these two pathways. After UV B irradiation, p53 was activated in both LXSN-infected cells and E5-expressing cells, and cell cycle analysis showed that nearly all cells in both cell populations were growth arrested. These data suggest that unlike HPV16 E6, which blocks apoptosis by inactivation of p53, HPV16 E5 protects cells from apoptosis by enhancing the PI3K-Akt and ERK1/2 MAPK signal pathways.     

Binding of the hepatitis C virus E2 glycoprotein to CD81 is strain specific and is modulated by a complex interplay between hypervariable regions 1 and 2

The envelope glycoprotein E2 of hepatitis C virus (HCV) is the target of neutralizing antibodies and is presently being evaluated as an HCV vaccine candidate. HCV binds to human cells through the interaction of E2 with the tetraspanin CD81, a putative viral receptor component. We have analyzed four different E2 proteins from 1a and 1b viral isolates for their ability to bind to recombinant CD81 in vitro and to the native receptor displayed on the surface of Molt-4 cells. A substantial difference in binding efficiency between these E2 variants was observed, with proteins derived from 1b subtypes showing significantly lower binding than the 1a protein. To elucidate the mechanism of E2-CD81 interaction and to identify critical regions responsible for the different binding efficiencies of the E2 variants, several mutants were generated in E2 protein regions predicted by computer modeling to be exposed on the protein surface. Functional analysis of these E2 derivatives revealed that at least two distinct domains are responsible for interaction with CD81. A first segment centered around amino acid residues 613 to 618 is essential for recognition, while a second element including the two hypervariable regions (HVRs) modulates E2 receptor binding. Binding inhibition experiments with anti-HVR monoclonal antibodies confirmed this mapping and supported the hypothesis that a complex interplay between the two HVRs of E2 is responsible for modulating receptor binding, possibly through intramolecular interactions. Finally, E2 proteins from different isolates displayed a profile of binding to human hepatic cells different from that observed on Molt-4 cells or isolated recombinant CD81, indicating that additional factors are involved in viral recognition by target liver cells.     

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.     

The role of progestin receptors and the mitogen-activated protein kinase pathway in delta opioid receptor facilitation of female reproductive behaviors

The present study investigated the role of the progestin receptor (PR) and the mitogen-activated protein kinase (MAPK) pathway in the facilitation of lordosis behavior by the delta opioid receptor agonist [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE). Ovariectomized, estrogen-primed rats were treated with the PR antagonist RU486 or the MAPK inhibitor PD98059 prior to intraventricular (icv) infusion of DPDPE. Both RU486 and PD98059 blocked receptive and proceptive behaviors induced by DPDPE at 60 min, and RU486 continued to inhibit estrous behavior at 90 min. Because delta opioid receptors can activate the p42/44 MAPKs, extracellular signal regulated kinases (ERK), we determined the effects of DPDPE on ERK phosphorylation. Icv infusion of DPDPE increased the levels of phosphorylated ERK in the hypothalamus and preoptic area of female rats, assessed by immunoblotting. These results support the participation of the PR and the MAPK pathway in the facilitation of lordosis behavior by delta opioid receptors.     

CD81 and claudin 1 coreceptor association: role in hepatitis C virus entry

Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.     

Transient Activation of the PI3K-AKT Pathway by Hepatitis C Virus to Enhance Viral Entry

The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Here we report that hepatitis C virus (HCV) transiently activates the PI3K-AKT pathway. This activation was observed as early as 15 min postinfection, peaked by 30 min, and became undetectable at 24 h postinfection. The activation of AKT could also be mediated by UV-inactivated HCV, HCV pseudoparticle, and the ectodomain of the HCV E2 envelope protein. Because antibodies directed against CD81 and claudin-1, but not antibodies directed against scavenger receptor class B type I or occludin, could also activate AKT, the interaction between HCV E2 and its two co-receptors CD81 and claudin-1 probably triggered the activation of AKT. This activation of AKT by HCV was important for HCV infectivity, because the silencing of AKT by siRNA or the treatment of cells with its inhibitors or with the inhibitor of its upstream regulator PI3K significantly inhibited HCV infection, whereas the expression of constitutively active AKT enhanced HCV infection. The PI3K-AKT pathway is probably involved in HCV entry, because the inhibition of this pathway could inhibit the entry of HCV pseudoparticle but not the VSV pseudoparticle into cells. Furthermore, the treatment of cells with the AKT inhibitor AKT-V prior to HCV infection inhibited HCV infection, whereas the treatment after HCV infection had no obvious effect. Taken together, our studies indicated that HCV transiently activates the PI3K-AKT pathway to facilitate its entry. These results provide important information for understanding HCV replication and pathogenesis and raised the possibility of targeting this cellular pathway to treat HCV patients.